InteractiveFly: GeneBrief

C-terminal binding protein : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homology | References

BIOLOGICAL OVERVIEW

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, a yeast interaction screen was carried out and a Hairy-interacting protein was identified, the Drosophila homolog of the human C-terminal-binding protein (CtBP) (Poortinga, 1998). CtBP is the subject of this overview, but to put its function in perspective, it will be necessary to first discuss Hairy.

The Hairy and Enhancer of split proteins are characterized by two conserved domains, the Orange domain and the C-terminal conserved tetrapeptide WRPW. Functional studies have shown that both of these domains, as well as the bHLH domain, are needed for the proper function of these proteins. The Orange domain contributes to the functional specificity of Hairy/E(spl) proteins (Dawson, 1995 and Giebel, 1997). The WRPW motif has been shown to be necessary and sufficient for the recruitment of Groucho, a WD repeat-containing protein that is not able to bind DNA on its own but, when brought to an endogenous or heterologous promoter, serves as a strong repressor of transcription. Together, these results have led to the prevailing view that Hairy functions as a promoter-bound repressor. An intact bHLH region is required for Hairy to bind to specific DNA sites; once bound, Hairy then recruits the Groucho co-repressor protein (Poortinga, 1998).

Recruitment of Groucho does not account for all of Hairy's repressor properties. Hairy can function genetically as a repressor in the absence of the WRPW motif, and presumably in the absence of the Groucho co-repressor. The Orange domain has been shown to be required for proper function of Hairy and E(spl)m8. This suggests that Hairy is involved in separable repression mechanisms: repression in some cases requiring the bHLH and Orange domains, and in other cases, requiring the bHLH and C-terminal WRPW motif (Dawson et al., 1995). Thus, Hairy may function modularly, with the scope and specificity of its interactions dependent on the proteins recruited to its various conserved domains. Based on the expectation that Hairy works as part of a multiprotein complex, the yeast two-hybrid protein interaction system was employed to identify a Hairy-interacting protein, the Drosophila homolog of human C-terminal binding protein (CtBP), that interacts with a small, previously uncharacterized C-terminal region of Hairy (Poortinga, 1998).

Drosophila CtBP plays a role in embryonic segmentation. Reduction of maternal dCtBP results in severe segmentation defects visualized by cuticle preparations or engrailed staining. Expression of the primary pair-rule genes, eve and runt, as well as expression of hairy itself, is disrupted in embryos lacking maternal dCtBP, whereas the level and spatial positioning of gap gene expression appears normal. The primary pair-rule genes are required to establish each other's expression as well as to direct the striped expression of downstream secondary pair-rule genes, such as fushi tarazu. Similar to what is seen in hairy mutant embryos, Ftz stripes are expanded throughout the trunk region in embryos lacking maternal dCtBP. The physical interaction of CtBP with Hairy and CtBP's genetic interaction with hairy indicate a role for dCtBP in Hairy-mediated repression. The domain of Hairy that interacts with CtBP contains a five amino acid repeat (PLSLV) that bears sequence similarity to a six amino acid motif in the E1a C-terminus (PXDLSX) which interacts with mammalian CtBP (Poortinga, 1998).

CtBP is involved in transcriptional repression mediated by Knirps and Snail. Knirps and Snail are thought of as short-range repressors, acting over distances of less than 100 base pairs to quench upstream activators or the core transcription complex. This form of repression allows enhancers to work independently of one another to direct complex, additive patterns of gene expression, including the seven-stripe patterns of even-skipped and hairy expression. A P-DLS-K sequence is present in the repression domains of Knirps and Snail, and the latter protein also contains the related sequence P-DLS-R. Gene dosage assays suggest that Knirps and Drosophila CtBP interact in vivo. Embryos that are heterozygous for the knirps⁹ null mutation exhibit occasional defects in the even-skipped expression pattern, including reduced staining of stripe 5. Combining the Drosophila CtBP and knirps⁹ mutation results in more severe disruptions in the eve pattern, including the fusion or loss of stripes 4 and 6. The latter knirps⁹/CtBP transheterozygous phenotype is virtually indistinguishable from that observed for knirps mutant embryos. These results raise the possiblility that Drosophila CtBP is part of a larger co-repressor complex that assembles on the Knirps/DNA template. Snail might also require CtBP, because embryos derived from dCrBP mutant oocytes exhibit dorsoventral patterning defects. The C-terminal repression domain of a third short-range repressor, Kruppel, contains a sequence that is related to the mammalian and Drosophila CtBP interaction sequence -- P-DLS-H. Mutations in this sequence nearly abolish Kruppel-mediated repression in human osteocarcinoma cells (Nibu, 1998a).

CtBP may mediate repression throught the enzymatic modification of chromatin because it sequence is related to D-isomer 2-hydroxy acid dehydrogenases. Despite this rather unexpected homology, immunolocalization assays indicate that the Drosophila CtBP protein accumulates in nuclei. Perhaps CtBP cause local changes in chromatin structure by introducing subtle changes in core histones. Alternatively, it is possible that CrBP is a component of an enzymatic cascade that modulates the activities of histone deacetylases or other co-repressor proteins (Nibu, 1998a).

CtBP-independent repression in the Drosophila embryo

There are three mechanisms of transcriptional repression in eukaryotes. The first is quenching, whereby repressors and activators co-occupy closely linked sites and then the repressor inhibits adjacent activators. The second is direct repression, in which repressors block the function of the core transcription complex. The third is competition, in which repressors compete with activators for a common DNA-binding site. Previous studies have shown that the Drosophila CtBP corepressor (dCtBP) is essential for the quenching activity of three short-range sequence-specific repressors in the early Drosophila embryo: Krüppel, Knirps, and Snail. This study demonstrates that dCtBP is dispensable for target enhancers that contain overlapping activator and repressor binding sites. However, it is essential when Krüppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter. These findings provide evidence that competition is distinct from quenching and direct repression. Quenching and direct repression depend on dCtBP, whereas competition does not (Nibu, 2003 ).

Krüppel is a zinc finger DNA-binding protein that is composed of 502 aa residues. The quenching activity of the C-terminal repression domain (aa 402 to 502) requires a dCtBP interaction motif located at amino acids (aa) 464 to 470. Another repression domain has been identified in cultured cells. It is located between aa 62 and 92 and does not contain a dCtBP interaction motif. A transgenic embryo assay was used to determine whether this N-terminal repression domain might be a source for CtBP-independent repression in early embryos (Nibu, 2003).

A Gal4-Krüppel fusion protein containing aa 402 to 502 created gaps in the staining patterns directed by st2.UAS-st3-lacZ, NEE.UAS-lacZ, and NEE.UAS-twi-lacZ. The st2.UAS-st3-lacZ reporter gene contains Gal4 UAS binding sites near the distal eve stripe 2 enhancer (st2). NEE.UAS-lacZ reporter gene is driven by a modified 200-bp rhomboid rhomboid lateral stripe neurectoderm (NEE) enhancer that contains three Gal4 binding sites and three Dorsal activator sites. This reporter gene is normally activated in the ventral mesoderm. For the st2.UAS-st3-lacZ (st3 is the eve stripe 3 enhancer) and NEE.UAS-twi-lacZ reporter genes, repression was observed only for the staining pattern produced by the enhancer containing UAS binding sites. For example, the binding of the Gal4-Krüppel fusion protein to the stripe 2 enhancer does not alter expression from the neighboring stripe 3 enhancer. Similarly, the binding of the fusion protein to the rhomboid NEE enhancer does not alter expression from the twist enhancer. Substitutions in three of the amino acid residues within the dCtBP interaction motif (PEDLSMH to AAALSMH) eliminate the repression activity of an otherwise normal Gal4-Krüppel fusion protein (Nibu, 2003).

There is a second potential dCtBP interaction motif, located between aa 414 and 420 (PLDLSED), that weakly binds dCtBP in vitro. However, this second motif is not sufficient to support discernible repression activity in vivo. These results suggest that most or all of the repression activity of the Gal4-Krüppel 402-502 fusion protein resides within the major dCtBP interaction motif between amino acid residues 464 and 470. Moreover, repression is not observed for a Gal4-Krüppel fusion protein that contains the N-terminal repression domain (aa 62 to 92). These results suggest that the C-terminal dCtBP motif mediates most or all of the quenching activity in the early embryo (Nibu, 2003).

The proximal UAS site within the NEE.UAS-lacZ reporter gene is located 120 bp 5' of the core promoter, slightly beyond the range of Krüppel-mediated repression. In contrast, the UAS sites map within 50 bp of critical Dorsal sites within the NEE. Thus, repression of the reporter gene is most likely due to quenching rather than the direct repression of the core promoter. Another lacZ reporter was created to investigate this issue, NEE-5xUAS-lacZ. The most distal UAS site is located 250 bp 5' of the most proximal Dorsal binding site within the modified 700-bp NEE enhancer, while the most proximal UAS site is located just 57 bp 5' of the transcription start site of the hsp70 promoter. The Gal4-Krüppel 402-502 fusion protein attenuates lacZ expression. This direct repression is not obtained with the mutagenized fusion protein lacking the dCtBP interaction motif or with a fusion protein containing the N-terminal repression domain. These results suggest that the C-terminal dCtBP interaction motif is essential for both quenching and direct repression (Nibu, 2003).

Previous studies suggest that Krüppel mediates quenching by recruiting dCtBP to distal enhancers, such as the eve stripe 2 enhancer. An NEE-lacZ reporter gene that contains two synthetic Krüppel recognition sequences located 50 bp 5' of the most distal Dorsal binding site and 50 bp 3' of the most proximal site was created. This enhancer lacks the native Snail repressor sites and therefore directs lacZ expression in both lateral and ventral regions of early embryos. lacZ staining was diminished in central regions due to the localized expression of the Krüppel repressor. This gap in the pattern was eliminated in Kr¹/Kr¹ mutant embryos. Krüppel also failed to repress the reporter gene in mutant embryos derived from dCtBP germ line clones. These results indicate that dCtBP⁺ gene activity is required for the quenching activity of the Krüppel repressor (Nibu, 2003).

Subsequent experiments were done to determine whether dCtBP is required for the direct-repression activity of Krüppel and another short-range repressor, Knirps. lacZ transgenes with either Krüppel or Knirps binding sites located near the core promoter were examined. Both transgenes contain two tandem copies of the 250-bp twist proximal enhancer placed either upstream or downstream of rhomboid lateral stripe enhancers (NEE). In wild-type embryos, the enhancers direct additive patterns of expression in the lateral neurogenic ectoderm and ventral mesoderm. A single Krüppel binding site located 75 bp 5' of the transcription start site was sufficient to create a central gap in both staining patterns. Staining directed by the tandem twist enhancers was nearly eliminated, whereas the lateral stripe produced by the rhomboid NEE was diminished. Repression of the twist pattern is almost certainly due to direct repression, since the solo Krüppel site maps more than 800 bp from the nearest Dorsal activator site in the twist enhancer. Krüppel-mediated repression is lost when the transgene is introduced into embryos obtained from dCtBP germ line clones. There is no longer a central gap in the staining pattern. Moreover, there is a fusion of the expression patterns directed by the twist and NEE enhancers due to a loss in the activity of the Snail repressor. Normally, Snail binds to the NEE enhancer and represses expression in the ventral mesoderm, thereby restricting the staining pattern to lateral stripes in the neurogenic ectoderm. The broad uniform staining pattern obtained in dCtBP mutants suggests that the dCtBP corepressor is required for the direct repression of the core promoter (Nibu, 2003).

Similar results were obtained with the Knirps repressor. In this case, two tandem Knirps binding sites were placed 55 bp 5' of the transcription start site. In wild-type embryos, there is a clean gap in both the NEE-mediated lateral stripes and the twist-mediated staining pattern in the ventral mesoderm. This gap coincides with the site of Knirps expression in the presumptive abdomen. As seen for Krüppel, the gap in the staining patterns disappears in dCtBP mutant embryos. These results suggest that dCtBP is required for the direct repression activities of both Krüppel and Knirps (Nibu, 2003).

The preceding experiments suggest that dCtBP is required for both quenching and the direct repression of the core promoter. A synthetic lacZ reporter gene was prepared to determine whether Krüppel can mediate repression by competition and, if so, whether dCtBP is required for this repression. A 14-bp oligonucleotide that contains overlapping Dorsal and Krüppel binding sites was synthesized. Each subunit of the Dorsal homodimer binds to an inverted half-site: GGG...CCC. Krüppel binds DNA as a monomer, and the core recognition sequence includes the CCC Dorsal half-site. This short sequence also contains an optimal Bicoid binding site (GGATTA). This motif is located between the two half-sites of the Dorsal recognition sequence and overlaps the Krüppel consensus sequence (Nibu, 2003).

Gel shift assays were done to determine whether Dorsal and Krüppel bind the synthetic 14-bp sequence in a mutually exclusive manner. A 30-bp fragment that contains the 14-bp sequence along with 8 bp of flanking sequence at each end was synthesized. In the first set of experiments, a full-length Krüppel protein produced in E. coli was mixed with the 30-bp fragment and fractionated on an agarose gel. A shifted Krüppel-DNA complex was observed. The addition of increasing amounts of the Dorsal DNA-binding domain (Dl DBD; aa 1 to 403) resulted in the gradual loss of this complex. A new complex that is identical in size to those obtained with the Dorsal protein alone was observed. These results suggest that high concentrations of the Dorsal DNA-binding domain can displace Krüppel (Nibu, 2003).

Similar results were obtained in reciprocal DNA-binding assays. In this case, the shifted Dorsal-DNA complex was formed in the absence of Krüppel. The addition of increasing amounts of the Krüppel protein resulted in the gradual loss of the Dorsal-DNA complex. A new complex was obtained that has the same size as the one observed with increasing amounts of Krüppel in the absence of the Dorsal protein. These results suggest that increasing amounts of Krüppel can displace Dorsal-DNA complexes. Thus, the gel shift assays indicate mutually exclusive binding of Dorsal and Krüppel to the overlapping binding sites contained within the 14-bp fragment (Nibu, 2003).

Transient-transfection assays were used to determine whether the Krüppel DNA-binding domain is sufficient to mediate transcriptional repression. Six tandem copies of the synthetic oligonucleotide used in the preceding DNA-binding assays were attached to an eve-luciferase reporter gene containing the minimal eve promoter. This reporter gene was introduced into mbn-2 cultured cells (a Drosophila blood cell line) along with various expression vectors containing Dorsal or Krüppel coding sequences. An expression vector containing the full-length Dorsal coding sequence (Dl FL) produced a 6 fold induction in luciferase activity. However, an expression vector containing the Krüppel DNA-binding domain (Kr DBD; aa 217 to 401) reduced luciferase activity to background levels. This reduction in reporter gene expression was not obtained with a Krüppel expression vector that contained a single amino acid substitution in the zinc finger DNA-binding domain (Kr⁹ DBD). These results suggest that Krüppel can repress the synthetic enhancer by simply binding DNA and excluding the Dorsal activator. Repression does not depend on Krüppel protein sequences that map outside the DNA-binding domain. Subsequent experiments were done to determine whether Krüppel can mediate repression by competition in transgenic embryos (Nibu, 2003).

Either 6 or 14 tandem copies of the 14-bp synthetic enhancer sequence were attached to a lacZ reporter gene containing the minimal, 42-bp eve promoter region. Similar results were obtained with both fusion genes, and most of the following results were obtained with individual strains carrying the transgene with six copies attached. The transgene exhibits a combinatorial pattern of lacZ staining in wild-type (yw) embryos. Staining is first detected in the anterior 40% of 120-min embryos, presumably in response to the broad Bicoid activator gradient and is also detected in both anterior regions and along the entire length of the ventral mesoderm. Mesoderm expression was first seen at the time when the maternal Dorsal protein is released from the cytoplasm and enters nuclei. During cellularization, staining is lost in central regions, presumably due to the onset of Krüppel expression. In addition, there is a refinement in the anterior staining pattern, so that it becomes restricted to the anterior one-fourth of the embryo and exhibits a reasonably sharp posterior border. This staining pattern persists during gastrulation and germ band elongation (Nibu, 2003).

The transgene was introduced into different mutant backgrounds in order to confirm that the synthetic enhancer is regulated by Bicoid, Dorsal, and Krüppel. The anterior staining pattern is eliminated when the transgene is introduced into embryos derived from females homozygous for a null mutation in bicoid. However, staining persists in ventral regions in response to the Dorsal gradient. The loss of staining in the anterior regions correlates with an anterior expansion of the Krüppel expression pattern in bicoid mutants. The maternal Dorsal gradient is eliminated in embryos derived from females that are homozygous for a null mutation in gastrulation defective (gd⁷/gd⁷). lacZ staining in the ventral mesoderm of these mutants is lost. However, staining persists in anterior regions, presumably in response to the Bicoid gradient, which is unaffected in gd mutants. The transgene was also crossed into Kr¹/Kr¹ mutant embryos. The central gap of repression seen in wild-type embryos is essentially abolished in Kr mutants. There may be a subtle attenuation in central regions due to the low levels of Krüppel protein that are retained in this mutant (Kr¹ is not quite a null allele. The anterior staining pattern directed by the Bicoid gradient may be a bit broader in Kr mutants than in wild-type embryos, suggesting that the Krüppel repressor might help refine the pattern. These results indicate that the artificial enhancer is activated by Bicoid and Dorsal but repressed by Krüppel. Competition is the likely form of repression since the Krüppel repressor sites directly overlap the Bicoid and Dorsal activator sites (Nibu, 2003).

One of the central goals of this study was to determine whether Krüppel requires dCtBP when it mediates repression by competition. This issue was investigated by crossing the transgene into mutant embryos derived from germ line clones produced in dCtBP/+ females. Krüppel continues to induce a central gap of repression in these mutants. In fact, the repression obtained in dCtBP mutants is comparable to that observed in wild-type embryos. These results provide a clear example of Krüppel-mediated repression in the absence of the dCtBP corepressor. In contrast, Krüppel fails to repress transcription in dCtBP mutants when Krüppel and Dorsal sites do not overlap (Nibu, 2003).

This study provides evidence for two distinct mechanisms of short-range repression, corepressor-dependent (quenching and direct repression) and corepressor-independent (competition) repression. In addition, this is the first demonstration that transcriptional repression by competition does not require a corepressor in transgenic Drosophila embryos. dCtBP is dispensable when Krüppel binding sites directly overlap Dorsal activator sites. However, dCtBP is essential for repression when the Krüppel and Dorsal sites are nonoverlapping and can be coordinately occupied. The previous analysis of eve stripe 2 regulation led to the proposal that the Krüppel repressor establishes the posterior stripe 2 border via competition. Two of the Krüppel repressor sites contained within the stripe 2 enhancer overlap Bicoid activator sites. Subsequent studies led to the surprising observation that Krüppel binding sites need not overlap activator sites in order to mediate transcriptional repression (Nibu, 2003).

There are three Krüppel binding sites in the minimal, 480-bp eve stripe 2 enhancer. Two of the sites directly overlap Bicoid activator sites. In both cases, it is likely that the binding of the Krüppel repressor precludes the binding of Bicoid. This type of simple competition is probably not restricted to the regulation of eve stripe 2. For example, one of the mixed Bicoid/Krüppel binding sites in the stripe 2 enhancer is conserved in a newly identified ftz enhancer, which appears to be activated by Bicoid but repressed by Krüppel (V. Calhoun and M. Levine, unpublished data reported in Nibu, 2003). The two enhancers contain the same composite recognition sequence, ACGGATTAA. Repression by competition probably governs, in part, the regulation of the rhomboid lateral stripe enhancer (NEE) since some of the Snail repressor sites directly overlap critical Dorsal and basic helix-loop-helix activator sites (Nibu, 2003).

An implication of this study is that the residual activity of the Krüppel repressor observed in dCtBP mutants might be due to repression by competition. For example, Krüppel can repress the hairy stripe 7 enhancer when misexpressed throughout early embryos using the heat-inducible hsp70 promoter. This repression is retained in dCtBP mutants. Moreover, a mutant form of Krüppel that lacks the dCtBP interaction motif can repress hairy stripe 7 expression. hairy stripe 7 is activated, at least in part, by Caudal and repressed by Krüppel. Interestingly, five Krüppel binding sites directly overlap Caudal activator sites within the hairy stripe 7 enhancer. Similar arguments apply to the Knirps repressor, which helps establish the posterior border of eve stripe 3. The stripe 3 pattern expands in kni^-/kni^- mutant embryos but is essentially unchanged in dCtBP mutants. Knirps repressor sites might overlap critical activator sites, such as binding sites for D-Stat or an unknown activator(s) within the stripe 3 enhancer. Previous studies suggest that Brinker can also function independently of corepressors when bound to sites that directly overlap critical Smad activator sites within cis regulatory regions of Dpp target genes. Direct evidence for simple competition was obtained in transient-transfection assays. The Krüppel DNA-binding domain is sufficient to inhibit activation of the synthetic enhancer by Dorsal in cultured mbn-2 cells (Nibu, 2003).

The results reported in this study exclude another possible explanation for the residual activity of the Krüppel and Knirps repressors in dCtBP mutants: direct repression of the core promoter. In principle, direct repression could involve distinct corepressor proteins. If so, then target genes that contain promoter-proximal Krüppel and Knirps binding sites might be repressed in dCtBP mutants. However, the lacZ fusion genes containing either a single Krüppel site or two tandem Knirps sites located near the transcription start site are no longer repressed in dCtBP mutants. Thus, the possibility is favored that the residual Krüppel and Knirps repression activities depend on competition between overlapping activator and repressor binding sites within selected target enhancers (Nibu, 2003).

The demonstration that both quenching and direct repression require dCtBP raises the possibility that these two seemingly distinct forms of repression employ similar mechanisms. At least three types of models come to mind. First, dCtBP could disrupt physical interactions between upstream activators and the RNA polymerase II transcription machinery/mediator complex at the core promoter. Perhaps dCtBP masks or modifies the activation domains of upstream activators. However, this model can account for quenching but not direct repression. A second type of model involves local chromatin modification. dCtBP contains a well-conserved dehydrogenase catalytic center and binds NADH. Perhaps dCtBP modifies proteins such as histones and helps condense DNA within the limits of a nucleosome. In Saccharomyces cerevisiae, the Rpd3 histone deacetylase (HDAC) causes histone deacetylation over a distance of just two nucleosomes. A third model is that dCtBP 'poisons' the RNA polymerase II transcription machinery and impedes its binding, assembly, or function at the core promoter. This poisoning can be accomplished by placing dCtBP-dependent repressors near the core promoter or by looping distal enhancers to the promoter. According to the latter model, the linkage requirement seen for short-range repressors (they must bind within 100 bp of adjacent activators) might reflect a reliance of the repressors on linked activators in order to loop to the core promoter (Nibu, 2003).

Adaptive evolution targets a piRNA precursor transcription network

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription (Parhad, 2020).

Transposable elements (TEs) are major genome components that can induce mutations and facilitate ectopic recombination, but transposons have also been co-opted for normal cellular functions, and transposon mobilization has rewired transcriptional networks to drive evolution. Species survival may therefore depend on a balance of transposon silencing and activation. The Piwi interacting RNA (piRNA) pathway transcriptionally and post-transcriptionally silences transposons in the germline. However, how this pathway is regulated is not completely understood (Parhad, 2020).

In Drosophila, piRNAs are produced from heterochromatic clusters composed of complex arrays of nested transposon fragments, which appear to provide genetic memory of past genome invaders. Adaptation to new genome invaders is proposed to involve transposition into a cluster, which leads to sequence incorporation into precursors that are processed into trans-silencing anti-sense piRNAs. However, a subset of isolated transposon insertions also produce sense and anti-sense piRNAs, providing an independent adaptation mechanism and epigenetic memory of genome invaders. Expression of piRNAs from these loci is disrupted by piwi mutations, but Piwi-bound piRNAs map to all insertions, not just the subset that function in piRNA biogenesis. The mechanism that defines these 'mini-cluster' thus remains to be determined (Parhad, 2020).

In Drosophila, germline piRNA clusters and transposon mini-clusters are bound by the RDC complex, which is composed of the HP1 homolog Rhino (Rhi), which co-localizes with the linker protein Deadlock (Del) and the Rai1 homolog Cutoff (Cuff). The three components of the RDC are co-dependent for localization to clusters and essential to germline piRNA production. Rhi is composed of chromo, hinge, and shadow domains. The chromo domain binds to H3K9me3-modified histones, and the shadow domain binds Del, which recruits Moonshiner (Moon) and TATA box binding protein-related factor 2 (TRF2), promoting 'non-canonical' transcription from both genomic strands (Parhad, 2020).

The third RDC component, Cuff, was identified in a screen for female sterile mutations and found to encode a homolog of the decapping exonuclease Rai1 required for transposon silencing and piRNA biogenesis. Critical residues in the catalytic pocket of Rai1 are not conserved in Cuff, but sidechains that bind the RNA backbone are retained, suggesting that Cuff may be an RNA 5' end-binding protein. Intriguingly, germline piRNAs in Drosophila are preferentially produced from unspliced transcripts, and cuff mutations significantly increase piRNA precursor splicing, and 5' cap binding by the nuclear cap binding complex (CBC) promotes splicing. Together, these findings suggest that that Cuff competes with the CBC for binding to capped cluster transcripts, suppressing splicing and promoting piRNA biogenesis. However, tethering Cuff to a reporter transcript increases read-through transcription, consistent with suppression of transcription termination. The molecular function for Cuff in piRNA biogenesis thus remains enigmatic (Parhad, 2020).

All three RDC genes are rapidly evolving under positive selection, suggesting that adaptive evolution of the complex is driven by a genetic conflict with the transposons the piRNA pathway silences, but other mechanisms are possible. Previous work found that rapid evolution has modified the Rhi-Del interface, producing orthologs that function as mutant alleles when moved across species. Analysis of these cross-species incompatibilities defined an interaction between the Rhi shadow domain and Del that prevents ectopic assembly of piRNA cluster chromatin. Crosses between Drosophila melanogaster and Drosophila simulans, which are sibling species, lead to hybrid sterility and are important model for genetic control of reproductive isolation. Significantly, sterile hybrids between these species phenocopy piRNA pathway mutations. Adaptive evolution of piRNA pathway genes may therefore contribute to reproductive isolation and speciation (Parhad, 2020).

These findings also suggest that cross-species analysis of rapidly evolving genes may provide a powerful genetic approach to structure-function analysis. This study applies this approach to cuff. These studies indicate that adaptive evolution has targeted direct or indirect interactions among Cuff, the Del-TRF2 non-canonical transcriptional complex, and the transcriptional co-repressor C-terminal binding protein (CtBP). CtBP was first identified as a host binding partner of Adenovirus E1A and subsequently implicated in diverse developmental pathways and cancer. This study shows that Drosophila CtBP suppresses canonical transcription from promoters in transposon terminal repeats and from promoters flanking two major germline piRNA clusters. Significantly, in both contexts, activation of canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution has therefore targeted interactions between Cuff and two transcription regulators, which coordinately control germline piRNA expression (Parhad, 2020).

Adaptive evolution is a hallmark of genes engaged in a genetic conflict, which typically leads to co-evolution of host-pathogen gene pairs that encode interacting proteins. However, pathogens can also produce mimics that target interactions within host defense systems, raising the possibility that adaptation can also remodel interaction between host proteins. Supporting the possibility, adaptive evolution has remodeled an interface between the Rhi and Del, which are core components of the host transposon defense machinery. These adaptive changes prevent gene function across closely relates species and define an interaction that is required to restrict the RDC to piRNA clusters, which defines the specificity of the transposon silencing machinery. These findings suggest that adaptive evolution targets important functional domains, which can be functionally analyzed using cross-species complementation. This approach is applied to the third RDC component, cuff and shows that adaptive evolution targets interactions between this Rai1 homolog and proteins that coordinate canonical and non-canonical piRNA cluster transcription and piRNA biogenesis (Parhad, 2020).

Transposon silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, and Cuff co-localizes with Rhi and Del at these piRNA source loci. Rhi binds to H3K9me3 marks and recruits Del. Del, in turn, binds Moon, which recruits TRF2 to initiate non-canonical transcription from both genomic strands. In contrast, the current data suggest that Cuff coordinates canonical and non-canonical cluster expression. D. simulans cuff ortholog fails to rescue D. melanogaster cuff mutations and leads to dominant sterility when overexpressed in wild-type flies. Significantly, these phenotypes are associated with stable binding to Del, TRF2, and CtBP. As noted above, Del and TRF2 function in non-canonical transcription of piRNA clusters. CtBP is a conserved transcriptional co-repressor, first identified as a host factor that binds to Adenovirus E1a, and subsequently shown to function in numerous developmental pathways. CtBP does not directly interact with DNA, but binds sequence specific transcription factors and recruits histone-modifying enzymes. This study shows that CtBP-kd activates canonical promoters linked to piRNA source loci. Adaptive evolution has therefore remodeled interactions between Cuff and factors that control both canonical and non-canonical transcription of piRNA precursors loci (Parhad, 2020).

Dominant phenotypes can result from mutations that produce new interactions or functions (neomorphic mutations) and assembly of complexes that are not formed by wild-type proteins. However, the current findings, with previous studies, suggest that substitutions in sim-Cuff stabilize normally transient complexes with both TRF2 and CtBP. In D. melanogaster, Cuff and Del do not co-precipitate, but the proteins co-localize to nuclear foci, interact in two-hybrid assays, and are co-dependent for association with piRNA clusters (Mohn, 2014). Del, in turn, co-precipitates with TRF2 and Moon, and all three proteins are required for non-canonical cluster transcription (Andersen, 2017), but TRF2 does not normally accumulate at clusters. In contrast, overexpression of sim-Cuff drives TRF2 co-localization with the RDC. Similarly, ChIP-seq shows that Cuff and Del localize to canonical promoters that are suppressed by CtBP, but CtBP does not accumulate at these promoters. Substitutions in the sim-Cuff ortholog thus appear to stabilize normally transient associations with Del and TRF2 and with CtBP (Parhad, 2020).

The majority of Drosophila germline clusters are transcribed from internal non-canonical initiation sites and do not have flanking canonical promoters. CtBP-kd does not significantly alter long RNA or piRNA expression from these loci. However, canonical promoters flank the right side of the 42AB cluster and both ends of the 38C cluster, and CtBP-kd increases transcription from these canonical promoters, which is associated with reduced transcription and piRNA production from downstream regions]. It has not been possible directly assay non-canonical transcription at most transposon insertions that produce piRNAs, as the inserted sequences are repeated, but CtBP-kd increases canonical transcription of transposons and is linked to collapse of piRNAs mapping to sequences flanking these insertions. In addition, deletion of the promoters flanking 42AB and 38C leads to spreading of piRNA production into flanking domains (Andersen, 2017). Together, these findings indicate that canonical transcription directly or indirectly represses non-canonical transcription and piRNA production (Parhad, 2020).

On the basis of these findings, it is proposed that Cuff coordinates canonical and non-canonical piRNA precursor transcription. By stabilizing Rhi, Del, Moon and TRF2, Cuff promotes non-canonical transcription. By contrast, Cuff appears to function with CtBP to control canonical transcription. Rescue of cuff mutants with sim-Cuff, which shows enhanced binding to CtBP, is phenocopied by CtBP-kd: both lead to increased canonical transcription. Formation of stable complexes with sim-Cuff thus appears to inhibit CtBP, activating canonical transcription and reducing downstream non-canonical transcription. Normally, the interaction between Cuff and CtBP is weak and free CtBP suppresses canonical promoters, while Cuff functions with Del-TRF2 to drive of non-canonical transcription. It is speculated that this balance may be altered in response to stress or environmental signals, which can activate transposons. Intriguingly, CtBP is also an NADH/NAD binding protein, suggesting that the balance between canonical and non-canonical piRNA precursor transcriptions may be regulated in response to metabolic state (Parhad, 2020).

The RDC proteins Moon and TRF2 are required for piRNA precursor transcription, and all of these factors are rapidly evolving. By contrast, CtBP is conserved from flies to humans, and a putative human oncogene. The data presented here, with earlier analysis of Rhi and Del (Parhad, 2017), indicate that rapid evolution has modified multiple interactions between rapidly evolving proteins in the piRNA biogenesis, and association of these proteins with a highly conserved transcriptional co-repressor. Rapidly evolving genes with specialized functions are frequently the most accessible to phenotype-based forward genetic approaches in model systems, and linking these specialized genes to conserved pathways can be a challenge. The studies reported in this study indicate that cross-species studies can help define these links, bridging the gap between genetically tractable model organisms and human biology (Parhad, 2020).

Pits and CtBP control tissue growth in Drosophila melanogaster with the Hippo pathway transcription repressor, Tgi

The Hippo pathway is an evolutionary conserved signalling network that regulates organ size, cell fate control and tumorigenesis. In the context of organ size control, the pathway incorporates a large variety of cellular cues such as cell polarity and adhesion into an integrated transcriptional response. The central Hippo signalling effector is the transcriptional co-activator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood and therefore this study sought to identify proteins that potentially operate together with it. Using an affinity purification and mass-spectrometry approach this study identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologues of Tgi, CtBP and Pits (VGLL4, CTBP2 and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution (Vissers, 2020).

The Hippo pathway was first discovered in Drosophila melanogaster genetic screens as an important regulator of organ growth. It has subsequently been shown to control the growth of multiple different tissues (epithelial, muscle, neural, and blood) in different species. It also controls cell fate choices in both D. melanogaster and mammals, while mutation of Hippo pathway genes underpins several human cancers. In the context of organ size control, the Hippo pathway responds to cell biological cues and its surrounding environment. For example, the Hippo pathway is regulated by cell-cell adhesion, cell-matrix contacts, cell polarity proteins, and by mechanical forces transmitted by the actin and spectrin cytoskeletons. These signals converge on a core signaling complex consisting of the Hippo (Hpo) and Warts (Wts) kinases and the adaptor proteins Salvador (Sav) and Mats. The central Hippo signaling effector is Yorkie (Yki), which is a transcriptional coactivator. Yki rapidly shuttles between the nucleus and cytoplasm and this is regulated by Wts-mediated phosphorylation at conserved amino acids, which limits access to the nucleus. When nuclear, Yki partners with sequence specific transcription factors to control expression of genes such as DIAP1, bantam, and cyclin E (Vissers, 2020).

The best-characterized Yki-interacting transcription factor is the TEA domain protein Scalloped (Sd). Yki and Sd promote transcription by interacting with chromatin-modifying proteins like the Mediator complex, the SWI-SNF complex, and the Trithorax-related histone methyltransferase complex (Zheng, 2019). The mammalian orthologs of Yki (YAP and TAZ) and Sd (TEAD1-TEAD4) regulate transcription by interacting with similar protein complexes. In human cells, YAP and TEAD regulate gene expression predominantly by binding to enhancers, as opposed to promoters. In addition, they promote transcriptional elongation by recruiting the Mediator complex and Cdk9 (Vissers, 2020).

The mechanism by which Sd and TEADs repress transcription is less well defined. Genetic evidence from tissues such as the D. melanogaster ovary indicates that Sd can act as a default repressor of transcription, and this activity is antagonized by Yki. Sd's default repressor function is mediated in part by the corepressor protein Tondu-domain-containing Growth Inhibitor (Tgi), also known as Sd-Binding Protein (Guo, 2013). To activate gene expression, Yki is thought to compete with Tgi for Sd binding and alleviate repression of target genes. Tgi interacts via its Tondu domains with Sd and via PY motifs with Yki's WW domains (Guo, 2013; Koontz, 2013). This relationship is conserved in mammals between YAP and TAZ, TEAD1-TEAD4, and VGLL4 (the Tgi ortholog). Sd can also regulate transcription together with the Zinc finger domain protein Nerfin-1. These proteins work in partnership to maintain the fate of D. melanogaster medulla neurons (Vissers, 2018) and they also influence cell competition in growing imaginal discs (Guo et al. 2019). Currently, the mechanism by which Tgi and VGLL4 cooperate with Sd/TEADs to control transcription and tissue growth is incompletely understood (Vissers, 2020).

To better understand how Tgi regulates transcription, proteomics approaches were used, and four high-confidence Tgi-interacting proteins were identified, all of which are transcriptional regulatory proteins. In addition to the known Tgi partners Yki and Sd, this study identified two previously unknown Tgi-interacting proteins, CG11138 (also known as protein interacting with Ttk69 and Sin3A, or Pits) and C-terminal binding protein (CtBP), both of which have been linked to transcriptional repression. Both gain and loss of function of pits and CtBP modified tissue growth aberrations caused by Hippo signaling defects. Furthermore, overexpression of Pits reduced expression of well-defined Hippo pathway target genes, thus highlighting the possibility that Pits and CtBP operate with Tgi to limit tissue growth and transcription (Vissers, 2020).

The transcriptional corepressor Tgi has emerged as an important regulator of transcription that is regulated by Yki and Sd, but the mechanism by which it does so is currently unclear. This study set out to address this by identifying Tgi-interacting proteins. Four such proteins were identified with high confidence, all of which have been ascribed functions as transcription regulators: the previously identified Hippo pathway proteins Yki and Sd, as well as Pits and CtBP. Previous structure-function studies showed that Tgi's growth inhibitory function fully depends on its ability to interact with Sd (Guo, 2013; Koontz, 2013), suggesting that Sd is the sole transcription factor that mediates Tgi's influence on transcription. The finding that Sd was the only sequence-specific transcription factor detected by mass spectrometry in Tgi purifications in S2 cells further supports this model. Additionally, biochemical data support the notion that Pits and CtBP function together with the Tgi corepressor to limit tissue growth, although genetic studies fall short of providing conclusive evidence for this. The fact that pits and ctbp were required for Tgi's ability to limit eye growth when overexpressed argues that Tgi requires them to repress gene expression through Sd. Further support for this idea comes from the finding that pits overexpression repressed the expression of the well-defined Yki/Sd/Tgi targets DIAP1 and ban. On the other hand, pits mutant flies were homozygous viable and displayed no obvious gross phenotypic abnormalities, and pits mutant larval eye imaginal discs cells expressed normal levels of DIAP1. However, it should be noted that loss of either sd or tgi in larval eye imaginal discs also has no obvious effect on expression of its target genes, while overexpression does. This suggests that in the growing larval eye imaginal disc Sd does not have a major role in gene repression. Alternatively, in the absence of sd, tgi, or pits, other proteins might compensate for them and regulate expression of their target genes (Vissers, 2020).

Strong genetic evidence that helped identify Tgi as a mediator of Sd's transcriptional repression activity was the finding that loss of tgi partially rescued the undergrowth of yki clones in larval eye imaginal discs (Koontz, 2013). Similarly, in the current study, loss of CtBP partially restored the undergrowth phenotype of yki larval eye clones. Therefore, CtBP might work in partnership with Tgi and Sd to repress target genes that are required for eye growth. Alternatively, CtBP might limit eye overgrowth by acting in parallel to Tgi and possibly also Sd. Indeed, a recent study provided evidence for both of these models with the discovery that CtBP represses transcription of the progrowth microRNA bantam in both Yki-dependent and independent manners. In contrast to CtBP loss, pits loss did not rescue the undergrowth phenotype of yki eye imaginal disc clones, which stands in apparent opposition to its requirement for growth inhibition induced by Tgi overexpression. The reason for this discrepancy is currently unclear. Future studies aimed at identifying the full suite of Yki/Sd/Tgi target genes that are required for mediating eye growth should help to provide clarity on the different roles of Yki, Sd, Tgi, CtBP and Pits on transcription and eye growth, but this genetic program is currently unknown (Vissers, 2020).

The mechanism by which Yki/Sd/Tgi regulate transcription in D. melanogaster appears to be largely conserved in mammals (Guo, 2013; Koontz, 2013; Zhang, 2014). This found that Pits and CtBP bind to Tgi through conserved protein motifs. Therefore, their biochemical relationship is also likely to be conserved in other species. Indeed, the interaction between the human CtBP and Tgi orthologs CTBP2 and VGLL4 has been reported, which inhibits adipogenesis of murine 3T3-L1 cells (Zhang, 2018). In addition, several studies have identified physical interactions between VGLL4 and the human Pits orthologs IRF2BP1, IRF2BP2, and IRF2BPL. A very recent study reported a functional interaction between these genes in the context of cancer: the Pits ortholog IRF2BP2 acted with VGLL4 to suppress liver tumor growth that was caused by YAP hyperactivity and also the expression of YAP-TEAD target genes (Feng, 2019). The D. melanogaster studies imply that Pits can act as a corepressor of Yki/Sd target genes, as opposed to an activator. In support of this, most studies of the three mammalian Pits orthologs IRF2BP1, IRF2BP2, and IRF2BPL indicate that they act as corepressors, although the mechanism of corepression is poorly characterized, and CtBP is generally considered a transcription repressor. Clearly, further studies are required to clarify the mechanism by which Hippo pathway target genes are regulated by Yki, Sd, and Tgi. To explore roles for Pits and CtBP on transcription in the growing eye, it will be important to examine their genome occupancy relative to Tgi and Sd, and also to better define the mechanism by which they regulate transcription. This should shed light on the control of Hippo pathway target genes and could also be valuable for defining the emerging role of the Tgi ortholog VGLL4 in human cancers (Vissers, 2020).

A prominent gene activation role for C-terminal binding protein in mediating PcG/trxG proteins through Hox gene regulation

The evolutionarily conserved C-terminal binding protein (CtBP) has been well characterized as a transcriptional co-repressor. This study reports a previously unreported function for CtBP, showing that lowering CtBP dosage genetically suppresses Polycomb group (PcG) loss-of-function phenotypes while enhancing that of trithorax group (trxG) in Drosophila, suggesting that the role of CtBP in gene activation is more pronounced in fly development than previously thought. In fly cells, this study shows that CtBP is required for the derepression of the most direct PcG target genes, which are highly enriched by homeobox transcription factors, including Hox genes. Using ChIP and co-IP assays, this study demonstrate that CtBP is directly required for the molecular switch between H3K27me3 and H3K27ac in the derepressed Hox loci. In addition, CtBP physically interacts with many proteins, such as UTX, CBP, Fs(1)h and RNA Pol II, that have activation roles, potentially assisting in their recruitment to promoters and Polycomb response elements that control Hox gene expression. Therefore, this study reveals a prominent activation function for CtBP that confers a major role for the epigenetic program of fly segmentation and development (Bi, 2022).

For over two decades, CtBP has been extensively studied in both invertebrate and mammalian model systems. CtBP physically interacts with a large number of sequence-specific DNA binding proteins and mediates their transcriptional activities. Although the prevailing model is that CtBP functions as a co-repressor for these DNA binding factors, its major biological function in vivo remains controversial, particularly during the course of animal development. In Drosophila, genetic studies have shown that CtBP may both enhance and repress gene expression in a transcription factor-dependent manner. In mice, the co-repressor role of CtBP does not sufficiently explain the deficits found in both CtBP1 and CtBP2 mutant mice. Rather, the study suggests that CtBPs may contribute equally to gene repression and activation . Thus, the question remains: which one is more important for the in vivo function of CtBP, repression or activation (Bi, 2022)?

Answering this question is not an easy task owing to the apparent pleiotropic nature of CtBP. In Drosophila, loss of CtBP severely disrupts the embryonic body plan and inhibits early embryonic regulators, including many of the pair-rule genes and potentially some gap genes, preventing further investigation on its role in later developmental stages. In particular, it is quite difficult to dissociate the direct action of CtBP on Hox from its role on pair-rule genes, which in turn affect Hox expression. This scenario might help to explain in part why genetic studies in mouse are not particularly informative (Bi, 2022).

To circumvent this difficulty, this study examined the role of CtBP in the epigenetic context. It was found that CtBP suppresses PcG homeotic transformations and enhances those of trxG . Therefore, CtBP may function as a dose-dependent modifier of the major epigenetic regulators, opposing its co-repressor role in fly development. Pc transformations have at least partially known gene bases, such as those demonstrated with ectopic expression of Ubx in the wing discs and Sex combs reduced in leg discs shown by other studies. Therefore, the fact that CtBP suppresses Pc transformations may suggest or reflect an altered embryonic Hox pattern that was too delicate to be observed. Importantly, the heteroallelic combination strategy that was employed in this study allowed dissociation of the role of CtBP in earlier embryonic development from later stages and led to the finding that activation may be a prominent function for CtBP during the course of development. Although it is hard to tell whether CtBP acts directly to modulate PcG and trxG functions in these in vivo experiments, it is most likely that CtBP may function in a parallel temporal fashion with Pc, as neither altered h their activities in a contex even skipped and fushi tarazu, nor any defect in adult flies was discernable for heterozygous CtBP alleles (Bi, 2022).

In agreement with the in vivo studies, this study demonstrated, using RNA-seq analyses in embryonic-derived Kc cells, that CtBP plays an essential activation role in the regulation of most direct PcG targets, having the highest enrichment of homeobox transcription factors. In sharp contrast, CtBP, in conjunction with PcG, co-represses a relatively smaller proportion of genes with diverse functions, among which only a handful may potentially direct PcG targets. These findings might suggest that CtBP has a prominent activation role that is highly selective for developmental transcription factors, including Hox genes, whereas the repressive role of CtBP is unspecific and possibly irrelevant with PcG repression. Using ChIP and co-IP assays, evidence is provided that CtBP physically interacts with CBP and UTX, helping to recruit these histone modifiers to several well-characterized regulatory sites in Hox loci. Accordingly, CtBP is required to coordinate the switch from a repressive H3K27me3 form to an active H3K27ac mark. For the sake of inspiration, a wider function of CtBP could be imagined in rewiring epigenetic networks composed by various kinds of histone modifications and other marks. For example, it has been proposed that H3K27ac signal coincides with H3K4me3, which constitutes a key part of the molecular mechanism of antagonizing Pc silencing. Indeed, the results might allow favoring of a model in which CtBP constitutes part of a large complex including many epigenetic writers and erasers, such as CBP and UTX, closely working with the core transcriptional machinery centered by RNA Pol II. Therefore, CtBP may play a profound role in PcG/trxG-mediated Hox gene regulation (Bi, 2022).

Although many biochemical studies have indicated that CtBPs are mainly associated with repressive histone modifiers, it is noteworthy that these studies were mostly performed in cell lines with a static gene expression status and so may not properly reflect the dynamic changes of transcriptional programs in vivo. Furthermore, the previously identified CtBP-interacting proteins are not dedicated transcriptional repressors/co-repressors. As such, there are examples illustrating the requirement of LSD1 and some of the HDACs for transactivation. However, CtBP has been reported by others to interact with proteins for gene activation. For example, it has been documented in detail that CtBP1 directly binds to the bromodomain of p300 (Bi, 2022).

Together, these findings highlight a direct and prominent activation role of CtBP in early fly development stages, probably no later than the determination of segment identities when Pc and Hox genes are acting. Mechanistically, CtBP could play its coactivator role in the dynamic switch or establishment of proper chromatin structures for prolonged gene activation by recruiting histone modifiers such as CBP and UTX. Alternatively, CtBP might play roles in both activation and repression, functioning in the balance between PcG and trxG actions. For example, it could be that CtBP activates in PREs or enhancers but represses at promoters, as shown by the unchanged CtBP occupation upon PcG depletion. Significantly, unlike previous findings that CtBP only activates genes in exceptional cases, this study has provided evidence that CtBP directly activates gene expression in the major developmental processes, such as the precise patterning of Hox genes, indicating that activation is probably an important role for CtBP in the context of development. This study has thus uncovered a gene co-activation role for CtBP in vivo that might have been previously masked due to its impact on the earlier developmental regulators (Bi, 2022).

It will be intriguing to know whether CtBP activates PcG targets beyond Hox genes or an opposite mechanism exists, as in mouse ESC differentiation, in which CtBP2 couples with NuRD-mediated deacetylation for identified CtBP-unassociated PcG targets. In mouse, the loss of CtBP2 causes incomplete ESC differentiation, a process known to be dependent upon gene activation. This study could provide an alternative interpretation, if the prominent gene activation role of CtBP were conserved in mouse. Given the complexity of the role of CtBP in cancers, these findings might provide additional data that can be used towards the development of novel strategies based on CtBP as a therapeutic target (Bi, 2022).

Widespread regulatory specificities between transcriptional co-repressors and enhancers in Drosophila

Gene expression is controlled by the precise activation and repression of transcription. Repression is mediated by specialized transcription factors (TFs) that recruit co-repressors (CoRs) to silence transcription, even in the presence of activating cues. However, whether CoRs can dominantly silence all enhancers or display distinct specificities is unclear. This work reports that most enhancers in Drosophila can be repressed by only a subset of CoRs, and enhancers classified by CoR sensitivity show distinct chromatin features, function, TF motifs, and binding. Distinct TF motifs render enhancers more resistant or sensitive to specific CoRs, as was demonstrated by motif mutagenesis and addition. These CoR-enhancer compatibilities constitute an additional layer of regulatory specificity that allows differential regulation at close genomic distances and is indicative of distinct mechanisms of transcriptional repression (Jacobs, 2023).

Animal development and homeostasis critically depend on differential gene expression, enabled by the precise regulation of transcriptional activation and repression. Although repression is often associated with heterochromatin, genes can also be silenced in transcriptionally permissive euchromatin by repressive transcription factors (TFs), also termed repressors, that bind to DNA and recruit corepressors (CoRs). As CoRs can suppress transcription even in the presence of activators, this mode of gene silencing is termed active transcriptional repression. Active repression is critical and its failure can cause developmental defects and diseases like cancer; and it is conceptually intriguing as it requires the fast and efficient overriding of activating cues. However, the modes and mechanisms of this process are unclear, and whether a regulatory code coordinates repression and activation, is unknown. Given that transcriptional activation can occur via distinct and mutually incompatible modes, it is intriguing to speculate whether distinct modes of active transcriptional repression exist. Examples of specificities between repressors and activators have indeed been observed. The CoR Retinoblastoma protein (Rb) as part of the DREAM complex for example can repress the TFs E2F, Mip120 and PU.1 but not others like SP-1, and repressors can have different activities in distinct transcriptional contexts. Comprehensive studies allowing to define different modes of active repression and uncover their regulatory rules are however lacking. This study determined the mutual compatibilities between five known CoRs and a genome-wide library of active enhancers by measuring enhancer-activity changes upon CoR tethering in otherwise unperturbed cells, similar to activator-bypass experiments. It was reasoned that testing each enhancer with each CoR in all possible combinations should reveal CoR-enhancer combinations that lead to decreased enhancer activity (enhancers are sensitive) and those that do not (resistant), indicative of compatible and incompatible pairings (Jacobs, 2023).

First, this study tested whether distinct specificities between CoRs and enhancers exist, whereby certain enhancers are sensitive to repression by a given CoR, while other enhancers are resistant. For this, it was necessary to systematically measure the effects that selected CoRs have on the activity of a large number of enhancers. The comprehensive mapping of CoR-enhancer compatibilities, by examining all combinations of CoRs and enhancers, requires highly controllable quantitative high-throughput assays. Therefore the massively parallel enhancer-activity assay STARR-seq was modified to enable the function-based testing of genome-wide enhancer candidate libraries with different CoRs. Briefly, four upstream-activating-sequence (UAS) motifs were introduced immediately downstream of the enhancer library, which leave the enhancer sequence intact yet allows for the direct tethering of selected CoRs via the Gal4 DNA-binding domain. The tethering of CoRs next to active enhancers directly assesses whether CoRs can override existing activating cues, a process akin to active repression (Jacobs, 2023).

Drosophila S2 cells were chosen as a model system, and a panel of five CoRs; CoRest, CtBP, Rbf, Rbf2 and Sin3A was tested. These CoRs represent different protein complexes, repressive pathways, enzymatic functions, and distinct groups with context-specific functions. Testing diverse CoRs casts a wide net and should increase the ability to detect compatible and incompatible CoR-enhancer pairs. For each CoR, two independent UAS-STARR- seq screens were performed where the UAS-STARR-seq library was co-transfect with a vector that expresses the Gal4-CoR (or Gal4-GFP as neutral control), and spike-in-controls were used for normalization. As spike-in controls a distinct STARR-seq library was used containing 18 Drosophila pseudoobscura enhancers, cloned without the UAS motifs, and hence not targeted by the Gal4- -CoRs. In all cases, the two independent replicates correlated well. In order to reliably assess repression (which requires a high baseline activity), it was decided to evaluate enhancer-activity changes for 3094 enhancers that were highly active in Gal4-GFP controls (Jacobs, 2023).

First, which of the 3094 enhancers could be silenced by the highly conserved CoR CtBP was determined, by assessing the enhancer activity changes when tethering Gal4-CtBP versus Gal4-GFP. This revealed that some enhancers, like the enhancers near Orct, Orct2 and Pka-C3, were reproducibly repressed by Gal4-CtBP, whereas others, like the enhancer near CG10516, were unaffected. A differential analysis based on the two replicates per condition using edgeR showed that CtBP significantly repressed 759 enhancers but not the remaining 2335. Thus, CtBP could only repress ~25% of the enhancers, suggesting that CtBP displays preferences or specificities towards some enhancers but not others (Jacobs, 2023).

The enhancer-activity changes upon recruiting Rbf2, a CoR from the retinoblastoma protein family was determined. Interestingly, Rbf2 was also able to repress only a subset of the enhancers (1733, 56%), including the enhancers near Orct and Pka-C3, which were also repressed by CtBP, and CG10516, which was not repressed by CtBP. In contrast, Rbf2 was unable to repress the aforementioned Orct2 enhancer and others, indicating that the specificities of Rbf2 differ from those of CtBP. Indeed, while 502 enhancers were repressed by CtBP and Rbf2, 1231 enhancers were repressed by Rbf2 but not by CtBP and, vice versa, 257 enhancers were repressed by CtBP but not by Rbf2. These enhancer-CoR specificities were validated in luciferase reporter assays, in which the CoRs were recruited upstream of the enhancer and promoter. This assay confirmed that an enhancer near serpent was specifically repressed by Gal4-CtBP but not Gal4-Rbf2, an enhancer near CG2116 was specifically repressed by Gal4-Rbf2 but not Gal4-CtBP, and an intronic enhancer of kay was repressed by both CoRs, each in agreement with the STARR-seq results. Taken together, the CoRs CtBP and Rbf2 are each able to repress a specific subset of enhancers but not others, indicating the existence of distinct CoR-enhancer specificities (Jacobs, 2023).

Screening three additional prominent CoRs; CoRest, Rbf and Sin3A revealed that each of them was able to repress a specific subset of enhancers. CoRest was the strongest repressor, repressing 1452 enhancers and often reducing their activity to background levels. The overall repression profiles of CoRest and Sin3A were similar to that of CtBP and clearly different from Rbf2. Rbf's repression profile was not similar to any of the other tested CoRs. In general, the five tested CoRs were each able to repress a subset of enhancers and displayed distinct specificities (Jacobs, 2023).

Given that enhancers were differentially sensitive to some of the tested CoRs, and resistant to others, whether these differential sensitivities were related to other enhancer properties was examined. For this, the enhancers were clustered into groups of similar repression patterns. A self-organizing tree algorithm (SOTA) was used with the PCC as distance metric to cluster the enhancers into five groups based on their sensitivity to the CoRs. Cluster 1 contains CoRest and CtBP resistant enhancers while enhancers in cluster 2 are resistant to CtBP and Sin3A. Cluster 3 contains enhancers that are resistant to Rbf2, enhancers in cluster 4 are sensitive to all CoRs while enhancers in cluster 5 are overall very sensitive to repression but resistant to Rbf. Hence, the enhancers of S2 cells could be divided into five groups, defined by their differential response to the tested CoRs (Jacobs, 2023).

Enhancers clustered by corepressor sensitivity differ in chromatin marks, transcription factor motif content and binding Next, whether these enhancer clusters differ in additional properties that correlate with their behaviour towards the CoRs was tested. Initial enhancer activity, as measured by UAS- STARR-seq with Gal4-GFP, was similar for all five clusters. Also H3K27ac, a histone modification that marks active enhancers, was similarly enriched at the endogenous enhancer loci of all clusters. It was inferred that the distinct specificities did not stem from differences in initial enhancer strengths. The histone modifications H3K4me1 and H3K4me3 did however show differential and complementary trends: H3K4me1 was the highest enriched at cluster 5, followed by 4 and 3, whereas H3K4me3 was more highly enriched at enhancer clusters 1 and 2. High H3K4me1 levels combined with high H3K27ac levels have been associated with distal regulatory regions or cell type-specific enhancers, suggesting that enhancers with the highest H3K4me1 levels (cluster 5) might be most specific to S2 cells. Analysing chromatin accessibility and H3K27ac levels in other cell types and tissues indeed revealed that enhancers from cluster 5 were highly cell type-specific. Interestingly, these enhancers were also the most strongly repressed by CoRest, CtBP and Rbf2, suggesting that developmental or cell type-specific enhancers might intrinsically be more sensitive to repression by certain CoRs, while more globally active or housekeeping enhancers might be more resistant. As active enhancers are known to function through a variety of different TFs, it was hypothesised that their differential response to the CoRs might be linked to the specific TFs they bind (Jacobs, 2023).

Using published TF ChIP-seq data, it was observed that prominent TFs were bound to enhancers from specific clusters and absent from others. The TFs DREF and M1BP for example bound almost exclusively to enhancers from clusters 1 and 2, with DREF preferring cluster 1 and M1BP cluster 2. Trithorax-like (Trl, also called GAGA factor or GAF) on the other hand was absent from these clusters and instead mainly bound to enhancers from cluster 3 and to a lower extent to cluster 4. The distribution of these three TFs suggests that differential CoR sensitivities might relate to distinct TFs. To identify associations between CoR sensitivities and TFs in a more comprehensive manner, TF-motif enrichment analyses was performed for the enhancers of the five clusters using the 6502 TF motifs from the iRegulon database. Consistent with the ChIP-seq results, motifs for DREF, M1BP and Trl were specifically enriched in clusters 1, 2 and 3, respectively. Additional motifs were also differentially enriched between the clusters: Rsc30 and E-box motifs were enriched in cluster 3, Sim motifs were enriched in cluster 4, while ETS and GATA motifs were enriched in cluster 5. Other prominent TF motifs were enriched over several clusters, including AP-1, Ato and CrebB. Taken together, the different enhancer clusters, defined by their sensitivity towards CoRs, associate with distinct chromatin features and TF motif content (Jacobs, 2023).

To directly test the association between TF motifs and sensitivity towards each of the CoRs,the enrichment of TF motifs within enhancers that were sensitive ) versus those that were resistant to each CoR were evaluated. For CtBP, for example, it was found that AP-1, Trl and GATA motifs were strongly enriched in sensitive enhancers, whereas DREF, Ohler1 and Mip120 motifs were enriched in the resistant enhancers. Motif enrichment profiles across all five CoRs revealed an intricate relationship between enhancer-CoR sensitivity and TF motifs with highly distinctive enrichments. Consistent with the conserved role of Rb proteins as part of the DREAM complex, E2F, DP, and Mip120 motifs were enriched in Rbf sensitive enhancers. Interestingly, these three motifs as well as DREF and M1BP motifs were enriched in enhancers that were resistant to CoRest, CtBP and Sin3A repression. On the other hand, motifs for the developmental TFs GATA, AP1 and TEAD were enriched in enhancers sensitive to CoRest, CtBP and Sin3A and resistant to Rbf and Rbf2. Furthermore, Trl and ETS motifs were specifically enriched in enhancers that were resistant to Rbf2 and Rbf respectively, while sensitive to all other CoRs. These results indicate that, for each CoR, certain TF motifs are specifically and strongly enriched in sensitive and resistant enhancers (Jacobs, 2023).

Given the differential motif enrichments, whether TF motif content is predictive of an enhancer's CoR sensitivity was tested. For this, a Generalized Linear Model (GLM) was trained using TF motif counts as features and enhancer sensitivity to a given CoR as response variable. For each CoR, using 10-fold cross-validation, the models based on motif counts performed well and were able to predict an enhancer's sensitivity and resistance to a given CoR. Overall, these results establish a strong association between CoR sensitivity and TF motif content that is predictive and might correspond to causal relationships (Jacobs, 2023). It was noticed that DREF TF motifs were enriched in enhancers that were resistant to CoRest and CtBP. Indeed, enhancers bound by DREF, such as enhancers near the RYBP and jupiter genes, are specifically resistant to repression by CoRest and CtBP but sensitive to repression by Rbf. Ranking all enhancers based on their sensitivity to repression by CoRest or CtBP confirmed that resistant enhancers were significantly enriched for DREF motifs and bound by DREF according to ChIP-seq. Given the correlation between CoRest and CtBP resistance and presence of DREF, it was considered that DREF might protect enhancers against CoRest and CtBP-mediated repression. To test whether DREF motifs are indeed required for the resistance, 65 DREF-motif containing enhancers, mutated the DREF motifs, were tested, and the enhancers' sensitivity to repression was assessed. The mutated enhancers were significantly more sensitive to repression by CoRest and CtBP, while their sensitivity towards Rbf did not change. It is inferred that DREF motifs are required for resistance to CoRest and CtBP. Several other TF motifs were predicted to confer resistance to repression by specific CoRs. ETS- family motifs, for example, were specifically enriched in enhancers that were resistant to Rbf repression was observed. The ETS motifs were mutated in 157 enhancers and indeed increased sensitivity towards Rbf-mediated repression. Remarkably, the opposite trend was observed for the other CoRs, which were all able to repress the wildtype enhancers containing ETS motifs better than their mutated counterparts, suggesting that ETS TFs are in general sensitive to repression. Similarly, Trl motifs were enriched in enhancers that were specifically resistant to Rbf2 repression and mutating these motifs in 127 enhancers led to a slight but specific increase in sensitivity towards Rbf2, but not towards the other CoRs (Jacobs, 2023).

In general, specific TF motifs are required to protect resistant enhancers against a given CoR. As these motifs and enhancers are however still sensitive to other CoRs, an intricate pattern emerges, which suggests that the interplay between repressors and activators can be highly specific (Jacobs, 2023).

Given that specific TF motifs are required for CoR resistance, it was asked whether these motifs are also sufficient to cause CoR resistance. To test this, two 'resistant' TF motifs (10 bp each) were introduced into CoR-sensitive enhancers at positions deemed unimportant for enhancer activity, and the enhancers' sensitivity to repression was evaluated. As a control for the addition of motifs, two neutral control TF motifs were introduced at the same positions (of the ACE2 and FOX TFs), which were predicted to not impact the sensitivity to repression. Since DREF motifs were required for resistance to CoRest and CtBP-mediated repression, whether they were sufficient to render CoRest and CtBP-sensitive enhancers resistant was tested. Overall, the introduction of two DREF motifs was sufficient to protect the enhancers from CoRest and CtBP-mediated silencing and this protection was specific and due to the DREF motifs, as control motifs had no effect on sensitivity. Similarly, ETS motifs were necessary and sufficient to desensitize enhancers from repression by Rbf, and Trl motifs were necessary and sufficient to specifically generate Rbf2-resistant enhancers (Jacobs, 2023).

In each case, the gain in resistance due to the addition of selected TF motifs was highly CoR specific, as these new motifs had little effect on the repression by other CoRs or could even sensitize the enhancers to other CoRs. The addition of ETS or Trl motifs for example made enhancers more sensitive to all other CoRs except for Rbf or Rbf2 respectively, while DREF motifs increased sensitivity towards Rbf. Hence, introducing specific TF motifs changed the enhancer's sensitivity to repression by a given CoR in a specific and predictable manner. Taken together, it is concluded that specific TFs are necessary and sufficient to confer resistance to repression by a given CoR, while other CoRs are able to repress these TFs, indicating that certain TFs can directly counteract some CoRs and their repressive function or that different modes of transcriptional activation require different modes of repression (Jacobs, 2023).

This work has shown that transcriptional enhancers are differentially susceptible to active repression by prominent CoRs. The regulatory specificities between active enhancers and five CoRs were mappedon a genome-wide scale, and it was discovered that enhancers across the genome are repressed by just a subset of specific CoRs. This additional layer of regulatory specificities enables the differential repression of closely spaced enhancers and genes as they frequently occur in the Drosophila genome. At the fs(1)h/mys locus for example, CoRest could repress the intronic mys enhancer without affecting the neighboring fs(1)h gene, whereas Rbf could do the opposite. Similarly, at the kay/fig locus some CoRs like CtBP could repress both closely spaced enhancers, whereas Rbf2 could specifically repress the enhancer closest to fos intronic gene (fig) but not the others (Jacobs, 2023).

The uncovered specificities between repressors and enhancers not only suggest that repression occurs via distinct mechanisms, but also reveal a previously unappreciated layer of 'resistance' against repression. Enhancers that are sensitive or resistant towards a given CoR display clear differences in their TF motif content, and motif mutagenesis experiments changed enhancer sensitivities in a predictable manner. Specific motif combinations of activating and repressive TFs, together with the regulatory proteins present in each cell type, will largely determine when and where a regulatory region is active or repressed. In addition, the ability of TFs to confer resistance also allows for enhancers and genes to be activated by de- repression via TF recruitment, as has been demonstrated by motif addition. This additional layer of regulation circumvents the requirement to remove the involved repressors and the interplay between TFs and CoRs provides more flexibility to the regulatory system. Importantly, the fact that one mode of activation can be sensitive to one repressor but resistant to another implies that distinct activation and repression mechanisms must converge. Active repression might directly interfere with specific factors or defined steps of transcriptional activation. Certain CoRs might for example inactivate specific TFs but not others (e.g. via posttranslational modifications), or counteract the TFs' downstream activating mechanisms. Alternatively, certain TFs might directly counteract a repressor's function or bypass the rate-limiting step of initiation- or elongation controlled by the CoR. Discerning the distinct mechanisms of activation and repression and how they intersect and coordinate will be of great future interest (Jacobs, 2023).

REGULATION

NADH regulates CtBP

The corepressor CtBP (carboxyl-terminal binding protein) is involved in transcriptional pathways important for development, cell cycle regulation, and transformation. CtBP binding to cellular and viral transcriptional repressors is regulated by the nicotinamide adenine dinucleotides NAD+ and NADH, with NADH being two to three orders of magnitude more effective. Levels of free nuclear nicotinamide adenine dinucleotides, determined using two-photon microscopy, correspond to the levels required for half-maximal CtBP binding and are considerably lower than those previously reported. Agents capable of increasing NADH levels stimulate CtBP binding to its partners in vivo and potentiate CtBP-mediated repression. It is proposed that this ability to detect changes in nuclear NAD+/NADH ratio allows CtBP to serve as a redox sensor for transcription (Zhang, 2002).

C-terminal-binding protein directly activates and represses Wnt transcriptional targets in Drosophila

Regulation of Wnt transcriptional targets is thought to occur by a transcriptional switch. In the absence of Wnt signaling, sequence-specific DNA-binding proteins of the TCF family repress Wnt target genes. Upon Wnt stimulation, stabilized β-catenin binds to TCFs, converting them into transcriptional activators. C-terminal-binding protein (CtBP) is a transcriptional corepressor that has been reported to inhibit Wnt signaling by binding to TCFs or by preventing -catenin from binding to TCF. This study shows that CtBP is also required for the activation of some Wnt targets in Drosophila. CtBP is recruited to Wnt-regulated enhancers in a Wnt-dependent manner, where it augments Armadillo (the fly β-catenin) transcriptional activation. CtBP is required for repression of a subset of Wnt targets in the absence of Wnt stimulation, but in a manner distinct from previously reported mechanisms. CtBP binds to Wnt-regulated enhancers in a TCF-independent manner and represses target genes in parallel with TCF. The data indicate dual roles for CtBP as a gene-specific activator and repressor of Wnt target gene transcription (Fang, 2006).

CtBP has previously been identified as a repressor of Wnt signaling, as measured by TCF reporter genes in cultured cells. Consistent with this, CtBP was identified in an overexpression screen via its ability to suppress Wg and Arm action in the developing eye. In wing imaginal discs, CtBP overexpression also inhibited the Wg target Senseless (Sens). Consistent with this overexpression data, the reduction of CtBP in cultured cells via RNAi is also consistent with a role for CtBP in repressing some Wnt targets (Fang, 2006).

The working model for CtBP repression of Wnt target gene expression holds that CtBP binds to the same area of the nkd and CG6234 loci as TCF, but this binding is TCF-independent. Consistent with this, knock down of CtBP and TCF or gro synergistically derepresses nkd expression. No synergism was seen with TCF/gro double depletions. The RNAi and ChIP data together favor a model where CtBP acts in parallel with TCF/Gro to repress nkd expression in the absence of Wg stimulation. Because CtBP has no detectable ability to bind nucleic acids, it is assumed that unknown DNA-binding protein(s) recruit CtBP to the WRE (Fang, 2006).

The existing models for CtBP antagonism of Wnt signaling cannot explain the data. TCF-independent recruitment of CtBP to WREs is not consistent with work suggesting direct binding of CtBP to TCF. The alternative mechanism, where a CtBP/APC complex diverts Arm/β-catenin away from TCF, also is inconsistent with the results. In this model, the activation of nkd expression after CtBP RNAi treatment would be dependent on TCF and arm. Because the derepression of nkd occurred when both CtBP and TCF were depleted and was not affected when arm was also inhibited, this model is not favored to explain the effects of CtBP depletion on nkd expression. These distinct mechanisms for CtBP repression are not mutually exclusive and may all occur in some contexts (Fang, 2006).

There is a qualitative difference in the amount of derepression found between the two Wg targets studied in Kc cells. Depletion of CtBP and TCF/gro causes a large (20- to 30-fold) increase in nkd basal expression, but has a much more modest (<3-fold) effect on CG6234. These differences may reflect a fundamental difference in the way TCF/Gro and CtBP act on various Wnt targets in unstimulated cells, but it is equally likely that the surrounding cis-elements in these targets have a strong influence on the degree of derepression that can be observed (Fang, 2006).

In addition to defining a novel mechanism for CtBP repression of Wg targets, strong evidence is provided for CtBP playing a role in Wg-mediated transcriptional activation. In the wing imaginal discs, loss of CtBP resulted in a lag in Wg-dependent activation of Sens and a reduction in Dll expression. In cultured Kc cells, CtBP depletion caused a two- to three-fold reduction in the ability of Wg to activate CG6234 expression. The ability of Gal4-Arm chimeras to activate a Gal4 reporter gene is also highly dependent on CtBP levels. In all these contexts, CtBP is not absolutely required for Wg signaling, but is necessary for maximal activation of Wg/Arm transcriptional activation (Fang, 2006).

The positive effect of CtBP on Wg signaling is direct, as judged by ChIP. Assuming that ChIP is measuring the degree of occupancy of CtBP on the chromatin, and not simply antigen accessibility, Wg stimulation promotes the association of CtBP with the CG6234 WRE. This increase in CtBP binding is not observed in TCF-depleted cells. Gal4-Arm recruits endogenous CtBP to a UASluc reporter. Taken together, these data support a model where TCF/Arm recruits CtBP to Wg targets. No binding between Arm and CtBP has been detected by co-immunoprecipitation, suggesting that another factor(s) may act as an adaptor between CtBP and the Arm bound to TCF (Fang, 2006).

Arm has transcriptional activation activity in both the N- and C-terminal portions of the protein. CtBP overexpression or RNAi depletion greatly effects the activity of the N-terminal half of Arm but has no effect on the C-terminal portion. Consistent with this, the N-terminal portion can recruit CtBP to a reporter gene, but not the C-terminus. Other factors that have been linked to the N-terminal portion of Arm include Lgs and Pygo and the ATPases Pontin and Reptin. It may be that CtBP acts in concert with one or more of these factors (Fang, 2006).

CtBPs have strong sequence similarity with D2-hydroxyacid dehydrogenases. hCtBP1 is a functional dehydrogenase and point mutations blocking CtBP1 dehydrogenase activity inhibit its ability to interact with binding partners and act as a transcriptional corepressor. However, another group found that similar mutations had no effect on the ability of CtBP to repress transcription. In this report, mutation of two residues (D290A and H312T) predicted to be essential for catalytic activity had no effect on the ability of fly CtBP to potentiate Gal4-Arm transcriptional activation. Further complicating the issue is data from experiments expressing the fly CtBP fused to Gal4DBD in mammalian cells. In some cells, Gal4-CtBP activated a UAS reporter, while the same reporter was repressed in other cell lines. Interestingly, conversion of CtBP's catalytic histidine to glutamine abolished transcriptional activation, but not repression. The heterologous nature of these experiments and the differences in the assays employed may explain the discrepancy between these studies, and further experiments will be needed on endogenous targets to determine how much dehydrogenase activity of CtBP contributes to repression and activation of Wnt targets (Fang, 2006).

Although CtBP is required for maximal activation of CG6234 expression and a Gal4-Arm-dependent reporter gene, Wg activation of nkd does not appear to require CtBP. The basis for this gene-specific requirement for CtBP is not clear. CtBP is recruited to the nkd WRE in a Wg-dependent manner, similar to what was observed for CG6234. It may be that CtBP is required for nkd activation, but this is masked by its role in repressing nkd expression. This hypothesis could be tested if were possible to separate CtBP's activator and repressor activities (Fang, 2006).

The requirement for CtBP in Wnt transcriptional activation may have been previously overlooked due to its well-characterized role as a co-repressor. For example, mouse embryos that lack CtBP2 have axial truncations and reduced Brachyury (T) expression that is reminiscent of Wnt3a mutants. These results suggest that the activating role for CtBP in Wnt signaling that was identified is evolutionarily conserved (Fang, 2006).

Involvement of co-repressors Groucho and CtBP in the regulation of single-minded in Drosophila

Dorso-ventral patterning results in the establishment of the two germ layers in the Drosophila embryo, mesoderm and mesectoderm, that are separated by a strip of cells giving rise to the mesectoderm and eventually to the ventral midline. The mesectoderm is specified by the expression of single-minded which is activated through the concerted action of Dorsal and Twist in addition to a Notch signal. In the mesoderm, sim is repressed by Snail together with the co-repressor C-terminal binding protein (CtBP). This study addresses the involvement of the two co-repressors CtBP and Groucho (Gro) in repression of sim in the neuroectoderm. It was shown earlier that sim is restricted in the neuroectoderm with help of Suppressor of Hairless [Su(H)] and Hairless. Using the female sterile technique, germ line clones deficient for Gro, CtBP or Hairless were generated, and sim mRNA was assayed relative to snail mRNA expression. sim repression requires both co-repressors Gro and CtBP to be fully repressed in the neuroectoderm, suggesting that a repression complex is assembled including Su(H) and Hairless as was shown for other Notch target genes before. Moreover, this work implies that Gro is important for the repression of sim specifically within the mesoderm anlagen, indicating that Snail and CtBP are insufficient to entirely silence sim in this germ layer (Nagel, 2007).

Protein Interactions

Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. The E1A sequence motif that interacts with CtBP, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K), is present in the repression domains of two unrelated short-range repressors in Drosophila (Knirps and Snail) and is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element-induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps, which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals (Nibu, 1998a).

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Two of the repressors, Hairy and Dorsal, are known to recruit a common co-repressor protein, Groucho. Evidence is presented that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, all of which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H (also termed P-DLS-R). This motif is essential for the repression activity of these proteins in transgenic embryos. It is proposed that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression (Nibu, 1998b).

A Gal4-Knirps fusion protein containing the C-terminal third of the Knirps protein (amino acid residues 255-429) has been shown to be able to repress a modified eve stripe 2-lacZ reporter gene in transgenic embryos. The fusion protein contains the Knirps P-DLS-K motif, and mutations in this sequence (PMDLSMK to AAAASMK) inactivate its repression activity. These results suggest that dCtBP is an important component of Knirps-mediated repression, but do not exclude the possibility that additional sequences in Knirps are also important for repression. To address this issue of sufficiency, the function of the P-DLS-K motif was examined in the context of the full-length, wild-type protein. Knirps is normally expressed in two domains, one anterior to eve stripe 1 and the other in the presumptive abdomen, spanning eve stripes 4, 5 and 6. The posterior border of stripe 3 is thought to depend on repression by Knirps. Ectopic expression of knirps with the eve stripe 2 enhancer results in the loss of stripe 3 expression and dominant lethality. It has been suggested that the endogenous stripe 3 pattern is repressed by the diffusion of ectopic Knirps products from stripe 2. A mutant form of Knirps that lacks the P-DLS-K motif does not repress stripe 3 expression. The mutant protein is identical to native Knirps except for four changes in the P-DLS-K motif (PMDLSMK to AAAASMK). The mutant protein is expressed at the same levels as the wild-type protein, but does not mediate efficient repression. Moreover, while the ectopic expression of the wild-type Knirps protein results in embryonic lethality, transgenic strains that misexpress similar levels of the mutant protein are fully viable. These results suggest that P-DLS-K represents the primary repression motif in the Knirps protein, although high levels of the mutant protein cause weak and variable disruptions in the stripe 3 pattern (Nibu, 1998b).

Similar assays were used to assess the significance of the P-DLS-K and P-DLS-R motifs in the Snail repressor. The eve stripe 2 enhancer was used to misexpress snail in transgenic embryos. snail is normally expressed in ventral regions where it helps establish the limits of the presumptive mesoderm by repressing various target genes such as rhomboid. The ectopic snail stripe results in an abnormal rhomboid pattern that contains a gap in the vicinity of eve stripe 2. This observation suggests that ectopic Snail products bind to the endogenous rhomboid NEE and repress its transcription. Point mutations in the P-DLS-K and P-DLS-R motifs eliminate the repression activity of an otherwise normal stripe 2-snail transgene. The mutant Snail mRNA is expressed at levels comparable with the wild-type RNA. Additional studies indicate that mutations in the P-DLS-K motif alone, with P-DLS-R intact, result in only weak repression of the rhomboid pattern. To assess the significance of the P-DLS-H motif contained in a repression domain in a C-terminal region of Krüppel, the activities were examined of a Gal4-Krüppel fusion protein that contains the C-terminal 101 amino acids residues from Krüppel. The chimeric coding sequence was expressed in ventral regions of transgenic embryos under the control of the twist promoter region. A lacZ reporter gene was introduced into embryos expressing this fusion protein. The reporter gene contains the eve stripe 2 and stripe 3 enhancers, and normally, it exhibits equally intense expression in both dorsal and ventral regions. The distal stripe 2 enhancer contains two tandem Gal4-binding sites (UAS), and when the lacZ reporter gene is crossed into embryos expressing the twist-gal4/Krüppel vector, it is repressed in the ventral mesoderm. The introduction of just three amino acid substitutions in the P-DLS-H motif (PEDLSMH to AAALSMH) eliminates the repression activity of the Krüppel fusion protein. The same substitutions also eliminate interactions between Krüppel and dCtBP in vitro. Similar results were obtained when the Gal4-Krüppel fusion protein was expressed in central regions of transgenic embryos using the Krüppel promoter region. The lacZ reporter gene used to assess the activity of this expression vector contains a modified rhomboid lateral stripe enhancer placed upstream of the proximal enhancer from the twist promoter. Normally, the reporter gene is expressed in lateral lines (mediated by the modified rhomboid enhancer) and the ventral mesoderm (twist enhancer). However, there is a gap in the lateral lines when the reporter gene is crossed into embryos expressing the Krüppel-gal4/Krüppel expression vector. This gap results from the binding of the Gal4-Krüppel fusion protein to UAS sites in the distal rhomboid enhancer. The gap is lost with a mutant fusion protein containing amino acid substitutions in the P-DLS-H motif, thereby indicating the importance of this motif in vivo (Nibu, 1998).

The mechanism by which dCtBP mediates transcriptional repression is unknown. However, the current study provides evidence against a previously proposed mechanism for Krüppel (Sauer, 1995). Krüppel activity is shown to be lost in dCtBP mutants, and the C-terminal region of the protein contains an essential P-DLS-H repression motif. Moreover, preliminary studies suggest that ectopic expression of the native Krüppel protein causes patterning defects in early embryos, which are reversed when the P-DLS-H motif is mutagenized. These results strongly suggest that Krüppel-mediated repression depends on the recruitment of the dCtBP co-repressor. The earlier study provided evidence that repression depends on the direct interactions of Krüppel with the beta-subunit of the TFIIE general transcription factor (Sauer, 1995). It is conceivable that this mechanism of repression is employed in other tissues at later stages in the Drosophila life cycle, although it is noted that a recent study provides strong evidence that a mammalian Krüppel-like protein also employs a CtBP co-repressor (Nibu, 1998b and references).

The BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain is an evolutionarily conserved protein-protein interaction motif. Many BTB-containing proteins are transcriptional regulators involved in a wide range of developmental processes. However, the significance of the BTB domain in development has not been evaluated. Evidence is presented that overexpression of the Tramtrack69 (Ttk69) protein not only blocks neuronal photoreceptor differentiation but also promotes nonneuronal cone cell specification in early Drosophila eye development. The Ttk69 BTB domain is critical for mediating interaction with the Drosophila homolog of C-terminal-binding protein (dCtBP) in vitro, and dCtBP minus mutations genetically interact with ttk69. Furthermore, the C-terminal region downstream of the DNA-binding zinc fingers has been shown to be essential for Ttk69 function. A dCtBP consensus binding motif in the C terminus appears to contribute to Ttk69 activity, but it cannot be fully responsible for the function of the C terminus (Wen, 2000).

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, a detailed structure-function analysis of the Knirps protein has been undertaken. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions have been identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities (Keller, 2000).

Knirps has been shown to repress transcription when bound adjacent to either basal promoters or activators within enhancer elements. These studies of Knirps activity when the protein binds close to the basal promoter reveals additional properties of the endogenous protein. (1) Repression by Knirps does not appear to be sensitive to phasing effects, as shown by equivalent activity of constructs with Knirps binding sites offset by 5 bp at -70 and -75 bp. (2) In this series of genes, the transcriptional repression activity appears to be directed at the basal promoter element, because the repression weakens as the distance from Knirps sites to the basal promoter is increased while the distance to the enhancer element is held constant. (3) While Knirps repression is limited to a relatively short distance, there is a measurable interval (from 100 to 130 bp) over which Knirps activity is attenuated but not entirely abolished (Keller, 2000).

This intermediate level of repression might be useful in adjusting the amount of repression imposed on a target gene or setting a target gene threshold, as has been demonstrated for the Drosophila Giant short-range repressor. With Giant, a less-than-twofold difference in posterior versus anterior protein levels is sufficient to switch a gene from on to off. Thus, two features of short-range repressors may allow for flexibility in genetic regulatory circuits: (1) short-range repressors allow modular enhancers to act independently, by avoiding regulatory cross talk, and (2) the exquisite distance dependence may contribute to the differential response of endogenous target genes to repressor gradients (Keller, 2000).

This study demonstrates that the Knirps protein contains two functionally distinct repression activities. The C-terminal region appears to mediate repression through recruitment of the dCtBP protein: it consists of a region contained within residues 202 to 358 (minimally, residues 248 to 291 and 313 to 358 including the PMDLSMK dCtBP binding motif. In contrast, the N-terminal repression region (minimally, residues 139 to 330) appears to function independently of dCtBP. Although this region contains some of the amino acid residues that are present in the dCtBP binding constructs, the two activities are clearly distinct based on dCtBP dependence. The N-terminal region does not bind to dCtBP and it can repress in a mutant embryo that lacks maternal dCtBP. Any residual amounts of dCtBP from maternal or zygotic expression are likely to be very low, because the loss of maternal dCtBP expression causes a loss of activity of Snail, Knirps, and Krüppel on a number of target genes, producing severe embryonic defects and early developmental arrest (Keller, 2000).

Knirps repression domains have been defined in the context of Gal4 fusion proteins, but several lines of evidence suggest that the native Knirps protein can also repress target genes independently of dCtBP. Most compellingly, an eve stripe 3 lacZ reporter gene that is derepressed in a knirps mutant background is not derepressed in a CtBP mutant. In addition, a frameshift mutation (kni^14F) that produces a protein lacking the dCtBP interaction motif retains partial activity, perhaps via the N-terminal repression activity that has been defined in this study. Finally, a study of ectopically expressed Knirps protein that lacks a dCtBP binding motif found that the protein has weak repression on eve stripe 3 (Keller, 2000).

A region of the Knirps protein containing an alanine-rich tract had been identified in earlier studies as a repression domain in cell culture studies but is neither necessary nor sufficient for repression in the embryo. The repression function of 189-254 protein may be specific to transfection assays, similar to findings for the non-Groucho binding region of the Engrailed repressor protein (Keller, 2000).

It is not yet clear whether repression by the N-terminal and C-terminal regions of Knirps contribute to quantitative or qualitative differences in repression, or if these two aspects of repression are indeed entirely separable. The eve stripe 3 enhancer is clearly repressed in the region of kni expression in the absence of maternal dCtBP, yet in previous experiments, ectopically expressed Knirps was able to repress the eve stripe 3 element effectively only when the dCtBP binding motif of the protein was still intact. The most likely explanation for these apparently contradictory results is that dCtBP contributes to a portion of the Knirps-mediated repression of stripe 3. Endogenous Knirps is abundant enough to repress expression of eve stripe 3 in dCtBP mutant embryos, but the levels of ectopically produced Knirps protein are apparently insufficient to repress effectively when binding to dCtBP is abolished. dCtBP may also have an effect on Knirps protein stability or targeting, which might contribute to the reduced activity of the mutant protein. Previous studies have indicated that in the absence of dCtBP, repression of a synthetic rho lacZ reporter gene by endogenous Knirps is reduced. However, close examination of the data indicates that some anterior repression is apparently present, consistent with the idea that Knirps retains a measurable level of activity in the dCtBP mutant (Keller, 2000).

The Gal4-Knirps chimeras containing only the N-terminal repression domain appear to have higher levels of activity on lacZ reporters than does full-length Knirps protein lacking the dCtBP binding motif. A test was performed to see whether this difference might be attributed to masking of the N-terminal repression region by the C terminus in the absence of dCtBP. The data indicate that this model is not correct; Gal4-Knirps chimeras containing the N-terminal repression domain linked to a C-terminal region lacking a dCtBP binding activity are highly effective repressors. Gal4-Knirps chimeras may be inherently more effective repressors if one role of dCtBP is to facilitate dimerization of Knirps proteins. With chimeras, this function would be provided by the Gal4 DNA binding domain, because Gal4 binds DNA as a dimer. Alternatively, autoinhibition of the Knirps DNA binding domain, similar to that seen with Ets-1, AML-1, and Pitx2, may be relieved by dCtBP binding, but Gal4 chimeras would not be subject to such regulation. However, the effective regulation of eve stripe 3 lacZ in a CtBP mutant argues for a simpler quantitative effect model. Loss of dCtBP binding might simply reduce the total repression activity of Knirps protein, so that the low levels of misexpressed Knirps would be unable to effect repression. The Gal4-Knirps repressor utilizing only one repression region might be more functional due to increased effectiveness of dimerized repressor proteins or to a greater sensitivity of the lacZ reporters used (Keller, 2000).

Multiple repression activities in a protein may allow for qualitative or quantitative effects on gene expression. Qualitatively, a repressor may operate selectively in distinct tissue types or on different promoters. Loss of maternal dCtBP protein does not affect eve stripe 3 regulation, but it does abolish repression of the eve stripe 4+6 enhancer element, suggesting that this element is dCtBP dependent. Quantitatively, dual activities may increase the overall level of repression, much as transcriptional activators have been suggested to employ multiple paths to achieve synergistic activation (Keller, 2000).

Examples of both qualitative and quantitative effects are seen with the ZEB repressor, a protein that contains two repression domains. One domain blocks activation by Myb and Ets factors of lymphocyte-specific promoters, while the second domain, which contains a conserved CtBP binding motif, blocks the activity of the muscle cell-specific MEF2C factor. In contrast to these activator-specific effects, a quantitative contribution of multiple repression domains has been observed with the murine ZEB homolog deltaEF-1. When CtBP binding residues are mutated in deltaEF-1, repression of a MyoD-activated promoter is impaired but not abolished (Keller, 2000 and references therein).

Other repressor proteins may also possess both CtBP-dependent and dCtBP-independent activities. In Drosophila, the Krüppel protein contains a C-terminal dCtBP binding repression domain and an N-terminal repression domain. The latter domain has only been characterized in cell culture assays, but genetic evidence indicates that Krüppel can repress hairy in a CtBP mutant, possibly by means of this N-terminal domain. The Wnt signaling pathway transcription factor Tcf-3 can interact with both the Groucho and CtBP proteins through separate repression domains in Xenopus laevis, and the CtBP-binding portion of XTcf-3 has potent repression activity in the frog embryo. The Rb retinoblastoma protein has been shown to interact with both histone deacetylases and CtBP, although the physiological relevance of the CtBP interactions is not yet clear. Net, an Ets protein family member that can repress transcription of the c-fos promoter, has also been shown to possess two independent repression domains, one of which interacts with CtBP1. Loss of the CtBP binding motif from Net reduces the repression activity of the protein in cell culture assays. Finally, the BKLF transcription factor, which can interact with CtBP2 to repress transcription in Drosophila cell culture, contains an additional CtBP-independent activity detectable in NIH 3T3 cells (Keller, 2000).

dCtBP and its homologs appear to be able to mediate repression directly when recruited to promoters by a heterologous DNA binding domain, both in cell culture systems and in the embryo. The dCtBP corepressor has homology to alpha-hydroxy acid dehydrogenases and contains a conserved NAD-binding domain. The protein binds to NAD, but no dehydrogenase activity has been detected in vitro, and mutation of a conserved histidine in the putative active site does not compromise the repression activity of a chimeric CtBP2 protein in cell culture assays. The dCtBP protein may contain other uncharacterized enzymatic activities. Recently it was reported that the Sir2 transcriptional repressor possesses ADP ribosylation activity, and furthermore, that NAD is important for histone deacetylase activity of the protein. Some evidence suggests that CtBP may function through histone deacetylase pathways, but pair-rule gene repression by gap proteins such as Knirps and Krüppel is not compromised by mutations in the Rpd3 histone deacetylase (Keller, 2000).

The physiological relevance of CtBP binding is not yet known for a number of proteins that have been found to interact in yeast two-hybrid assays, but genetic evidence from Drosophila clearly indicates that dCtBP is an important repression cofactor. These data demonstrate that for at least one Knirps target gene, another pathway of repression is also utilized. A considerable body of evidence, including genetic and biochemical data, indicates that repressors may have multiple lines of communication with the transcriptional machinery, just as transcriptional activators have been found to contain multiple activation domains that act on multiple targets. Further genetic and biochemical characterization of Knirps will help elucidate the pathways utilized by this short-range repressor (Keller, 2000).

Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators; these proteins are required to maintain active states of Hox gene expression during development. A trithorax acetylation complex (TAC1) has been purified that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression (Petruk, 2001).

Notch signal transduction involves the presenilin-dependent intracellular processing of Notch and the nuclear translocation of the intracellular domain of Notch, NICD. NICD associates with Suppressor of Hairless [Su(H)], a DNA binding protein, and Mastermind (Mam), a transcriptional coactivator. In the absence of Notch signaling, Su(H) acts as a transcriptional repressor. Repression by Su(H) is relieved by the activation of Notch. In the Drosophila embryo, this transcriptional switch from repression to activation is important for patterning the expression of the single-minded (sim) gene along the dorsoventral axis. The mechanisms by which Su(H) inhibits the expression of Notch target genes in Drosophila has been investigated. Hairless, an antagonist of Notch signaling, is required to repress the transcription of the sim gene. Hairless forms a DNA-bound complex with Su(H). Furthermore, it directly binds the Drosophila C-terminal Binding Protein (dCtBP), which acts as a transcriptional corepressor. The dCtBP binding motif of Hairless is essential for the function of Hairless in vivo. It is proposed that Hairless mediates transcriptional repression by Su(H) via the recruitment of dCtBP (Morel, 2001).

The mechanism by which Hairless might regulate transcription was investigated. Sequence analysis of Hairless identifies a putative binding site for the Drosophila C-terminal Binding Protein (dCtBP). This site is located at the very C terminus of the Hairless protein. In Drosophila and mammals, CtBP is a transcriptional corepressor. It was therefore tested whether Hairless binds to dCtBP. The full-length Hairless protein, H[1-1076], interacts with dCtBP in a yeast two-hybrid assay. In contrast, a truncated version of Hairless in which the last 15 amino acids had been deleted, H[1-1061], does not bind to dCtBP. This shows that the Hairless-dCtBP interaction strictly depends on the conserved C-terminal part of Hairless that contains the dCtBP binding site. Furthermore, a small C-terminal peptide, H[1052-1076], is sufficient to bind dCtBP. Finally, a specific interaction between Hairless and dCtBP is also observed in vitro with a GST pull-down assay. H[1-1076], but not H[1-710] or H[1-1061], is efficiently retained by a GST-dCtBP fusion protein. This in vitro interaction indicates that the Hairless-dCtBP interaction is likely to be direct. It is concluded that the conserved C-terminal part of Hairless contains a motif necessary and sufficient to bind dCtBP (Morel, 2001).

To test the functional significance of this binding site, an in vivo assay was used. The expression of a truncated version of Hairless that does not bind dCtBP, H[1-1061], does not lead to an increased density of sense organs and does not rescue the loss of Hairless function. Thus, the last 15 amino acids of Hairless are required for the activity of the protein. Interestingly, flies overexpressing both H[1-1061] and Su(H) display a wild-type phenotype. This shows that H[1-1061] is unable to cooperate with Su(H) to block Notch signaling. Nevertheless, H[1-1061] expression suppresses the loss-of-bristle phenotype associated with increased levels of Su(H). Since H[1-1061] binds Su(H), it is possible that H[1-1061] proteins form nonproductive complexes with Su(H). Accordingly, the residual activity of the mutant H^RP1 protein, which carries a 68 amino acid C-terminal deletion, might result from its ability to sequester Su(H) without actively repressing transcription. These results therefore suggest that Hairless requires the binding of dCtBP to repress the expression of Notch target genes (Morel, 2001).

In summary, these findings indicate that Hairless links Su(H) to the dCtBP corepressor. It is therefore proposed that Hairless antagonizes Notch signaling activity by recruiting dCtBP to repress Notch target gene expression. The activation of the Notch receptor would then lead to a competition between NICD and Hairless to assemble DNA-bound regulatory complexes of opposite activities (Morel, 2001).

Two corepressors have been identified in the early Drosophila embryo: Groucho and dCtBP. Both proteins are recruited to the DNA template by interacting with short peptide motifs conserved in a variety of sequence-specific transcriptional repressors. Once bound to DNA, Groucho appears to mediate long-range repression, while dCtBP directs short-range repression. The short-range Krüppel repressor is converted into a long-range repressor by replacing the dCtBP interaction motif (PxDLSxH) with a Groucho motif (WRPW). The resulting chimeric repressor causes a different mutant phenotype from that of the native Krüppel protein when misexpressed in transgenic embryos. The different patterning activities can be explained on the basis of long-range silencing within the hairy 5' regulatory region. The analysis of a variety of synthetic transgenes provides evidence that Groucho-dependent long-range repressors do not always cause the dominant silencing of linked enhancers within a complex cis-regulatory region. A 'hot chromatin' model is suggested, whereby repressors require activators to bind DNA (Nibu, 2001a).

Complex enhancers direct stripes and bands of gene expression in the early Drosophila embryo. These enhancers are typically 300 bp-1 kb in length and contain clustered binding sites for transcriptional activators and repressors. Different enhancers can work independently of one another within a common cis-regulatory region to direct composite patterns of gene expression. For example, the seven-stripe even-skipped (eve) expression pattern is activated by five separate enhancers located 5' and 3' of the transcription unit. The ability of these enhancers to function in an autonomous fashion depends on short-range transcriptional repressors that work over distances of <100 bp to inhibit, or quench, upstream activators. The binding of the Krüppel repressor to the stripe 2 enhancer does not interfere with the activity of the stripe 3 enhancer since Krüppel mediates repression only when positioned near upstream activators. Consquently, Krüppel quenches Bicoid activators within the stripe 2 enhancer without interfering with the D-Stat activators bound to the stripe 3 enhancer (Nibu, 2001a).

There are several short-range repressors in the early embryo, including Krüppel, Snail, Knirps and Giant. Most or all of these repressors interact with a common corepressor protein, dCtBP, which is the Drosophila homolog of a human protein that was found to attenuate the oncogenic activities of the adenovirus E1A protein. dCtBP is maternally expressed and ubiquitously distributed throughout early embryos. A variety of studies suggest that the dCtBP corepressor protein is recruited to the DNA template by interacting with a conserved sequence motif contained in most or all sequence-specific short-range repressors: PxDLSxK/R/H. There is emerging evidence that mammalian CtBP proteins also function as corepressors, although it is not known currently whether the mammalian repressors (e.g. bKLF, Ikaros and ZEB-1) only function over short distances (Nibu, 2001a).

A number of repressors can work when positioned far from upstream activators and the core promoter. For example, the binding of the Hairy repressor to a modified rhomboid lateral stripe enhancer (NEE) can cause the dominant silencing of a linked mesoderm-specific enhancer, even when the two enhancers are separated by >1 kb in the 5' cis -regulatory region. Hairy interacts with a second ubiquitous corepressor protein, Groucho. Hairy-Groucho interactions depend on a conserved sequence motif at the Hairy C-terminus: WRPW. These studies suggest that the dCtBP corepressor protein mediates short-range repression, while Groucho mediates long-range repression. The present study provides additional support for this possibility (Nibu, 2001a).

The long-range action of the Groucho corepressor poses a potential problem with regard to enhancer autonomy in complex promoter regions. In principle, the binding of a Groucho-dependent repressor could result in the dominant silencing of all enhancers located in the 5' and 3' regulatory regions of a target gene. This imposes a potentially severe constraint on the evolution of complex patterns of gene activity. To investigate this issue, the activities have been examined of chimeric repressor proteins that contain the DNA-binding domains of the short-range Krüppel or Snail repressors and the Groucho interaction sequences in the long-range Hairy repressor. These chimeric repressors were expressed in specific regions of transgenic embryos using defined, heterologous enhancers. The Krüppel-Hairy fusion protein causes altered patterns of segmentation gene expression that are consistent with the notion that Hairy-Groucho interactions convert Krüppel into a long-range repressor. However, the abnormal rhomboid expression pattern obtained with a similar Snail-Hairy fusion protein suggests that it does not function as a dominant silencer, but instead causes the local repression of a single enhancer. The subsequent analysis of a number of synthetic transgenes provides direct evidence that the long-range Hairy repressor does not always cause the dominant silencing of linked enhancers (Nibu, 2001a).

A comparison of the altered patterns of hairy expression obtained with the twi-Krüppel and twi-Krüppel-hairy transgenes provides evidence that dCtBP and Groucho mediate short- and long-range repression, respectively. The twi-Krüppel transgene causes the repression of hairy stripe 6, but not stripe 5. Previous studies have shown that the stripe 6 enhancer contains optimal, high-affinity Krüppel operator sites that can be occupied by the low levels of Krüppel produced in ventral regions by the twi-Krüppel transgene. These low levels appear to be insufficient to bind the low-affinity sites within the hairy stripe 5 enhancer and, consequently, the native Krüppel protein works as a short-range repressor to inhibit stripe 6 expression without affecting stripe 5 expression. In contrast, the twi-Krüppel-hairy transgene leads to the repression of both stripes 5 and 6. The binding of the Krüppel-Hairy fusion repressor to the stripe 6 enhancer appears to cause the dominant silencing of the neighboring stripe 5 enhancer over a distance of ~2.5 kb in the hairy 5' regulatory region. An implication of these observations is that different repression domains exert distinct influences on embryonic patterning. Replacing the PxDLSxH motif (native Krüppel) with WRPW (Krüppel-Hairy) changes the regulatory activity of the Krüppel repressor (Nibu, 2001a).

The Snail-Hairy fusion protein represses the rhomboid lateral stripes, but fails to repress the amnioserosa pattern. In contrast, the same Hairy repression domain permits Krüppel to function as a dominant silencer within the hairy 5' regulatory region. There are several possible explanations for the failure of the Snail-Hairy repressor to silence rhomboid expression in the amnioserosa. Perhaps there is competition between dCtBP bound to the Snail moiety and Groucho bound to the Hairy moiety within the fusion protein. The Krüppel-Hairy fusion protein was mutagenized to eliminate the dCtBP motif (PEDLSMH), whereas the Snail-Hairy fusion protein retains both dCtBP sequences. Previous studies suggest that the conversion of the weak dCtBP interaction motif near the Hairy C-terminus, PLSLVIK, into an optimal motif, PLDLSIK, disrupts the repressor function of an otherwise normal Hairy protein. This result was taken as evidence that the dCtBP and Groucho corepressors interfere with one another when bound to closely linked motifs within the Hairy C-terminus. An argument against this explanation for the behavior of the Snail-Hairy fusion protein stems from the observation that the binding of Hairy to a modified NEE is sufficient to repress a linked mesoderm enhancer (twist PE), but not a similarly spaced race enhancer. Similarly, the binding of Hairy to a modified race enhancer fails to silence the mesoderm enhancer (Nibu, 2001a).

It is proposed that Hairy can only bind active or 'open' enhancers. The NEE is activated by the maternal Dorsal nuclear gradient and, consequently, it might contain activator proteins in both ventral and lateral regions of early embryos. As a result, the binding of Hairy to the modified h-NEE-h enhancer can lead to the dominant silencing of a linked mesoderm enhancer (twist PE). In contrast, there is no Dorsal activator in dorsal regions of the early embryo, thereby rendering the h-NEE-h enhancer in a closed or condensed state. This absence of activator might preclude the binding of Hairy so that the race enhancer is not silenced. Similarly, the race enhancer is probably activated by transcription factors that are restricted to dorsal regions, such as Zen and Smads. These activators are absent in ventral regions and, consequently, Hairy may be unable to bind the h-race-h enhancer and silence linked enhancers such as the twist PE (Nibu, 2001a).

The altered pattern of hairy expression caused by the Krüppel-Hairy fusion protein can be interpreted in the context of this 'hot chromatin' model. There is evidence that hairy stripes 5, 6 and 7 are activated by a posterior gradient of the Caudal activator. The binding of the Krüppel-Hairy fusion protein to the optimal Krüppel operator sites in the stripe 6 enhancer would be expected to silence the neighboring stripe 5 enhancer due to the open conformation of the stripe 6 enhancer in those regions of the embryo where stripe 5 is expressed. Thus, the Caudal activator might bind to both enhancers in the position of stripe 5, thereby rendering the stripe 6 enhancer accessible to the Krüppel-Hairy fusion protein (Nibu, 2001a).

The dependence of repressors on activators might restrain long-range repressors and permit enhancer autonomy. This dependence might reflect the inherent properties of activators and repressors. Some activators recruit enzymes that decondense chromatin, and this may be essential for the binding of repressors in vivo. Short-range repression has been put forward as an important mechanism for enhancer autonomy. It is suggested that a second mechanism involves the reliance of repressors on activators for binding to target enhancers (Nibu, 2001a).

There are at least three short-range gap repressors in the precellular Drosophila embryo: Krüppel, Knirps, and Giant. Krüppel and Knirps contain related repression motifs, PxDLSxH and PxDLSxK, respectively, which mediate interactions with the dCtBP corepressor protein. Giant might also interact with dCtBP. The misexpression of Giant in ventral regions of transgenic embryos results in the selective repression of eve stripe 5. A stripe5-lacZ transgene exhibits an abnormal staining pattern in dCtBP mutants that is consistent with attenuated repression by Giant. The analysis of Gal4-Giant fusion proteins has identified a minimal repression domain that contains a sequence motif, VLDLS, which is conserved in at least two other sequence-specific repressors. Removal of this sequence from the native Giant protein does not impair its repression activity in transgenic embryos. It is proposed that Giant-dCtBP interactions might be indirect and mediated by an unknown bZIP subunit that forms a heteromeric complex with Giant (Nibu, 2001b).

The minimal Giant repression domain spans amino acid residues 60-133. Alignment of this sequence with the Drosophila database identifies significant homology with the zinc finger repressor, Odd-skipped (Odd). Odd represses the expression of engrailed within the even-numbered parasegments and thereby defines which of the Ftz-expressing cells activate engrailed. Giant and Odd share the following sequence: VLDLSxxxxSxExP. A third transcriptional repressor in the early embryo, Tailless, also contains the VLDLS motif. Tailless is important for repressing segmentation gene expression in the anterior and posterior poles. It is unclear whether this sequence participates in Giant-dCtBP interactions, even though it is related to the dCtBP motif (PxDLSxR/K/H). Perhaps VLDLS helps recruit an unknown corepressor protein that mediates the residual repression activity of Gal4-Giant fusion proteins in dCtBP mutants (Nibu, 2001b).

The low levels of Giant produced by an twi-giant transgene are sufficient to repress the endogenous eve stripe 5 pattern but not stripe 2. The failure to repress stripe 2 is consistent with previous studies, which suggested that Giant might interact with a localized 'partner' in anterior regions of the early embryo. It is also possible that stripe 2 regulation depends on high concentration of the Giant protein. There are two alternative explanations for the sufficiency of low levels of Giant to repress stripe 5. First, the stripe 5 enhancer might contain optimal high-affinity Giant operator sites. Alternatively, Giant might interact with an unknown bZIP subunit, X, that is broadly expressed in the early embryo (Nibu, 2001b).

The second possibility, whereby Giant-X heterodimers regulate stripe 5 expression, is favored. Putative Giant operator sites in the stripe 5 enhancer lack obvious dyad symmetry, which might be expected for Giant-Giant homodimers. Moreover, the VLDLS motif is essential for the repression activity of Gal4-Giant fusion proteins but is dispensable in the context of the twi-giant transgene. For example, a deletion that removes the entire minimal repression domain (amino acids 60-133) does not significantly impair the ability of a twi-giant transgene to repress eve stripe 5 and hairy stripes 3, 4, and 5. Presumably, Gal4-Giant fusion proteins function as homomultimers, so that mutations in the repression domain attenuate or eliminate activity. In contrast, the same mutations might not disrupt the activities of a heterodimeric Giant-X complex because of the ability of subunit X to recruit dCtBP. Future studies will focus on the identification of subunit X and the corepressor(s) that interact with the conserved VLDLS motif (Nibu, 2001b).

The Giant protein is a short-range transcriptional repressor that refines the expression pattern of gap and pair-rule genes in the Drosophila blastoderm embryo. Short-range repressors including Knirps, Krüppel, and Snail utilize the CtBP cofactor for repression, but it is not known whether a functional interaction with CtBP is a general property of all short-range repressors. Giant repression activity was studied in a CtBP mutant and it has been found that this cofactor is required for Giant repression of some, but not all, genes. While targets of Giant such as the even-skipped stripe 2 enhancer and a synthetic lacZ reporter show clear derepression in the CtBP mutant, another Giant target, the hunchback gene, is expressed normally. A more complex situation is seen with regulation of the Krüppel gene, in which one enhancer is repressed by Giant in a CtBP-dependent manner, while another is repressed in a CtBP-independent manner. These results demonstrate that Giant can repress both via CtBP-dependent and CtBP-independent pathways, and that promoter context is critical for determining giant-CtBP functional interaction. To initiate mechanistic studies of the Giant repression activity, a minimal repression domain within Giant has been identified that encompasses residues 89-205, including an evolutionarily conserved region bearing a putative CtBP binding motif (Stunk, 2001).

The deletional analysis of Gal4-Giant chimeras indicates that Giant repression function can be localized to residues 89-205, an area of the protein that contains several tracts of highly conserved residues. Chimeras containing other portions of the Giant protein do not exhibit significant repression activity, suggesting that these regions cannot act autonomously to mediate repression, and might instead contribute to protein stability or expression. In particular, residues 266-322 appear to correlate with significantly higher repression activity of these proteins. The low levels of chimeric protein expression in the embryo precluded direct quantitation of each protein, thus this analysis is based primarily on those that did show significant activity (Stunk, 2001).

No significant physical interaction could be detected between Giant and CtBP in vitro, and the Giant protein lacks a perfect match to the consensus CtBP binding motif P-DLS-K/R/H found in the Knirps, Krüppel, and Snail proteins. However, a partial match is present: VLDLSRR (residues 98-104). The motif is evolutionarily conserved and is found within the minimal repression domain defined, consistent with a possible role in repression. Indeed, deletion of residues 89-107 inactivates the chimeric repressor. This region is clearly not sufficient for high-level repression, however, suggesting that other portions of the protein play important structural or functional roles. If CtBP directly contacts Giant in vivo, the lack of strong interaction in vitro may indicate that Giant must be posttranscriptionally modified to facilitate interaction with CtBP, perhaps via phosphorylation. Posttranslational modifications are known to play a role in CtBP binding in some instances; E1A-CtBP interactions have been shown to be regulated by acetylation of a conserved lysine residue in the CtBP binding motif. Alternatively, Giant may bind CtBP indirectly through a cofactor, much as BRCA1 has been suggested to bind CtBP through CtIP, or CtBP might be recruited via a heterodimeric basic-zipper partner of Giant. To determine whether CtBP-dependent and CtBP-independent repression activities are mediated by the same or distinct portions of the Giant protein, future studies will need to focus on identifying mutant proteins that are deficient in each of these activities (Stunk, 2001).

What characteristics of a regulatory region dictate CtBP-dependent or CtBP-independent repression? In considering which features of a gene determine CtBP-dependence or -independence, the structure of the basal promoter cannot be the deciding factor, for the same Kr promoter is regulated by distinct elements, some that exhibit CtBP-dependence and some that show CtBP-independence. Similarly, the eve gene is repressed by Knirps via CtBP-dependent and CtBP-independent regulatory elements. While the eve enhancers in question are kilobases apart, the Kr regulatory elements driving anterior and central domain (CD) expression are closely intertwined, and appear to share at least some of the same activator binding sites, suggesting that subtle differences in enhancer architecture or differences in levels of regulatory proteins interacting with those elements may dictate CtBP dependence. The Giant binding site in the Kr CD2 enhancer site was shown to be of higher affinity than the gt1 site in the eve stripe 2 enhancer. Thus, there may be a correlation between Giant binding site affinity and the requirement for CtBP, with elements containing Giant sites of lower affinity showing CtBP-dependence. A consensus has been derived for the Giant protein by aligning binding sites for Giant from eve, Kr, and the recently identified abdA iab-2 enhancer site. The consensus features an extended half-site inverted repeat TNTTAC, consistent with the dimeric nature of basic zipper proteins, and a central ACGT core common to recognition motifs for many basic zipper proteins. The higher affinity sequences from the CtBP-independent Kr CD element are closer to the consensus than those of the CtBP-dependent eve stripe 2 enhancer. Weaker sites may only be partially occupied, resulting in an overall lower level of Giant mediated repression. A loss of CtBP might further depress repression activity below a critical threshold, leading to the derepression observed. Repression of the lacZ reporter containing the giant CD1 site from Kr is CtBP-dependent, a result that contrasts with the CtBP independence of the CD itself, but this particular site may not be optimal, since it contains two mismatches. Full Giant activity may also be mediated on the native CD element through the additional high-affinity CD2 site (Stunk, 2001).

Other factors besides binding site affinity can affect Giant’s activity, and possibly its CtBP-dependence. Small alterations in the location of Giant binding sites is sufficient to strongly affect the ability of Giant to repress in transgenic embryo assays. Thus, location and affinity of Giant sites needs to be considered in studying CtBP-dependent repression. It is not believed that differences in the nature of the activators explain CtBP-dependence or -independence, because both AD and CD enhancers of Kr are activated by Bicoid protein, as is the eve stripe 2 enhancer. Detailed studies illuminating how the general properties of short-range transcriptional repressors are integrated into the design of promoter elements will promote an understanding of the control of complex developmentally regulated genes (Stunk, 2001).

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).

Transcriptional repressors can be classified as short- or long-range, according to their range of activity. Functional analysis of identified short-range repressors has been carried out largely in transgenic Drosophila, but it is not known whether general properties of short-range repressors are evident in other types of assays. To study short-range transcriptional repressors in cultured cells, chimeric tetracycline repressors were created based on Drosophila transcriptional repressors Giant, Drosophila C-terminal-binding protein (dCtBP), and Knirps. Giant and dCtBP are found to be efficient repressors in Drosophila and mammalian cells, whereas Knirps is active only in insect cells. The restricted activity of Knirps, in contrast to that of Giant, suggests that not all short-range repressors possess identical activities, consistent with recent findings showing that short-range repressors act through multiple pathways. The mammalian repressor Kid is more effective than either Giant or dCtBP in mammalian cells but is inactive in Drosophila cells. These results indicate that species-specific factors are important for the function of the Knirps and Kid repressors. Giant and dCtBP repress reporter genes in a variety of contexts, including genes that are introduced by transient transfection, carried on episomal elements, or stably integrated. This broad activity indicates that the context of the target gene is not critical for the ability of short-range repressors to block transcription, in contrast to other repressors that act only on stably integrated genes (Ryu, 2002).

The regulation of inducible promoters via chimeric tetracycline repressor (TetR) proteins has attracted considerable interest for use in ectopic expression systems in cell culture, microbes, plants, and whole animals. In these systems, a chimeric protein consisting of the Escherichia coli TetR protein fused to an activation domain binds to promoters containing Tet response elements (TREs). On addition of tetracycline or doxycycline, the chimeric protein is released from the promoter and the gene is inactivated. TetR DNA-binding domains with reverse specificity have been developed to permit activation of target genes on addition of the drug. Although this system can be highly regulated, low-level basal expression can be a problem in the case of potentially toxic gene products. To overcome this problem, higher specificity Tet DNA-binding domains have been recently developed. Many endogenous genes accomplished tight regulation by the coordinated action of repressors and activators. To mimic such composite systems, a Tet repressor can be combined with a Tet activator to give repression and activation in the absence and presence of doxycycline, respectively. Such combined Tet-based activation/repression systems have been developed for yeast and mammalian systems. Most of these systems use the KRAB repressor domain. Whether KRAB repressors can work in nonvertebrate cell types has not been reported, however. In this study, a panel of transcriptional repressors has been created based on well characterized short-range repressors from Drosophila. The chimeric proteins show reproducible repression activity in the Tet system in a variety of cell types and on stably integrated or transiently introduced reporter genes. Compared with the mammalian Kid repressor, these repressors may be the preferred alternative for regulation of expression in some cell types and with certain transgene configurations (Ryu, 2002).

CtBP and Hairy

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, a yeast interaction screen was carried out and a Hairy-interacting protein was identified, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy in the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. A P-element insertion was identified within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De (Poortinga, 1998).

To target protein interactions with specific conserved regions of the Hairy protein, a two-hybrid screen was carried out using a LexA-tagged Hairy partial protein bait. This strategy also allowed the circumvention of the reporter system repression that was encountered when using full-length Hairy protein as a bait. A VP16-tagged Drosophila library constructed from 0-4 h embryonic mRNAs was screened with a bait that encodes the Hairy Orange domain through to the C-terminus (h-C, amino acids 93-343. In directed yeast two-hybrid assays, this fragment, h-C28, interacts weakly with full-length Hairy, but more strongly with Hairy partial proteins and with E(spl)m, another member of Hairy-class bHLH proteins. It does not interact with Dpn and interacts poorly, if at all, with E(spl)m3, -m5, -m8, -mbeta -mgamma. h-C28 does not show interaction with proteins from other HLH classes (i.e. Scute, Emc). The region of Hairy required for interaction with h-C28 was mapped using a series of Hairy deletions and partial proteins fused to LexA. h-C28 interacts strongly with a 25 amino sequence immediately upstream of, but not including, the C-terminal WRPW motif. This identifies a previously undefined protein interaction domain within Hairy. dCtBP also interacts with itself (Poortinga, 1998).

The precellular Drosophila embryo contains approximately 10 well characterized transcriptional repressors. At least half are short-range repressors that must bind within 100 bp of either upstream activators or the core transcription complex to inhibit (or quench) gene expression. The two long-range repressors can function over distances of 1 kilobase or more to silence transcription. Previous studies have shown that three of the five short-range repressors interact with a common corepressor protein, dCtBP. In contrast, the two long-range repressors, Hairy and Dorsal, recruit a different corepressor protein, Groucho. Hairy also was shown to interact with dCtBP, thereby raising the possibility that Groucho and dCtBP are components of a common corepressor complex. To investigate this issue, wild-type and mutant forms of Hairy were misexpressed in transgenic embryos. Evidence is presented that Hairy-mediated repression depends on the Groucho interaction sequence (WRPW) but not the weak dCtBP motif (PLSLV) present in the native protein. Conversion of the PLSLV motif into an optimal dCtBP interaction sequence (PLDLS) disrupts the activity of an otherwise normal Hairy protein. These results suggest that dCtBP and Groucho mediate separate pathways of transcriptional repression and that the two proteins can inhibit one another when both bind the same repressor (Zhang, 1999).

The removal of the weak dCtBP interaction motif (PLSLV) does not impair Hairy-mediated repression of Sxl, fkh, hkb, and tll. If anything, removal of this motif augments Hairy function. This observation suggests that the binding of dCtBP somehow interferes with Groucho-mediated repression. Additional support for this view stems from the observation that the PLDLS/WRPW protein, which contains an optimal dCtBP motif, is inactive and fails to repress any of the target genes that were examined. The simplest interpretation of these results is that the dCtBP and Groucho corepressors interfere with one another when both are bound to Hairy. Such antagonistic interactions are supported by previous genetic studies, which suggest that lowering the dose of maternal dCtBP products can partially suppress the embryonic phenotypes of hairy mutants. The P-SLV-K and WRPW motifs are separated by just nine amino acid residues within the C terminus of the Hairy protein. When dCtBP and Groucho both bind, they might be unable to interact with additional corepressors or with their target proteins in the core transcription complex (Zhang, 1999).

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. mSin3A-histone deacetylase complexes are altered in the presence of dCtBP and dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, it is proposed that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function (Phippen, 2000).

Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. The chromatin profiling method DamID was used to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. DamID profiling was adapted such that tightly gated collections of embryos (2-6 h) could be used, and 20 Hairy targets related to early embryogenesis were found. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. Fifty-nine putative transcriptional targets of Hairy were identified. In addition to finding putative targets for Hairy in segmentation, groups of targets were found suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development (Bianchi-Frias, 2004).

The 59 putative Hairy targets identified correspond to bands of Hairy immunostaining on polytene chromosomes, suggesting that the polytene chromosome staining faithfully represents Hairy binding. Polytene chromosomes are functionally similar in transcriptional activity and display factor/cofactor binding properties similar to chromatin of diploid interphase cells, despite their DNA endoreplication (Bianchi-Frias, 2004).

Since the microarray chips used contained roughly half of Drosophila cDNAs, the actual number of Hairy targets was estimaed to be approximately twice that number (i.e., 118 targets). This predicted number of Hairy targets is close to the approximately 120 strongly staining sites observed on polytene chromosomes. Of the 59 putative Hairy targets identified in both the Kc cell and embryo DamID experiments, 58 correspond to bands of Hairy staining on the polytene chromosomes, suggesting that polytene chromosome staining is representing Hairy binding sites without regard to tissue specificity. It is not yet clear what is limiting Hairy accessibility in different tissues or why Hairy's access does not appear to be limited in salivary glands. It may be that polytene chromosome organization necessitates a looser chromatin structure or that the large number of factors that seem to be endogenously expressed in salivary glands affects accessibility. Ultimately, additional confirmation of the DamID and polytene staining correspondence will require microarray tiling chips containing overlapping genomic DNA fragments; however, such genomic DNA tiling chips are currently unavailable (Bianchi-Frias, 2004).

DNA methylation by tethered Dam has been shown to spread up to a few kilobases from the point where it is brought to the DNA. It was of concern in the beginning that Hairy targets might be missed if the DNA fragments of 2.5 kb or less that were recovered for probes were far away from the start of the transcribed region, especially since the Drosophila microarray chip used was generated using full-length cDNAs. Indeed, Hairy has been described as a long-range repressor; it is likely to bind at a distance from the transcription start site. However, the targets identified by DamID in both Kc cells and in embryos correspond closely to the Hairy staining pattern on polytene chromosomes. As is the case for Hairy, the distribution of DamID-identified loci that recruit the long-range repression-mediating Groucho corepressor corresponds well with the distribution of Groucho binding sites on polytene chromosomes. These results suggest that there is a higher-order structure to the promoter that is allowing factors that bind far upstream of the transcription start site to have physical access to the transcribed region (i.e., DNA looping) or that Hairy does not bind as far away from the transcription start site as it had been proposed to do (Bianchi-Frias, 2004).

Hairy is needed at multiple times during development, where it has primarily been associated with the regulation of cell fate decisions. During embryonic segmentation, ftz has long been thought to be a direct Hairy target. However, the order of appearance of ftz stripes is not inversely correlated with those of Hairy, as would be expected if ftz stripes are generated by Hairy repression. While it was not possible to assess ftz as a direct Hairy target using DamID, no evidence was found for ftz being a direct Hairy target based on the association of Hairy with polytene chromosomes. Indeed, the evidence suggesting that ftz is a direct target of Hairy is based on timing, i.e., that there is not enough time for another factor to be involved. Since the half-life of the pair-rule gene products is very short (less than 5 min), it is possible that additional factors could be acting and that the interaction between Hairy and ftz is indirect (Bianchi-Frias, 2004).

Interestingly, one of the Hairy targets identified in embryos is the homeobox-containing transcriptional regulator, prd. Pair-rule genes have been split into two groups: primary pair-rule genes mediate the transition from nonperiodic to reiterated patterns via positional cues received directly from the gap genes, whereas secondary pair-rule genes take their patterning cues from the primary pair-rule genes and in turn regulate the segment polarity and homeotic gene expression. The transcriptional regulator prd was originally categorized as a secondary pair-rule gene since its expression is affected by mutations in all other known pair-rule genes. However, prd stripes were subsequently shown to require gap gene products for their establishment, and the prd locus has the modular promoter structure associated with primary pair-rule genes. Thus prd has properties of both primary and secondary pair-rule genes and is a good candidate to directly mediate Hairy's effects on segmentation. Hairy can specifically bind to C-box sequences in the prd promoter and interacts genetically with prd. Further experiments will be required to determine if Paired in turn binds to the ftz promoter, such that the order of regulation would be Hairy > prd > ftz (Bianchi-Frias, 2004).

In addition to identifying potential targets for Hairy in segmentation, targets were identified that implicate Hairy in other processes including cell cycle, cell growth, and morphogenesis. The group of targets implicating Hairy in the regulation of morphogenesis includes: concertina, a G-alpha protein involved in regulating cell shape changes during gastrulation; kayak, the Drosophila Fos homolog involved in morphogenetic processes such as follicle cell migration, dorsal closure, and wound healing; pointed and mae, both of which function in the ras signaling pathway to control aspects of epithelial morphogenesis; egh, a novel, putative secreted or transmembrane protein proposed to play a role in epithelial morphogenesis, and Mipp1, a phosphatase required for proper tracheal development (Bianchi-Frias, 2004).

Hairy has been thought to be involved mostly in the regulation of cell fate decisions. However, mosaic experiments in the eye imaginal disc have suggested that Hairy may also play a role in the regulation of cell cycle or cell growth. Consistent with this, another group of Hairy targets implicates Hairy in the regulation of cell cycle or cell growth; this group includes stg, the Drosophila Cdc25 homolog; dacapo, a cyclin-dependent kinase inhibitor related to mammalian p27^kip1/p21^waf1; IDGF2, a member of a newly identified family of growth-promoting glycoproteins, and ImpL2, a steroid-responsive gene of the secreted immunoglobulin superfamily that functions as a negative regulator of insulin signaling. Consistent with a role for Hairy in growth signaling, mammalian HES family proteins have been linked to insulin signaling (Bianchi-Frias, 2004).

Since cells that are dividing or proliferating cannot simultaneously undergo the cell shape changes and cell migrations required for morphogenetic movements, Hairy may be required to transiently pause the cell cycle in a spatially and temporally defined manner, thereby allowing the cell fate decisions regulated by the transcription cascade to be completed. Since Hairy is itself spatially and temporally expressed, Hairy must be only one of several genes necessary to orchestrate these processes. While much progress has been made in understanding the regulatory networks governing pattern formation, cell proliferation, and morphogenesis, and while it is clear that they must be integrated, the details surrounding their coordination have not yet been elucidated. Thus, the putative Hairy targets identified are consistent with known processes involving Hairy and suggest that in addition to regulating pattern formation, Hairy plays a role in transiently repressing other events, perhaps in order to coordinate cell cycle events with the segmentation cascade. Further experiments will be needed to determine how these different roles for Hairy fit together (Bianchi-Frias, 2004).

The numbers of loci that recruit Groucho, dCtBP, and dSir2 cofactors are consistent with the breadth of interaction they have been shown to exhibit. One hundred and fifth-five loci were identified that recruit Groucho and, as expected, roughly twice as many sites were found on polytene chromosomes. Although Groucho was the first Hairy cofactor identified and its interaction site is often described as Hairy's 'major' repression motif, Groucho is associated with only a minority of Hairy targets in Kc cells. Groucho's dominance as a cofactor during segmentation may reflect a preference for Groucho in the reporter assays used previously to assess corepressor activity, or it may be more heavily recruited to Hairy's targets during segmentation. In the future it will be interesting to determine the loci that recruit Groucho in early embryos and, because Groucho binds a number of other repressors, which, if any, of these factors recruits Groucho as its major cofactor (Bianchi-Frias, 2004).

CtBP was identified as a repressive co-factor, first on the basis of its binding to the C-terminal region of E1A, and in Drosophila by its association with the developmental repressors Hairy and Knirps. CtBP is an integral component in a variety of multiprotein transcriptional complexes. It has been shown to function as a context-dependent cofactor, having both positive and negative effects on transcriptional repression depending upon the repressor to which it is recruited. More than 40 different repressors have been shown to recruit CtBP. Consistent with this wide recruitment of CtBP, 496 loci that recruit dCtBP were found by DamID profiling and roughly twice that many sites on polytene chromosomes. A global protein-protein interaction study has shown that the binding partners for Groucho and dCtBP are largely nonoverlapping. This, along with the near exclusivity of Groucho and dCtBP binding as assayed by DamID and polytene chromosome staining, makes it unlikely that both cofactors work together as a general rule and strengthens the possibility that the binding of each of these factors assembles different protein complexes that are, for the most part, mutually exclusive (Bianchi-Frias, 2004).

dSir2 was only very recently identified as a corepressor for Hairy and other HES family members. 107 loci were identified by DamID profiling that recruit dSir2 and roughly twice that many sites on polytene chromosomes. Surprisingly, the distribution of loci recruiting dSir2 identified by DamID profiling, as well as dSir2's staining on polytene chromosomes, shows regional binding specificity. This binding specificity may be a reflection of the different nuclear compartments in which these regions of the chromosomes are found. Sir2 has been described mostly as a protein involved in heterochromatic silencing rather than in euchromatic repression. The number of dSir2 euchromatic sites observed is similar to that of Groucho, suggesting that euchromatic repressors (in addition to HES family members) are likely to recruit Sir2. Consistent with this, a recent report has described a role for mammalian Sir2 in repressing the muscle cell differentiation program. The region-specific binding of dSir2 might reflect a difference in the types of factors it can associate with, or the association of dSir2 with particular chromosomal regions or nuclear domains (Bianchi-Frias, 2004).

Interestingly, dCtBP and dSir2 recruitment are largely overlapping, and this association continues outside of those loci where Hairy binds: 90% of dSir2-recruiting loci also recruit dCtBP. dCtBP and dSir2 are unique among transcriptional coregulators in that they both encode NAD⁺-dependent enzymatic activities. As NAD and NADH levels within the cell exist in closely regulated equilibrium, it is possible that dCtBP and dSir2 function as NAD/NADH redox sensors. In this way, the cell could use coenzyme metabolites to coordinate the transcriptional activity of differentiation-specific genes with the cellular redox state (Bianchi-Frias, 2004).

CtBP and Brinker

Responses to graded Dpp activity requires an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, it has been demonstrated that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. These results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters (Hasson, 2001).

That putative Brk target genes are repressed in clones of cells with increased gro dosage strongly suggests that Brk is a Gro-dependent repressor. Accordingly, Brk's proposed repression domain (RD) harbours a potential Gro recruitment motif (FKPY), similar to the Gro-binding domains defined in the repressors Hairy (WRPW), Runt (WRPY) and Huckebein (FRPW), and identical to that in Even-skipped (Eve). Brk also contains a CtBP-binding domain (PMDLSLG. Brk is shown, in fact, to be able to interact physically with both Gro and CtBP, and the functional relevance of these associations to Brk's in vivo repressor capacity are addressed (Hasson, 2001).

To demonstrate Brk's ability to associate with the two corepressors in vitro, the protein's putative RD (amino acids 369-541) was fused to glutathione S-transferase (GST), and it was incubated with radioactively labelled Gro or CtBP. In GST pull-down assays, Brk's RD (Brk^RD) readily retains [³⁵S]methionine-labelled Gro. To test further the specificity of this interaction, three mutant derivatives of the Brk^RD, fused to the GST moiety, were generated in which the Gro recruitment domain (Brk^RDmutG; FKPY to FEAY), the core of the CtBP-binding motif (Brk^RDmutC; DLS to AAA) or both (Brk^RDmutC/G) were altered. Brk's binding to Gro is impaired by the modifications in the FKPY motif. Significantly, however, Gro associates with the GST- Brk^RDmutC construct as strongly as it does with the native GST-Brk^RD fusion. GST-Brk^RD also binds labelled CtBP in vitro and, although the binding of Brk to CtBP is weak in this assay, the specificity of the interaction is clearly evident: the association between the two proteins is abolished by mutations in the CtBP recruitment domain but is unaffected by alterations in the Gro recruitment motif (Hasson, 2001).

Brk has been reported to negate transcription by competing with activators, such as Mad/Medea, for overlapping DNA target sites, thereby preventing activators' access to target promoters. The direct interactions of Brk with Gro and CtBP, however, suggest that Brk acts in a more instructive manner. While in the former 'passive' mechanism Brk is expected to rely solely on its competitive DNA-binding activity, the latter 'active' mechanism predicts that it accommodates an innate RD that depends on the recruitment of corepressors (Hasson, 2001).

To establish whether Brk contains a functional RD that can silence gene expression, separable from its DNA-binding domain, an in vivo assay was employed that relies on repression of the sex-determining Sex-lethal (Sxl) gene by ectopic expression of the pair-rule gene hairy. Sxl is normally expressed only in female embryos whereas, in males, it is repressed by Deadpan (Dpn), an autosomally encoded Hairy-related repressor protein. When Hairy is expressed prematurely, under the hunchback (hb) promoter, it mimics Dpn's repressor function and eradicates Sxl transcription in the anterior of syncytial blastoderm female embryos. Because Sxl is essential for dosage compensation in females, this repression subsequently leads to female-specific lethality. A form of Hairy, lacking its own RD, is inert in this assay. However, fusion of heterologous RDs to the truncated Hairy protein restores its ability to repress Sxl. Indeed, the equivalent expression of a hb-Hairy-Brk^RD transgene results in an effective repression of Sxl in the anterior halves of female embryos and female-specific lethality ensues. Thus, the region in Brk spanning the Gro- and CtBP-binding domains promotes potent repression in embryos (Hasson, 2001).

The ability to selectively disrupt Brk binding to each individual corepressor allowed the exploration of the dependence of its repressor potential on Gro and/or CtBP in vivo. Since both Gro- and CtBP-mediated repression can be detected in the Sxl-repression assay, truncated Hairy was fused to the three derivatives of the Brk RD, mutated in the Gro, CtBP or both recruitment motifs and placed under hb promoter regulation. In female embryos expressing Hairy-Brk^RDmutC, Sxl is substantially repressed, although not as effectively as by Hairy-Brk^RD. Furthermore, this repression still leads to statistically significant female-specific lethality. Thus, blocking CtBP binding does not completely abolish activity of the Brk RD. In comparison, mutating the Gro recruitment domain causes only residual Sxl repression and no apparent female-specific lethality. Finally, Sxl expression is seen throughout female embryos expressing hb-Hairy-Brk^RDmutC/G, and no female-specific lethality is observed. Thus, Brk relies mainly on Gro for repressing Sxl. Nevertheless, since mutating the CtBP recruitment motif in Brk's RD attenuates Sxl repression, it is concluded that, for full potency as a negative transcriptional regulator, Brk requires both corepressors (Hasson, 2001).

These data indicate that the interactions between Brk and the corepressors Gro and CtBP are indispensable for maximal repression of Sxl in vivo. Whether Brk requires both cofactors for repression of its endogenous target genes was examined. For repression of distinct target genes, Brk requires Gro and/or CtBP differentially, presumably as a function of specific promoter topology and architecture (Hasson, 2001).

Brk competes with an activator for binding to an omb wing enhancer, suggesting that, for this promoter, Brk should act independently of corepressors. Consistent with this, omb-lacZ is not ectopically expressed in cells homozygous for gro^E48 (hereafter referred to as gro^- clones), nor is it affected by CtBP loss-of-function clones, generated using the l(3)87De-10 allele (CtBP^-), or by CtBP^-, gro^- double mutant clones). Thus, single and double mutant clones for gro and CtBP do not phenocopy the omb derepression seen in brk^- clones, implying that Brk can repress omb even in the absence of these corepressors. Repression of the Dpp target gene spalt (sal) is also independent of Gro and CtBP. Nonetheless, in gro overexpression clones, omb is repressed, suggesting that, even for the omb promoter, Gro reinforces Brk repressor function (Hasson, 2001).

To establish whether Brk represses vgQ via Gro, CtBP or both, vgQ-lacZ expression was monitored in gro^- and CtBP^- single, and CtBP^-; gro^- double mutant clones. In this instance, a mandatory requirement for Gro, but not for CtBP is found; in gro^- clones, vgQ is upregulated. Importantly, as is the case for brk^- clones, the cell-autonomous upregulation of vgQ is seen only in gro^- clones close to the periphery of the disc, suggesting that the observed effects are Brk dependent. In contrast, in CtBP^- mutant clones vgQ expression is downregulated, in the Brk territory but also outside it, at the centre of the disc, indicating that these effects are Brk independent and that CtBP is positively required for vg expression. CtBP^-;gro^- double mutant clones show a composite effect: ectopic expression and upregulation of vgQ in clones in the brk expression domain, and a phenotype resembling that of CtBP^- clones at the middle of the disc, where brk is not expressed. Thus, Brk repression of vgQ is Gro- but not CtBP-dependent (Hasson, 2001).

omb and vgQ expression is completely shut off in clones of cells overexpressing gro, whereas that of brk is only reduced, suggesting that Brk might be repressing its own transcription via a negative autoregulatory loop. To establish whether, in negating its own expression, Brk is assisted by Gro and/or CtBP, gro^- and CtBP^- single, or CtBP^-, gro^- double mutant clones were stained for brk-lacZ expression. brk is never ectopically expressed in any of the single mutant clones, whereas ectopic brk expression is clearly observable in double mutant clones. Thus, in the absence of one corepressor, repression is adequately mediated by the other, suggesting that negative autoregulation by Brk is robust, relying on either Gro or CtBP (Hasson, 2001).

Strikingly, the effects on brk expression are seen only in double mutant clones found at the periphery of the disc, but not at the center where Shn is active, supporting the notion that the effects are, indeed, Brk- but not Shn-dependent. Furthermore, the fact that double mutant clones at the middle of the disc do not ectopically express brk suggests that Shn-mediated repression of brk transcription must be taking place even in the absence of both corepressors (Hasson, 2001).

To be able to compare Brk's dependence on Gro and CtBP in the embryo, full-length Brk was expressed in its native form or with its corepressor-binding domains mutated, using UAS-brk transgenes driven by maternal GAL4 . This experimental design is inapplicable for studying Brk's targets in the wing, since ectopic expression of brk prevents proliferation and survival of imaginal disc cells, but is nevertheless effective in the embryo. Ectopic Brk represses zen and dpp in mid- to late-cellularizing embryos but not earlier, so endogenous Brk targets were analyzed in transgenic embryos at comparable stages of development. Ectopic expression of all three mutant forms of Brk in embryos brings about repression of zen to the same extent as does native Brk. This result suggests that Brk represses zen independently of corepressors. In contrast, Brk requires corepressors for negating transcription of both tolloid (tld) and dpp. Thus, abolishing Brk's interactions with Gro (Brk^mutG), but not with CtBP (Brk^mutC), completely relieves tld repression, indicating that Brk repression of tld is strictly Gro dependent, as is repression of pannier (pnr). Similarly, dpp is repressed in embryos expressing Brk^mutC, but is still transcribed in embryos expressing Brk with its Gro recruitment motif mutated. In the case of dpp, however, CtBP must also be contributing to Brk repression, since the level of dpp expression is significantly lower in Brk^mutG-expressing embryos, in comparison with wild-type embryos or embryos expressing Brk^mutC/G. Thus it is concluded that, for repression of dpp, Brk rests mainly on Gro, yet for maximal repressor activity it also requires CtBP. The data indicate that Brk utilizes different means of repression for silencing its downstream targets in the embryo, as in the adult (Hasson, 2001).

Gro and CtBP mediate gene silencing in qualitatively different ways. Gro potentiates long-range repressors that function at a distance and that are able to block, in a dominant fashion, complex modular promoters consisting of multiple enhancer elements. In contrast, CtBP-dependent short-range repressors inhibit activators only locally, thereby permitting enhancer autonomy in a compound promoter. By virtue of its ability to recruit both Gro and CtBP, together with its capacity to outcompete pMad and other activators from binding DNA, Brk is competent to repress a multitude of complex Dpp target promoters, which receive positive inputs from manifold signaling pathways. It is proposed that, for promoters with low-affinity Mad-binding sites, the driving repressor force is direct competition between Brk and pMad for DNA binding, whereas for Dpp target promoters that contain high-affinity Mad-binding sites, corepressors are essential for mediating Brk repression. For this latter class of promoters, Brk relies on one or both of its cognate corepressors, depending on the particular promoter topology (Hasson, 2001).

Brk utilizes a self-reliant mechanism, which need not depend on tethered corepressors, by competing with activators over coinciding DNA-binding sites. In the absence of both Gro and CtBP, Brk represses not only omb and zen, but also sal, suggesting that the Brk-binding site(s) in the sal promoter overlap with those employed by activators. Transcription of both sal and vgQ requires activation by Mad, yet, although both promoters are exposed to identical levels of pMad, the sal expression domain is spatially more restricted than that of vgQ, presumably because activation of sal requires higher levels of pMad than that of vgQ. Hence, 'passive' competition-based repression should efficiently block activation of sal but may not be sufficient for promoters like vgQ, which are activated even by low amounts of Mad. For silencing such promoters, alternative mechanisms such as recruitment of corepressors have evolved and are employed (Hasson, 2001).

Brk represses its distinct endogenous target genes by recruiting Gro and/or CtBP differentially. For the silencing of many target promoters, Gro alone is sufficient (vg, tld and pnr) but, for fully repressing others, Brk depends on both corepressors. Thus, in the case of dpp and Sxl, when CtBP is lacking, a decrease in Brk's overall repressor capacity is apparent and, in the absence of Gro, repression is almost completely impaired. Importantly, for negating its own transcription, Brk can utilize either corepressor (Hasson, 2001).

The majority of activator and repressor binding sites in most Dpp-responsive enhancers have yet to be precisely mapped. It is nevertheless proposed that lengthy and complex promoters, which respond to several signaling inputs, will be found to be strictly silenced in a Gro-dependent manner. Thus, in repressing the vgQ enhancer, a composite cis-acting regulatory sequence with multiple elements that integrate information relayed by the dpp, wingless and EGF receptor signaling pathways, Brk is fully reliant on Gro, but not on CtBP. For other more simple promoters, short-range repression should be adequate and will be mediated by either corepressor, as exemplified by the robust Brk autoregulation, for which either Gro or CtBP is sufficient; CtBP and Gro are presumably interchangeable in this context, compensating for each other's absence (Hasson, 2001).

Significantly, the overexpression of gro results in ectopic omb repression, suggesting that, even for promoters that are switched off in a 'passive', competitive manner, excess Gro can over-potentiate Brk-mediated negative transcriptional regulation. Thus, Gro and/or CtBP might reinforce Brk repression of those promoters on which it initially acts by competing with activators for binding to DNA, via recruitment of histone deacetylases and alterations to chromatin structure, or by some other mechanism (Hasson, 2001).

In the embryonic midgut of Drosophila, Wingless (Wg) signaling elicits threshold-specific transcriptional response, that is, low-signaling levels activate target genes, whereas high-signaling levels repress them. Wg-mediated repression of the HOX gene Ultrabithorax (Ubx) is conferred by a response sequence within the Ubx B midgut enhancer, called WRS-R. It further depends on the Teashirt (Tsh) repressor, which acts through the WRS-R without binding to it. Wg-mediated repression of Ubx B depends on Brinker, which binds to the WRS-R. Brinker binds to a site distinct from that occupied by the Wg effector, the Pangolin/Armadillo activator complex. Brinker thus acts at short range to block the activity of this complex. Furthermore, Brinker blocks transcriptional activation by ubiquitous Wg signaling. Brinker binds to Tsh in vitro, recruits Tsh to the WRS-R, and mutual physical interactions are found between Brinker, Tsh, and the corepressor dCtBP. This suggests that the three proteins may form a ternary repressor complex at the WRS-R to quench the activity of the nearby-bound Pangolin/Armadillo transcription complex. Finally, brinker and tsh produce similar mutant phenotypes in the ventral epidermis, and double mutants mimic overactive Wg signaling in this tissue. This suggests that Brinker, which was initially discovered as an antagonist of Dpp signaling, may have a widespread function in antagonizing Wg signaling (Saller, 2002).

Most likely, Brinker uses a mechanism called quenching to block Pangolin/Armadillo. Quenching involves interaction of repressors (and the corepressors they recruit) with activators bound to nearby sites. Brinker is known to be able to quench target genes by recruiting the corepressor Groucho, which is involved in multiple quenching processes. groucho antagonizes wg, and TCF factors can bind to Groucho proteins directly, so Pangolin may thus be able to recruit Groucho unassisted. However, these findings do not rule out the possibility that Pangolin relies on cooperation with Brinker to achieve Groucho recruitment (Saller, 2002 and references therein).

Brinker can bind to the corepressor dCtBP, so Brinker may recruit dCtBP instead of, or in addition to, Groucho. Recall that Tsh plays a critical role in the Wg-mediated repression in the midgut. Moreover, Tsh can bind to Brinker as well as to dCtBP, so it seems plausible that Tsh plays a pivotal role in assisting Brinker in the recruitment of dCtBP. Like Groucho, dCtBP is a corepressor with quenching activity. In addition, Tsh may itself be involved in the quenching process. It has been suggested that quenching may be based on obstruction of the interaction between the activation domain of a transcriptional activator and the general transcription machinery -- intriguingly, hypophosphorylated Tsh binds to the carboxy-terminal activation domain of Armadillo to modulate Wg signaling (Saller, 2002 and references therein).

CtBP and Teashirt

Drosophila teashirt functions as a region-specific homeotic gene that specifies trunk identity during embryogenesis. Based on sequence homology, three tsh-like (Tsh) genes have been identified in the mouse. Their expression patterns in specific regions of the trunk, limbs and gut raise the possibility that they may play similar roles to tsh in flies. By expressing the putative mouse Tsh genes in flies, evidence is provided that they behave in a very similar way to the fly tsh gene: (1) ectopic expression of any of the three mouse Tsh genes, like that of tsh, induces head to trunk homeotic transformation; (2) mouse Tsh proteins can rescue both the homeotic and the segment polarity phenotypes of a tsh null mutant; (3) following ectopic expression, the three mouse Tsh genes affect the expression of the same target genes as tsh in the Drosophila embryo; (4) mouse Tsh genes, like tsh, are able to induce ectopic eyes in adult flies; (5) all Tsh proteins contain a motif that recruits the C-terminal binding protein and contributes to their repression function. As no other vertebrate or fly protein has been shown to induce such effects upon ectopic expression, these results are consistent with the idea that the three mouse Tsh genes are functionally equivalent to the Drosophila tsh gene when expressed in developing Drosophila embryos (Manfroid, 2004).

Comparison of the organization of Tsh with Tsh-related proteins in mouse and humans suggests that common functional features are probably defined by the region encompassing the three zinc-finger motifs and by the presence of a motif known to interact with CtBP. Interestingly, mouse and Drosophila Tsh proteins display intrinsic transcriptional repression activity. The repression ability of Tsh proteins is partly due to their interaction with the co-repressor CtBP. In the visceral mesoderm, Tsh is recruited to the Ubx enhancer in a repressor complex containing Brk and CtBP, wherein Tsh does not seem to bind directly to DNA, but rather Brk is the DNA-binding partner. In the ectoderm, however, Tsh directly binds to the modulo enhancer and represses transcription in vivo. The association of CtBP with Tsh is dependent on the CtBP-interacting motif (PLDLS) located in the N-terminal part of Tsh, and this CtBP/Tsh complex contributes to the observed repression. An analogous motif (PIDLT) is found in the C-terminal part of the three mouse Tsh proteins. Despite the different context encompassing the PIDLT motif in the mouse proteins (C-terminal), this motif is functional and essential for the repressor function of mouse Tsh1. Although the role of this motif was addressed only for only mouse Tsh1, repression activity of Tsh2 and Tsh3 is equally potentiated by mouse Ctbp1, suggesting that mouse Ctbp1 is a co-repressor acting with all mouse Tsh proteins. Interestingly, the PIDLT motif lies within a region of the three mouse Tsh proteins where the sequence similarity is low and thus appears to be a highly conserved functional domain in a variable region. In addition, it is worth noting that, in mammalian cells, some repression activity persists in mouse Tsh1 after deletion of the CtBP-interacting motif, implying that other mechanisms of transcriptional repression are used by mouse Tsh. In contrast to Tsh, which contains a repressor domain rich in Ala, analysis of the mouse Tsh protein sequences fail to reveal a comparable feature or any known motif that could account for the mouse Tsh1DeltaPIDLT repressor activity (Manfroid, 2004).

In the Drosophila eye the retinal determination (RD) network controls both tissue specification and cell proliferation. Mutations in network members result in severe reductions in the size of the eye primordium and the transformation of the eye field into head cuticle. The zinc-finger transcription factor Teashirt (Tsh) plays a role in promoting cell proliferation in the anterior most portions of the eye field as well as in inducing ectopic eye formation in forced expression assays. Tiptop (Tio) is a recently discovered paralog of Tsh. It is distributed in an identical pattern to Tsh within the retina and can also promote ectopic eye development. A previous study demonstrated that Tio can induce ectopic eye formation in a broader range of cell populations than Tsh and is also a more potent inducer of cell proliferation. This study focused on understanding the molecular and biochemical basis that underlies these differences. The two paralogs are structurally similar but differ in one significant aspect: Tsh contains three zinc finger motifs while Tio has four such domains. A series of deletion and chimeric proteins were used to identify the zinc finger domains that are selectively used for either promoting cell proliferation or inducing eye formation. These results indicate that for both proteins the second zinc finger is essential to the proper functioning of the protein while the remaining zinc finger domains appear to contribute but are not absolutely required. Interestingly, these domains antagonize each other to balance the overall activity of the protein. This appears to be a novel internal mechanism for regulating the activity of a transcription factor. It was also demonstrated that both Tsh and Tio bind to C-terminal Binding Protein (CtBP) and that this interaction is important for promoting both cell proliferation and eye development. It was also shown that the physical interaction that has been described for Tsh and Homothorax (Hth) do not occur through the zinc finger domains (Datta, 2011).

Finally, the binding of Tsh and Tio to the transcriptional co-repressor CtBP appears crucial for both proteins, since removal of this domain interferes with the ability of Tsh and Tio to induce ectopic eyes and promote proliferation. Furthermore, the loss of CtBP expression ahead of the furrow relieves the repression of dac expression by Tsh. These results indicate that CtBP is a member of transcriptional repressive complexes that include Tsh and Tio and plays a critical role within the retinal determination network (Datta, 2011).

CtBP and Hairless

The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). In vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, these findings indicate that 'default repression' of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development (Barolo, 2002b).

H is a novel protein, with no known vertebrate homologs. However, the H gene has been identified in three members of the order Diptera: Drosophila melanogaster, D. hydei, and the mosquito Anopheles gambiae. H is surprisingly poorly conserved among these three species: It shares 63% identity between D. melanogaster and D. hydei (diverged ~65 Mya), and 33% identity between Drosophila and Anopheles (diverged ~260 Mya). The rapid divergence of the H protein sequence readily allows the identification of short conserved motifs, which are presumably important for H function. Two such regions occur in a part of H that is required for its interaction with Su(H) in vitro (Barolo, 2002b).

Another conserved motif in the H protein is YSIxxLLG, which is perfectly conserved from Drosophila to Anopheles. This sequence resembles certain examples of the 'eh1' type of Gro-binding domain found in many transcriptional repressor proteins. Among eh1 domains, the 'octapeptide' motifs in the Pax 2/5/8 proteins, which have been shown to directly mediate repression by recruiting Gro-family corepressors, show the greatest similarity to this region of H. In addition, the extreme C-terminal sequence of H, PLNLSKH, includes a match to the consensus binding site for the CtBP corepressor, Px(D/N)LS. The PLNLS motif, fully conserved from Drosophila to Anopheles, exactly matches motifs found in four vertebrate CtBP-binding transcription factors. H also contains three lengthy alanine-repeat domains: AAAVAAAAAAAAA, AAAAAAAAAA, and AAVAAA AAAAAA. Alanine repeats and alanine-rich regions are common in transcriptional repression domains, and are found in many repressor proteins. However, these repeats are reduced or absent in the D. hydei and A. gambiae H proteins: this suggests that they may not make an essential contribution to H function (Barolo, 2002b).

A gel retardation experiment reported by Brou (1994), indicating that H can inhibit the binding of Su(H) to DNA in vitro, has strongly influenced interpretations of genetic studies of H, Su(H), and N. A DNA-binding-inhibition model of H function is indeed consistent with both loss- and gain-of-function genetic data demonstrating that H affects cell fate in a manner antagonistic to N signaling, including the N-stimulated transcriptional activation function of Su(H). However, the recent discovery of Su(H)-mediated transcriptional repression has forced a reconsideration of this simple model, since it makes incorrect predictions about the effect of H on a cell fate that is dependent on the repression function of Su(H). It is proposed that the genetic data on cell fate are instead consistent with a different role for H: facilitating transcriptional repression by Su(H) (Barolo, 2002b).

During the socket/shaft cell fate decision in adult mechanosensory bristle development, the cell that responds to N signaling takes the socket fate, while its sister cell, in which N signal transduction is blocked by the Numb protein, takes the shaft fate. Overexpression of Su(H), or loss of H function, during the socket/shaft decision causes both cells to adopt the socket fate; conversely, overexpression of H, or loss of Su(H) function, results in two shaft cells. Autorepression by Su(H) in shaft cells is important for maintaining the shaft cell fate. The corepressors Gro and dCtBP are important for specification of the shaft cell, a fate that is inhibited by N signaling and depends on both H activity and Su(H)-mediated repression. Reduction of gro or dCtBP function strongly enhances the effects of both reduction of H activity and loss of Su(H) repression, and suppresses the effects of reduced N signaling in the bristle lineage. It is therefore concluded that Gro and dCtBP, along with H and transcriptional repression mediated by Su(H), act in the opposite direction from the N signaling pathway during the socket/shaft cell fate decision, in that they promote the fate (shaft) that is inhibited by N signaling. The observation that both gro and dCtBP heterozygotes show a weak dominant (haploinsufficient) shaft-to-socket cell fate conversion phenotype is further confirmation of an important role for both corepressors in promoting the shaft cell fate. These results represent the first in vivo functional evidence for the involvement of Gro and dCtBP in transcriptional repression mediated by Su(H) (Barolo, 2002b).

Genetic analyses show that gro loss-of-function mutations enhance the effects of reduced H activity on two N-mediated cell fate decisions, the socket/shaft decision and the epidermal/SOP decision, while reduction of gro activity suppresses the effects of N loss of function on the socket/shaft and pIIA/pIIB cell fate decisions. In addition, gro has a weak haploinsufficient bristle loss phenotype, resembling an excess of N signaling. A role for gro in promoting the SOP cell fate is surprising, because gro was originally identified as a 'neurogenic' gene that acts to inhibit the SOP fate downstream of N signaling, in its capacity as a corepressor for bHLH transcriptional repressor proteins encoded by N target genes in the Enhancer of split gene complex [E(spl)-C]. In fact, gro was named after the phenotype of flies homozygous for gro¹, a weak hypomorphic allele: bushy tufts of bristles over the eyes caused by a failure of N-mediated lateral inhibition of the SOP fate. At least one E(spl)-C bHLH repressor gene appears to be directly repressed by Su(H) in SOPs; the proposal that Gro promotes the SOP fate by cooperating with H to repress N target genes in this cell is currently being tested. If proved, this would represent a novel and complex form of regulation, in which Gro inhibits the SOP fate in all but one cell of the proneural cluster by partnering with the E(spl)-C bHLH repressors, and simultaneously promotes the SOP fate in one neighboring cell by preventing the expression of its own partners (Barolo, 2002b).

The current results support the hypothesis that H antagonizes N signaling by acting as an adaptor molecule between the transcription factor Su(H) and the corepressor proteins Gro and dCtBP. This model entails an unusual mechanism of repression: DNA-binding transcriptional repressors that recruit CtBP or the Gro family of corepressors generally do so via direct protein-protein interactions, although evidence for CtBP recruitment by non-DNA-binding proteins has been reported. In mammalian cells, the corepressors SMRT and CIR bind directly to the Su(H) homolog CBF1 (Barolo, 2002b).

In contrast to a DNA-binding inhibition model for H function, an adaptor/corepressor model explains why H counters N^IC/Su(H)-mediated activation, but not Su(H)-mediated repression. Like previous views of H function, this model presumes competition between Su(H)-binding partners, in this case between N^IC-containing activation complexes and H/Gro/dCtBP repression complexes. N^IC activation complexes are likely to include the Mastermind (Mam) protein, and may also include the p300 coactivator. In the presence of N signaling, Su(H)/N^IC/Mam complexes presumably replace Su(H)/H/Gro/dCtBP complexes on target genes, and convert Su(H) from a repressor to an activator. Whether this occurs by simple affinity-based competition for binding to Su(H), or by a mechanism involving active impairment of the H/Su(H) interaction, is unknown. Under an adaptor/corepressor model, the H mutant phenotype results from derepression of Su(H)/N target genes in cells lacking N pathway activity, thus mimicking an increase in N signaling. The H overexpression phenotype may be explained by the displacement of N^IC-containing activation complexes by an excess of H-containing repression complexes, thus repressing N^IC/Su(H) target genes in cells that respond to the N signal (Barolo, 2002b).

It has recently become apparent that the transcriptional target genes of at least six major developmental signaling pathways are in many cases subject to 'default repression'; that is, binding sites for signal-regulated transcription factors, which mediate activation during signaling events, mediate repression in the absence of signaling (for review, see Barolo, 2002a). Each of these pathways uses a different mechanism to switch from repression to activation upon stimulation of the pathway, but in each case, the effect seems to be the same: restricting the expression of pathway target genes to cells that receive active signaling. The results of this study strongly suggest that H contributes to default repression in the N pathway by directly recruiting the corepressors Gro and dCtBP to Su(H), and that formation of H/Su(H) repression complexes is crucial for the establishment of two N-inhibited cell fates, the SOP and shaft cell fates. Default repression, therefore, appears to be as important as signal-dependent activation for proper cell fate specification in this developmental context (Barolo, 2002b).

Lateral inhibition, wherein a single cell signals to its neighbors to prevent them from adopting its own fate, is the best-known setting for cell-cell communication via the Notch (N) pathway. During peripheral neurogenesis in Drosophila, sensory organ precursor (SOP) cells arise within proneural clusters (PNCs), small groups of cells endowed with SOP fate potential by their expression of proneural transcriptional activators. SOPs use N signaling to activate in neighboring PNC cells the expression of multiple genes that inhibit the SOP fate. These genes respond transcriptionally to direct regulation by both the proneural proteins and the N pathway transcription factor Suppressor of Hairless [Su(H)], and their activation is generally highly asymmetric; i.e., only in the inhibited (non-SOP) cells of the PNC, and not in SOPs. The substantially higher proneural protein levels in the SOP put this cell at risk of inappropriately activating the SOP-inhibitory genes, even without input from N-activated Su(H). This is prevented by direct 'default' repression of these genes by Su(H), acting through the same binding sites Su(H) uses for activation in non-SOPs. Derepression of even a single N pathway target gene in the SOP can extinguish the SOP cell fate. Finally, crucial roles are defined for the adaptor protein Hairless and the co-repressors Groucho and CtBP in conferring repressive activity on Su(H) in the SOP. This work elucidates the regulatory logic by which N signaling and the proneural proteins cooperate to create the neural precursor/epidermal cell fate distinction during lateral inhibition (Castro, 2005).

Su(H) is known to act as a transcriptional repressor in another context during sensory organ development; namely, the socket/shaft sister cell fate decision in the bristle lineage. Auto-repression of Su(H) is necessary to prevent inappropriate high-level activation of the gene in the shaft cell, which in turn can cause this cell (which does not respond to N signaling) to adopt the N-responsive socket cell fate. The biochemical basis of transcriptional repression by Su(H) has been studied in some detail in this setting. Specifically, the Hairless (H) protein has been shown to act as an adaptor that recruits the transcriptional corepressor proteins Gro and CtBP to Su(H), thus conferring repressive activity (Castro, 2005).

Earlier work can be interpreted to suggest that a similar protein complex might mediate repression by Su(H) in the SOP. At several macrochaete and many microchaete positions on the adult fly, simultaneous reduction of the doses of Hairless and gro in an otherwise wild-type background leads to significant bristle loss; this is due to a failure of commitment to the SOP cell fate. A plausible interpretation of these findings is that H and Gro are normally part of a repressive Su(H)-containing complex in the SOP, and that reduction of their doses sufficiently compromises the repressive activity as to partially de-repress N pathway target genes like E(spl)m8, leading to failure of SOP specification. As a test of this model, it was thought that it might be possible to detect such de-repression of a suitable reporter gene. This expectation was borne out. Late third-instar wing discs from wild-type larvae or larvae heterozygous for null alleles of either Hairless or gro only rarely exhibit detectable activity of an E(spl)malpha-GFP reporter transgene in SOPs. By contrast, wing discs from larvae doubly heterozygous for null alleles of both Hairless and gro show substantial frequencies of ectopic GFP expression in SOPs. Moreover, the SOP expression observed in the double heterozygotes is considerably stronger than that detected rarely in a wild-type background. These results demonstrate that normal levels of Hairless and gro activity are required for the Su(H)-dependent repression of N pathway target genes in SOPs, and are consistent with the participation of a Su(H)-H-Gro-containing protein complex in this repression (Castro, 2005).

Broad overexpression of Hairless (including in proneural clusters) during lateral inhibition causes a 'neurogenic' phenotype; that is, the appearance of supernumerary bristles surrounding normal bristles. This phenotype is readily understood in light of the model described above; namely, that Hairless normally serves to recruit Gro and CtBP to Su(H) for its repressive activity in the SOP. Overexpression of Hairless in the N-responsive non-SOP cells of the PNC would be expected to elevate their levels of the repressive form of Su(H), causing repression of N pathway target genes that would normally be activated by the Su(H)-NIC-Mam complex. This in turn would result in a partial failure of lateral inhibition and the commitment of additional cells in the PNC to the SOP fate, giving rise to ectopic bristles in the adult (Castro, 2005).

A key prediction of the model is that the ability of Hairless to bind Gro (via the motif YSIHSLLG) and CtBP (via the motif PLNLSKH) should be required for the SOP fate-promoting activity of H. This prediction was tested by using an E(spl)malpha GAL4 driver to express different forms of H specifically in the non-SOP cells of the PNCs. The orbital region of the adult fly head is a particularly favorable territory in which to assay the production of supernumerary bristles by H overexpression. Expression of a wild-type UAS-Hairless transgene results in the appearance of an average of approximately four ectopic bristles in the orbital region. This activity is significantly impaired by mutating either the Gro recruitment motif (UAS-H[Gm]) or the CtBP-binding motif (UAS-H deltaC), suggesting that both co-repressors make a functional contribution. Loss of both motifs (UAS-H[Gm] deltaC) essentially abolishes the capacity of Hairless to promote ectopic bristle development in this assay. These results are strongly consistent with the interpretation that the SOP cell's requirement for Hairless activity is based on the recruitment by Hairless of Gro and CtBP to confer repressive activity on Su(H), thus preventing inappropriate expression of inhibitory N pathway target genes (Castro, 2005).

It is concluded that discrete transcriptional cis-regulatory modules, bearing binding sites for both Su(H) and the proneural proteins, direct the non-SOP-only expression pattern of E(spl)-C genes in PNCs. Mutation of the Su(H) sites in these modules results in an inversion of this pattern of activity, including both the loss of most non-SOP expression and the appearance of strong ectopic expression in SOPs. These observations reveal a dual role for Su(H) in the PNC: as a direct, N-activated transcriptional activator of E(spl)-C genes in non-SOP cells, and as a direct transcriptional repressor of the same genes in the SOP. The issue was addressed as to whether Su(H)-mediated repression of E(spl)-C genes in the SOP is important developmentally. The experiments with wild-type and Sm versions of an E(spl)m8 genomic DNA transgene demonstrate that it is. Failure to repress this single bHLH repressor gene is sufficient to extinguish the SOP fate (marked by Sens) at a frequency significantly greater than that observed with a repressible (wild-type) transgene. Evidence is provided that the Hairless protein is responsible for conferring repressive activity on Su(H) in the SOP, by recruiting the co-repressors Gro and CtBP. It is suggested that the Hairless null phenotype widespread, irreversible loss of the SOP fate in an E(spl)-C-dependent manner, offers the best indication of the developmental consequences of relieving Su(H)-mediated repression of all E(spl)-C genes in the SOP (Castro, 2005).

Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. This study investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, it was found that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, this study demonstrates that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding (Nagel, 2005).

To test the repressive effects of Hairless in the absence of N^ICD, Hairless ability to inhibit transcription in the presence of Grainyhead (Grh) was tested. The Notch response (NRE) reporter contains binding sites for the transcriptional activator Grh that stimulate transcription fourfold in the absence of N^ICD and increase the stimulation seen in the presence of N^ICD. Addition of full-length Hairless inhibits these effects, reducing transcription in the presence of Grh alone by 50%. Furthermore, this inhibitory effect is dependent on Su(H), as indicated by a lack of repression of HDeltaS, and requires both CtBP and Gro, since Hairless proteins with either interaction domain mutated (HDeltaC, H*C, HDeltaG, H*G) lose most of their repressive activity. Again, the levels of activity with the single mutants are similar to the levels seen with the double-mutant forms of the protein (HDeltaGC, H*GC) and all resulted in >90% of the expression seen with Grh. These experiments suggest that Hairless has two modes of repression, one that operates by repressing the transcriptional machinery through its recruitment of global corepressors and a second that operates by directly antagonizing N^ICD (Nagel, 2005).

These data confirm therefore that both Gro and CtBP can function as corepressors with Hairless, and indeed both factors are necessary for full repression by Hairless on the NRE; preventing the interaction with one or the other factor severely compromises Hairless activity. This is in apparent contrast to the effects on vg^BE-LacZ, for which only Gro appears essential. Furthermore, the two cofactors appear to act together, since Hairless proteins lacking both interaction motifs retains a level of repression that is comparable to the results seen upon removing either alone (Nagel, 2005).

Previous studies of CtBP and Gro have argued that they mediate repression in qualitatively different ways, although both are thought to recruit histone deacetylases. Gro has predominantly been associated with so-called long-range repression, as it operates to dominantly silence modular enhancers. In contrast, CtBP appears to act in a local way to inhibit activators that are bound nearby. However, these models do not appear compatible with a combined requirement for Gro and CtBP in Hairless-mediated repression. Furthermore, direct fusion of a Gro interaction domain to the Su(H) protein is sufficient to convert it into a potent repressor, as described for other transcriptional regulators. Why should Gro and CtBP therefore be interdependent in the context of Hairless recruitment? One simple explanation would be that one or the other corepressor is needed to specifically counteract N^ICD activation. For example, CtBP interferes with recruitment of p300, a histone acetyltransferase that is reported to interact with mammalian N^ICD. However, the data suggest that CtBP and Gro are both needed to repress Grh even in the absence of N_ICD, arguing that each corepressor can only perform a subset of its functions in the context of Hairless. Maybe the two corepressors recruit different enzymatic activities that are needed together to promote repression. If the Hairless complex were incompatible with oligomerization of Gro, which is reported to be important for stable repression, Gro might be able to recruit histone deacetylases but not to promote spreading of the repression complex. And if CtBP, which in mammals has been found complexed with methyl transferases as well as deacetylases, could recruit only histone methyl transferases, the corepressors would each confer a critical component on the Hairless complex. A more complete understanding of the molecular functions of Gro and CtBP in the context of chromatin dynamics and transcription complexes will be needed to determine why Hairless requires their coordinate activities in many developmental scenarios, as was shown in this study (Nagel, 2005).

CtBP and Snail

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. The structures of synthetic peptides identical to the CtBP binding sites on these proteins have been investigated using NMR spectroscopy. Peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity [K(i) of 1.04, 1.34 and 0.52 microM, respectively]. However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides are markedly reduced [K(i) of 332 and 56 microM, respectively] whilst the Hairy peptide binds much more strongly [K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP]. In addition peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. It is concluded that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data constitute evidence for a multiplicity of CtBPs and partner proteins (Molloy, 2001).

The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of Prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the members of the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string (Ashraf, 2001).

A clearly demonstrated in vivo function of Snail is transcriptional repression. The repression function is mediated through the recruitment of dCtBP (Drosophila C-terminal binding protein), which acts as a co-repressor for Snail to regulate target genes such as rhomboid, lethal of scute and single-minded. There are two conserved P-DLS-R/K motifs in Snail, as well as in Worniu and Escargot, and they have been shown to be critical for recruiting dCtBP. Mutations of these motifs abolish the repressor function of Snail in the blastoderm. To gain insight into the molecular mechanism of how Snail regulates CNS development, transgenic copies of snail, which had the dCtBP interaction motifs mutated were introduced into the osp29 deletion background. M1 contains the N-terminal motif mutation and M2 contains the C-terminal motif mutation. The expression of inscuteable and ftz was examined. The assay shows that the double mutant (M12) lost most of the ability to rescue, and M1 has lost some ability to rescue. However, M2 functioned quite efficiently, closer to that of the wild-type protein, to rescue inscuteable and ftz expression. These results demonstrate that the dCtBP interaction motifs are essential for the Snail function in the CNS, consistent with the idea that Snail acts as a repressor in neuroblasts to regulate gene expression. Thus, the activation of inscuteable and string by the Snail family may be indirect (Ashraf, 2001).

The Snail repressor positions Notch signaling in the Drosophila embryo

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. The subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) has been investigated. These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This 'ventral dominance' includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. The significance of ventral dominance is discussed with respect to the patterning of the vertebrate neural tube, and ventral dominance is compared with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos (Cowden, 2003).

Further support for ventral dominance of the Snail repressor was obtained by analyzing mutant embryos derived from CtBP germline clones. CtBP is a maternally deposited corepressor protein essential for snail-mediated repression. Removal of this corepressor results in ventral derepression of sim and vnd into the presumptive mesoderm due to loss of Snail mediated repression. However, this ventral expansion of vnd does not result in a transformation of mesoderm into medial neuroblasts. Instead, the expanded vnd pattern is lost at slightly later stages, and expression becomes restricted to lateral regions, similar to the endogenous expression pattern. This lateral restriction is consistent with the observation that neuroblasts are formed in lateral regions of CtBP^- mutants, and not in ventral regions that normally form the mesoderm. Neuroblast segregation can be visualized using a snail antisense RNA probe, which stains all neuroblasts following gastrulation. Sim may be responsible for the late repression of vnd, because vnd expands into the ventral midline of sim mutant embryos. Repression of vnd by Sim is probably indirect because a Krüppel-sim transgene does not alter vnd expression in the lateral neuroectoderm. Perhaps Sim activates an unknown repressor that ultimately inhibits vnd expression in the midline (Cowden, 2003).

'Ventral dominance' might govern the patterning of the ventral nerve cord in older embryos, in addition to the prepatterning of the neuroectoderm in pregastrulating embryos. Sim might exclude vnd, ind, and msh expression in the ventral midline. In embryos lacking maternal CtBP products, Snail fails to act as a repressor, allowing the ventral expansion of sim and vnd into the presumptive mesoderm. However, vnd expression is ultimately lost from ventral regions, while sim expression persists. As a result, ventral regions form an expanded mesectoderm, while neuroblasts arise from lateral regions. These observations suggest that Sim excludes vnd expression from ventral regions in CtBP mutants, either directly by acting through a CNS specific enhancer or indirectly by activating an unknown repressor. This putative repressor probably does not rely on the CtBP corepressor, as it is still capable of repressing vnd in CtBP germ line clones. According to a ventral dominance scenario, the misexpression of this unknown repressor should inhibit the expression of vnd, ind, and msh in the ventral midline. One potential target for the indirect repressor could be the EGF pathway. The ventral midline is a well-characterized source of EGF signaling and both vnd and ind rely upon EGF signaling for maintenance of expression. By eliminating EGF activation, this midline repressor could prevent vnd and ind expression (Cowden, 2003).

Mammalian YY1 functions as a PcG protein in Drosophila and requires CtBP for repression

Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, Pleiohomeotic. YY1 might therefore be a vertebrate PcG protein. Drosophila embryo and larval/imaginal disc transcriptional repression systems were used to determine whether YY1 represses transcription in a manner consistent with PcG function in vivo. YY1 represses transcription in Drosophila, and this repression is stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablate YY1 repression, and YY1 can substitute for Pho in repressing transcription in wing imaginal discs. YY1 functionally compensates for loss of PHO in pho mutant flies and partially corrects mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, YY1, as well as Polycomb, was found to require the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1 (Atchison, 2003).

Most biochemical studies have not revealed a physical association of YY1 with the known PcG complexes, although substoichiometric levels are observed in human Pc complexes, and some associations have been documented for Drosophila Pho. The transient nature of the Drosophila associations suggest that an intermediary protein exists. This study demonstrates genetic and physical associations between YY1 and CtBP, which link YY1 to PcG function and provide a mechanism for the recruitment of vertebrate PcG complexes to DNA. Since CtBP is able to homodimerize, it may interact with Pc by one dimer partner and with YY1 by the other dimer partner. These interactions could define the mechanism by which YY1 functions to repress transcription in both a PcG- and CtBP-dependent fashion. In addition, the CtBP and Pc experiments indicate that CtBP plays a more direct role in PcG repression. Thus, CtBP may perform more than one function in the repression mechanism (Atchison, 2003).

YY1 DNA binding and PcG recruitment requires CtBP

Mammalian Polycomb group (PcG) protein YY1 (see Drosophila Pleiohomeotic) can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA (Srinivasan, 2004).

To determine whether YY1 can recruit PcG proteins to DNA, chromatin immunoprecipitation (ChIP) assays were performed in a transgenic Drosophila embryo system consisting of hsp70-driven GALYY1 and a reporter construct containing the LacZ gene under control of the Ultrabithorax (Ubx) BXD enhancer and the Ubx promoter adjacent to GAL4-binding sites (BGUZ). The BGUZ reporter is expressed ubiquitously during embryogenesis but is selectively repressed in a PcG-dependent manner by GALYY1 and GALPc. Embryos were either left untreated or heat shocked to induce GALYY1 expression. After immunoprecipitation with various antibodies, the region surrounding the GAL4-binding sites in the BGUZ reporter was detected by PCR. Prior to heat shock, no GALYY1 could be observed at the reporter gene. After heat shock, GALYY1 binding to the reporter gene was easily detected. Interestingly, concomitant with GALYY1 binding, there was an increase in binding of the Polycomb (Pc) and Polyhomeiotic (Ph) proteins. Thus, YY1 DNA binding results in PcG recruitment to DNA (Srinivasan, 2004).

Binding of PcG proteins to PRE sequences is known to cause deacetylation of histone H3 and methylation on Lys 9 and Lys 27. Interestingly, induction of GALYY1 binding to the reporter gene resulted in loss of histone H3 acetylation on K9 and K14. Simultaneously, there was a gain of methylation on histone H3 Lys 9 and Lys 27. Therefore, YY1 binding to the BGUZ reporter results in the recruitment of PcG proteins to DNA and subsequent post-translational modifications of histones characteristic of PcG complexes (Srinivasan, 2004).

The presence of PcG proteins and the status of histone H3 modifications at the Ubx promoter region, which is 4 kb downstream of the GALYY1-binding site, were determined. To avoid amplification of the endogenous Ubx promoter, immunoprecipitated samples were amplified with primers spanning the Ubx-LacZ boundary. Interestingly, the presence of Pc and Ph was detected at the promoter after GALYY1 induction. The presence of GALYY1 at this site was also detected. The GAL4 protein alone does not bind to the Ubx promoter region, indicating specificity for YY1 sequences. The induced GAL4 protein was functional, however, because it efficiently bound to the GAL4-binding site in the BGUZ reporter. Binding by GALYY1 could, therefore, be due to either cryptic YY1-binding sites present at the promoter, physical association of GALYY1 with other proteins bound at the promoter, or interactions via looping of DNA between the GAL4-binding sites and the Ubx promoter. Again, induction of GALYY1 resulted in loss of acetylation of H3K9 and H3K14 and simultaneous gain of methylation on H3K9 and H3K27. These results are consistent with studies that have reported spreading of PcG proteins and histone modifications to flanking DNA (Srinivasan, 2004).

PHO and YY1 bind to the same DNA sequence, and PHO-binding sites have been identified in multiple PREs. Therefore, it was reasoned that YY1 would bind to endogenous PREs and perhaps increase recruitment of PcG proteins. For this, the major Ubx PRE (PRE_D), that contains multiple PHO-binding sites located in the bxd region, was examined. As expected, upon GALYY1 induction, GALYY1 was detected at this endogenous PRE site. In addition, YY1 binding was accompanied by an increase in Pc and Ph signals when compared with no heat shock controls and a loss of H3 K9 and H3 K14 acetylation and gain of H3 K9 and H3 K27 methylation. Thus, YY1 can bind to an endogenous PRE and can augment PcG recruitment (Srinivasan, 2004).

These results clearly indicated that YY1 DNA binding results in recruitment of PcG proteins, histone deacetylases (HDACs), and histone methyltransferases (HMTases) to DNA. To determine whether the Drosophila E(z) protein (which possesses HMTase activity) was involved, whether YY1 transcriptional repression was lost in an E(z) mutant background was examined. The results are consistent with the observation that E(z) specifically methylates histone H3 on Lys 27, which creates a binding site for the chromodomain of Pc. Thus, the repression observed with GALYY1 requires function of the E(z) PcG protein (Srinivasan, 2004).

It has been shown that YY1 interacts with Drosophila CtBP, a well-characterized corepressor molecule. CtBP can also interact with Pc in vivo. These associations led to a proposal that CtBP might play a bridging function between YY1 and PcG proteins. If true, one would expect loss of PcG recruitment to DNA in a CtBP mutant background. Indeed, ChIP experiments in a CtBP⁰³⁴⁶³/+ background showed greatly reduced Pc and Ph recruitment to the BGUZ reporter. In addition, histone H3 remained acetylated and unmethylated. Surprisingly, in a CtBP mutant background, a dramatic loss of GALYY1 DNA binding was observed. However, full-length GAL4 protein was able to bind to DNA equally well in wild-type and CtBP mutant backgrounds, indicating that the effect of CtBP mutation was specific for YY1. This is a very unexpected result because CtBP has never been demonstrated to control DNA binding of another protein. The absence of GALYY1 and PcG proteins bound to the BGUZ reporter in the CtBP mutant background suggested that expression of the LacZ gene should be increased. Indeed, LacZ expression was increased in CtBP mutant as compared with wild-type embryos. Thus, in a CtBP mutant background, GALYY1 does not bind DNA, PcG proteins are not recruited, histones remain acetylated and unmethylated, and transcription is derepressed (Srinivasan, 2004).

To be certain that this effect is not peculiar to the BGUZ reporter, the effect of CtBP mutation on GALYY1 and PcG binding at endogenous PREs was examined. For this, the Ubx PRE_D, engrailed (en) PRE, and sex combs reduced (scr) PRE were chosen. Strikingly, GALYY1 and Pc binding to all three PREs was greatly reduced in the CtBP mutant background. Reduction in GALYY1 and Pc DNA binding correlated with H3 K9 acetylation at the PRE_D and En PREs. In contrast, H3 K9 acetylation at the Scr PRE was lost in a CtBP mutant background. These results clearly indicate an essential role for Drosophila CtBP in PcG recruitment to DNA (Srinivasan, 2004).

Collectively, these studies clearly demonstrate PcG recruitment function by the multifunctional transcription factor YY1. This establishes YY1 DNA binding as a key mechanism for targeting PcG proteins to DNA. The loss of YY1 DNA binding and concomitant loss of PcG recruitment to reporters and endogenous PRE sequences in CtBP mutants underscores this mechanism. A model of YY1 and CtBP function is presented. It is proposed that in a CtBP mutant background, YY1 is sequestered by a protein that inhibits its ability to bind to DNA. In a CtBP wild-type background, YY1 is released from this protein, thus enabling it to bind to DNA. DNA binding by YY1 results in recruitment of PcG complexes that cause deacetylation of histones and methylation of histone H3 at Lys 9 and Lys 27. Deacetylation may also be mediated by HDACs directly recruited by interaction with YY1 (Srinivasan, 2004).

The ablation of YY1 DNA binding in a CtBP mutant background was totally unexpected. This represents a new mechanism for controlling YY1 DNA binding and PcG recruitment. The mechanism appears to be exquisitely sensitive to CtBP dose because YY1 DNA binding and PcG recruitment are greatly reduced in heterozygous mutant backgrounds. Heterozygous effects by CtBP on knirps and hairy mutant phenotypes have been observed in other systems, suggesting that CtBP levels are limiting in vivo (Srinivasan, 2004).

The exact role of CtBP in PcG-mediated repression is yet to be elucidated. The results suggest that CtBP is required for the function of a large subset of PREs that require YY1/PHO for PcG recruitment. Like PcG mutants, CtBP mutants in flies show segmentation defects, but homeotic derepression has not been observed. Heterozygous ctbp mutants can reverse pair-rule phenotypes observed in hairy mutants, and homozygotes show bristle and cuticle defects. Furthermore, embryos that are trans-heterozygous for wimp and the ctpb⁰³⁴⁶³ allele die and their cuticle preparations show severe segmentation defects. Similarly, mouse ctbp1 and ctbp2 null mutants show a variety of defects including skeletal abnormalities, but these defects do not precisely match the skeletal posterior transformations seen with mammalian PcG mutants. Based on the multiple PREs affected by CtBP mutation, it is unclear why a more severe CtBP heterozygous mutant phenotype is not observed. Perhaps a low level of PcG binding to DNA remains that is below detection in immunostains of polytene chromosomes, but which is sufficient to mediate biological effects. In support of this possibility, polytene spreads were occasionally observed that stained with Pc antibodies nearly as well as wild-type spreads. This suggests a possible threshold effect for CtBP involvement in PcG recruitment. ChIP studies on many more PRE sequences will be needed to clarify this issue (Srinivasan, 2004).

These results show that modulation of YY1 DNA binding by CtBP is a critical step in the recruitment of PcG proteins to DNA. This mechanism might be differentially used during development to control PcG assembly on PREs. The demonstration of recruitment of PcG proteins by YY1 should assist in the identification of mammalian PREs since the YY1 recognition sequence is well characterized (Srinivasan, 2004).

Quantitative contributions of CtBP-dependent and -independent repression activities of Knirps

The Drosophila Knirps protein is a short-range transcriptional repressor that locally inhibits activators by recruiting the CtBP co-repressor. Knirps also possesses CtBP-independent repression activity. The functional importance of multiple repression activities is not well understood, but the finding that Knirps does not repress some cis-regulatory elements in the absence of CtBP suggested that the co-factor may supply a unique function essential to repress certain types of activators. CtBP-dependent and -independent repression domains of Knirps were assayed in Drosophila embryos; the CtBP-independent activity, when provided at higher than normal levels, can repress an eve regulatory element that normally requires CtBP. Dose response analysis has revealed that the activity of Knirps containing both CtBP-dependent and -independent repression activities is higher than that of the CtBP-independent domain alone. The requirement for CtBP at certain enhancers appears to reflect the need for overall higher levels of repression, rather than a requirement for an activity unique to CtBP. Thus, CtBP contributes quantitatively, rather than qualitatively, to overall repression function. The finding that both repression activities are simultaneously deployed suggests that the multiple repression activities do not function as cryptic 'backup' systems, but that each contributes quantitatively to total repressor output (Struffi, 2004).

The expression of the endogenous eve gene is strongly perturbed by a loss of CtBP, consistent with the important role of this co-repressor in the activity of gap repressors Giant, Krüppel, and Knirps. To study the effectiveness of Knirps repression of individual eve regulatory elements, the expression of eve-lacZ reporter genes was examined. Knirps is required for correct regulation of the eve stripe 3/7 and 4/6 enhancers, as demonstrated by the expression patterns of lacZ reporter genes in kni mutant embryos. The posterior border of eve stripe 3 was not derepressed in a CtBP mutant, consistent with the CtBP-independent activity of Knirps on this enhancer. By contrast, Knirps repression of eve stripe 4/6 is compromised in a CtBP mutant background, indicating that the CtBP-independent repression activity of Knirps is insufficient to regulate this enhancer. Therefore, depending on which part of the eve gene is bound by the Knirps protein, its repression activity is either dependent or independent of the CtBP co-factor (Struffi, 2004).

Previous studies of Krüppel, Giant and Knirps have indicated that CtBP dependence or independence of their repression activities varies according to the specific cis regulatory element involved, suggesting that there are particular enhancer architectures that necessitate CtBP activity. The clearest example of enhancer specific requirements for CtBP is shown in the case of eve enhancers. In nuclei situated between eve stripes 4 and 6, the stripe 4/6 and 3/7 enhancers are both repressed by Knirps in the same nuclei, yet this repression is independent of CtBP on the 3/7 element and dependent on CtBP on the 4/6 element. By expressing increasing levels of the CtBP-independent form of Knirps, the requirement for CtBP is obviated. These results suggest that distinct requirements for the CtBP co-factor at different genes or cis regulatory elements can be based on the quantitative levels of repression activity. Indeed, the combination of the CtBP-dependent and CtBP-independent activities make a particularly powerful repressor, as judged by comparison of repression activities of Knirps 1-429 versus Knirps 1-330 on eve and other pair-rule genes. These results suggest that both repression domains can be simultaneously engaged on a given cis regulatory element, rather than a particular repression activity being selectively engaged at particular enhancers. Consistent with this picture, when they are assayed separately as Gal4 fusion proteins in embryos, both CtBP-dependent and CtBP-independent repression domains of Knirps have equal, modestly effective repression activities. By contrast, a Gal4 protein containing both domains is much more effective at repressing a strongly activated promoter (Struffi, 2004).

A model is presented that explains the quantitative contribution of the CtBP co-repressor to Knirps repression activity. At a relatively low level of Knirps protein activity, the eve 3/7 enhancer is repressed, and this level of repression activity is achieved at similar levels of Knirps, regardless of whether or not CtBP contributes to repression. Thus, in the absence of CtBP, the positions at which the stripe 3/7 boundaries form shift very little. The much higher level of repression required by the stripe 4/6 element is achieved only near the peak of Knirps protein levels. If CtBP is not complexed with Knirps, the intercept shifts sharply to the right, to a level of Knirps not normally present in the embryo. The sufficient level of repression in the absence of CtBP activity or protein is only achieved under conditions where Knirps is overexpressed (Struffi, 2004).

The threshold model explains how the contributions of separate repression activities act in a quantitative fashion to meet given thresholds, but what is the basis for distinct repression thresholds? There are at least two variables involved in dictating a threshold, namely, regulatory protein levels and the nature (number, affinity, and placement) of the relevant binding sites within a regulatory element. Varying intranuclear activator levels can influence repression thresholds, as suggested by regulation of the Krüppel gene: Giant requires CtBP for repression of this gene only in nuclei containing peak levels of the Bicoid activator. Varying intranuclear repressor levels will dictate how easily those thresholds are met, with or without multiple repression activities. Gap genes, including knirps, generate protein gradients that have properties of morphogens, i.e., they trigger differential responses at different threshold levels. The stripe 4/6 and 3/7 modular enhancers of the even-skipped gene are designed to respond to different levels of Knirps protein, allowing the embryo to establish multiple stripe boundaries with a single protein gradient. The short-range activity of Knirps allows the two enhancers to act independently, so that activators bound to the stripe 4/6 enhancer activate the gene in nuclei where the levels of Knirps are already sufficiently high to inhibit the stripe 3/7 enhancer (Struffi, 2004).

Binding site affinity and number have been clearly established to influence threshold responses in the case of transcriptional activators, such as Bicoid and Dorsal. A similar effect is likely to be true for repressors. Sequence analysis of the eve gene indicates that there are more high-affinity Knirps binding sites within the eve stripe 3/7 element than in the 4/6 enhancer, consistent with relative sensitivities of these elements that were determined experimentally. Removal of some of the Knirps binding sites in the eve stripe 3/7 enhancer reduces the sensitivity of this element to the Knirps gradient. However, the number of predicted high-affinity binding sites alone is not sufficient information to predict relative sensitivity to Knirps. If it were, one would expect the eve stripe 2 enhancer, with three predicted Knirps sites, to be more sensitive to Knirps than eve stripe 4/6, with only a single site, yet the reverse is true. This lack of correlation might be partly attributable to errors in the prediction of binding sites; however, additional factors, such as affinity of binding sites and relative placement with respect to other proteins, are likely to make the decisive difference in determining enhancer sensitivity to Knirps. In the case of the Giant repressor, small shifts in the placement of the binding site allows detection of less than two-fold differences in repressor concentrations, a 'gene tuning' mechanism that seems to have been invoked during internal evolution of the eve stripe 2 enhancer. The stoichiometry of activators to repressors has also been suggested to be a crucial factor in determining repression levels, and direct tests indicate that Giant and Knirps respond sensitively to differences in activator binding site number and affinity on defined regulatory elements (Struffi, 2004).

eve stripe 1 lies just posterior to the weak anterior domain of knirps expression, suggesting a possible role for Knirps in regulating that element, but it is not clear whether the relative sensitivity of other eve stripe enhancers normally active outside of the main posterior domain of Knirps expression is of physiological significance. The eve stripe 2 pattern lies outside of the normal area of Knirps expression, and is only repressed at the highest levels of Knirps, suggesting that repression might be through cryptic Knirps sites in the element. The robust activity of the eve stripe 5 enhancer even under conditions of high levels of Knirps misexpression emphasizes that this regulatory element has been designed to function in nuclei containing peak levels of Knirps protein. Similarly, runt stripe 5 also resists peak levels of ectopic Knirps. Both of these regulatory elements have few or no predicted Knirps-binding sites. These elements would provide a useful platform to test the number and placement of novel Knirps binding sites required to bring the element under the control of this repressor (Struffi, 2004).

The effects of Knirps misexpression on other endogenous pair rule genes reinforce the lessons learned from eve, regarding the relative potency of the Knirps repression domains and the sensitivity of different enhancers. Both the CtBP-independent region of Knirps as well as the intact protein are capable of repressing the hunchback parasegment 4 stripe, a highly sensitive target of Knirps. However, hairy, runt and ftz, which have been previously noted to have a higher threshold to Knirps repression, are noticeably less affected by Knirps 1-330 compared with Knirps 1-429. Thus, it is likely that CtBP activity contributes quantitatively to repression of other Knirps target genes in addition to eve (Struffi, 2004).

Repression of central runt stripes is consistent with previous findings of direct repression by Knirps and the greater sensitivity of stripes 2-4 relative to stripe 1. A greater effect of ectopic expression of Knirps is observed on hairy than noted in previous experiments, probably on account of higher levels of expression. Knirps expressed under the control of an eve stripe 2 enhancer was previously found to have little effect on anterior hairy expression, except for a delay in stripe 3/4 separation. Heat shock expression of full-length Knirps 1-429, by contrast, results in strong repression of hairy stripes 3, 4 and 7. The hairy stripe 3, 4 and 7 enhancers are predicted to contain Knirps-binding sites, in contrast to the unrepressed stripe 1 and 5 enhancers. The weaker Knirps 1-330 protein had an effect similar to that of full-length Knirps expressed from an eve stripe 2 expression construct, i.e., a delay of stripe 3/4 separation. Interestingly, knirps is important for activation of hairy stripe 6, and the protein can bind to the stripe 6 enhancer directly in vitro. No evidence of activation is seen upon overexpression, however, suggesting that such activation might be indirect (Struffi, 2004).

The derepression of ftz observed between stripes 2-4 and 6-7 is likely due to indirect effects of repression of hairy and eve expression; both of these genes are thought to repress ftz directly. By contrast, previous work involving lower levels of anteriorly expressed Knirps observed only weakened ftz stripes 2 and 3, rather than stripe fusion. This lower level of Knirps had a much less profound effect on upstream regulators hairy and eve, suggesting that Knirps might be a direct gap gene input to this pair-rule gene (Struffi, 2004).

This study suggests that the multiple repression activities of Knirps can be simultaneously mobilized to provide quantitatively correct levels of repression activity, and that the design of cis regulatory elements can elicit CtBP dependence. CtBP-independent activity can in some cases be directly attributed to direct competition with activator for DNA binding; however, the CtBP-independent activity of Knirps can repress activators on elements where sites are not overlapping, and overexpression of the DNA-binding domain of Knirps (Knirps1-105) is insufficient to mediate repression of endogenous eve enhancers. Cell culture and transgenic embryo assays indicate that both CtBP-dependent and independent repression activities of Knirps have very similar characteristics with respect to activator specificity, distance dependence and overall potency, thus the targets and molecular mechanisms might well be similar in each case. Key to a deeper understanding of the molecular circuitry controlled by short-range repressors such as Knirps will be biochemical knowledge of the mechanisms of repression employed on these developmentally regulated enhancers (Struffi, 2004).

The APC tumor suppressor binds to C-terminal binding protein to divert nuclear ß-Catenin from TCF

Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector ß-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. This study reports a third function of APC in antagonizing ß-catenin involving C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of ß-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. The evidence indicates that APC is an adaptor between ß-catenin and CtBP and that CtBP lowers the availability of free nuclear ß-catenin for binding to TCF by sequestering APC/ß-catenin complexes (Hamada, 2004).

To identify proteins that bind to APC in Drosophila embryos, crude embryonic extracts were incubated with bacterially expressed Drosophila E-APC fused to glutathione-S-transferase (GST). Analysis of associated proteins by MALDI mass spectrometry reveals dCtBP as an unexpected binding partner of E-APC. CtBP was initially discovered as a cellular protein binding to the C terminus of the adenovirus E1A protein, which suppresses its transformation potential. CtBP is a transcriptional corepressor in mammals and binds to various DNA binding proteins via a short conserved motif P-h-D-L-S-x-R/K. Mammals have a second CtBP relative, CtBP2, which also recognizes this motif and whose function overlaps that of CtBP (Hamada, 2004).

Intriguingly, a motif similar to P-h-D-L-S-x-R/K is found in each of the 15 amino acid repeats (15R) of APC and of Drosophila E-APC. These repeats can bind to ß-catenin but cannot promote its proteasomal destruction; the latter requires the Axin binding motifs of APC. Therefore, there is no known function of the 15Rs in the downregulation of ß-catenin. The interaction between an individual 15R and ß-catenin has been characterized at the structural level. The presumed CtBP binding motif shares some but not all of the residues in the C-terminal half of the 15R that are engaged in the interaction with ß-catenin (Hamada, 2004).

Binding between E-APC and dCtBP was confirmed in vitro by pull-down assays between bacterially expressed GST-dCtBP and in vitro translated E-APC. This binding is comparable to that between E-APC and Armadillo (Drosophila ß-catenin); however, Armadillo does not bind directly to GST-dCtBP. A small region spanning the two 15Rs of E-APC fused to GST is sufficient for binding to in vitro translated dCtBP, while a triple alanine substitution ('AxAxA') in the P-h-D-L-S motif of each 15R (in the context of the C-terminal half of E-APC) almost completely abolishes binding to dCtBP. The same is true for the binding between human CtBP and a central fragment of APC (residues 918-1698) that binds efficiently to GST-CtBP, while its mutant version AxAxA binds poorly. APC(918-1698) contains two further putative CtBP binding motifs that were substituted in addition ('AxAxAplus'). This further reduced the binding to GST-CtBP (by >16%); no binding whatsoever was detectable with a GST-LEF-1 control. Importantly, both APC mutants bind to ß-catenin equally well as the wild-type. Likewise, both mutants retain the ability to reduce the overall levels of coexpressed HA-tagged ß-catenin in transfected APC mutant cancer cells, though a low level of endogenous ß-catenin can still be detected by immunofluoresence in these transfected cells. Thus, the binding between APC and CtBP is specific and conserved and neither appears to affect APC's binding to ß-catenin nor its ability to promote the destruction of cytoplasmic ß-catenin (Hamada, 2004).

APC is also associated with CtBP in mammalian cells: endogenous CtBP can be coimmunoprecipitated with endogenous APC, and vice versa, in 293T cells and in HCT116 colorectal cancer cells that express wild-type APC. Furthermore, in APC mutant cancer cells, the resident APC truncations can be coimmunoprecipitated in SW480 cells, but not in COLO320 cells. Notably, the 15Rs are retained only in the APC truncation of the former, but not of the latter. Thus, the association of APC with CtBP in mammalian cells depends on its 15Rs (Hamada, 2004).

Few colorectal carcinomas express APC truncations that lack the 15Rs. COLO320 is one of the rare colorectal cancer cell line of this type. Interestingly, this line exhibits exceptionally high TCF-mediated transcription. This suggests that the 15Rs may harbor an activity that is critical for the downregulation of the transcriptional activity of TCF (Hamada, 2004).

To test whether the binding of CtBP to the 15Rs is functionally relevant, a complementation assay was used of APC mutant cancer cells based on a luciferase reporter linked to TCF binding sites (pTOPFLASH). This quantitative assay is highly specific for TCF-mediated transcription and serves as a fairly direct readout of exogenous APC function in restoring low levels of TCF transcription. COLO320 cells show very high TOPFLASH values, >2× higher than those of SW480 cells and up to 5× higher than those of other APC mutant colorectal cancer cells. These values are reduced substantially after cotransfection with APC(918-1698), which spans the 15Rs and the 5'-most nuclear export signal (NES1506) and Axin binding site. Similar APC fragments have previously been found to efficiently reduce the ß-catenin levels in SW480 cells. In contrast, the AxAxA mutant is less active in reducing TOPFLASH values, and AxAxAplus is even less active. The control values of pFOPFLASH (containing mutant TCF sites) are low and unchanged by the mutants. It is concluded that the binding between APC and CtBP is critical for the APC-mediated downregulation of the transcriptional activity of ß-catenin. The residual activities of AxAxA and AxAxAplus in this assay are likely to reflect their ability to promote Axin-mediated destruction and nuclear export of ß-catenin; note that APC(918-1698) and its mutant versions shuttle in and out of the nucleus, as judged by their nuclear accumulation after exposure to leptomycin B (Hamada, 2004).

Evidence has indicated that APC can sequester nuclear ß-catenin and keep it from binding to TCF and activating transcription. This sequestration can be demonstrated experimentally if an APC fragment is targeted to the nucleus by linkage to a nuclear localization signal (NLS): this causes a dramatic nuclear accumulation of endogenous ß-catenin, but these high levels of nuclear ß-catenin are ineffective in stimulating TCF-mediated transcription. This therefore provides an assay for measuring the sequestration of nuclear ß-catenin by APC (Hamada, 2004).

NLS-fusions of the AxAxA and AxAxAplus mutants were tested in this sequestration assay. Interestingly, the mutant NLS-fusions are less active in reducing TOPFLASH values than their wild-type controls. These differences are significant since the expression levels of wild-type and mutant NLS-fusions are essentially the same. Notably, the loss of function of the AxAxA and AxAxAplus mutants in reducing ß-catenin activity is exacerbated in this sequestration assay where the levels of nuclear ß-catenin are high. This suggests a role of the APC-CtBP interaction in sequestering nuclear ß-catenin (Hamada, 2004).

A possible model is that APC binds to free nuclear ß-catenin in competition with TCF and targets ß-catenin to CtBP (by being an adaptor between these two proteins), thus diverting ß-catenin away from TCF. CtBP, being anchored at specific sites within the nucleus, could act as a "sink" for APC/ß-catenin complexes, thus shifting the binding equilibrium of ß-catenin yet further away from TCF (Hamada, 2004).

Three lines of evidence support this model: (1) ß-catenin can be detected in a complex with CtBP in SW480, but not in COLO320 cells, whose APC truncation can bind neither CtBP nor ß-catenin; (2) in COLO320 cells transfected with NLS-fusions of APC, it is estimated that the levels of endogenous TCF-4/ß-catenin complexes are 1.5×-2× higher in the case of AxAxAplus compared to the wild-type control. These increased levels of TCF-4/ß-catenin complexes are likely to be the basis for the high TCF-mediated transcription in the complementation assays. (3) In CtBP mutant mouse cells expressing tagged LEF-1, 2×-3× more endogenous ß-catenin can be coimmunoprecipitated with LEF-1 than in the corresponding parental control cells (heterozygous for both alleles). The total levels of ß-catenin are the same in the two cell lines, as are the amounts of APC bound ß-catenin. The latter two lines of evidence indicate that CtBP reduces the availability of ß-catenin for binding to TCF (Hamada, 2004).

If so, absence of CtBP should result in elevated levels of TCF-mediated transcription. Indeed, the basal TOPFLASH activity (due to endogenous TCF/ß-catenin) in CtBP mutant cells is increased ~3.7× compared to their control cells. Furthermore, cotransfection of activates ß-catenin (S33A mutant) and Lef-1 stimulate TOPFLASH activity to higher levels in CtBP mutant cells compared to the control. By comparison, <2× differences are detected in transcriptional activity between mutant and wild-type cells if FOPFLASH or an SV40-based control reporter (pRL-SV) are tested. Indeed, the activity levels of the internal control renilla reporter (pRL-CMV) are the same in both cell lines. Therefore, Lef-1-mediated transcription is more sensitive to CtBP loss than the transcription mediated by other transcription factors. Thus, CtBP appears to antagonize TCF-mediated transcription in a relatively specific way (Hamada, 2004).

It has been reported that Xenopus CtBP can bind to XTcf-3 and antagonize the transcription of TCF target genes in the early Xenopus embryo. It was noted that TCF-3 and TCF-4 factors possess CtBP binding motifs and suggested that CtBP may be a corepressor of these TCFs. Potentially, this could explain the increased basal levels of TCF-mediated transcription in CtBP mutant cells compared to their parental controls. However, it is unlikely to explain the increased levels of Lef-1-stimulated transcription, given that Lef-1 is a TCF factor that lacks CtBP binding motifs (Hamada, 2004).

In vivo association between CtBP and TCF had never been demonstrated, so this was examined in comparison to the in vivo association between CtBP and APC. First, it was asked whether endogenous CtBP and TCF-4 coimmunoprecipitate in colorectal cancer cells, given that TCF-4 is expressed in these cells. ß-catenin coimmunoprecipitates with TCF-4, as expected; however, CtBP is not detectable in the same TCF-4 immunoprecipitate. Conversely, while APC coimmunoprecipitates with CtBP, TCF-4 does not. Thus, endogenous CtBP is associated with APC, but not with TCF, in colorectal cancer cells. Notably, the same is true in 293T cells in which TCF is transcriptionally inactive: endogenous CtBP is associated with APC and ß-catenin, but not with endogenous TCF-4. It is concluded that TCF is not detectable in a complex with CtBP, regardless of cell type and transcriptional activity (Hamada, 2004).

It has been reported that exogenous TCF-4 can repress TOPFLASH transcription in transfected simian COS cells (that lack E1A expression) in a CtBP-dependent manner, while a C-terminal truncation of TCF-4 without the CtBP binding motifs (such as those arising from frameshift mutations in TCF-4 in some microsatellite-unstable colorectal carcinomas) does not respond to overexpressed CtBP in this assay. These experiments were repeated by comparing the activities of mutant TCF-4, whose two CtBP binding motifs were mutated in the same way as those of APC (TCF-4 AxAxA with triple alanine substitutions in residues 1, 3, and 5 of the P-h-D-L-S-x-R/K motif) and its wild-type control in TOPFLASH assays, and in their response to overexpressed CtBP. Overexpressed TCF-4 can repress TOPFLASH transcription in a dose-dependent manner in transfected SW480 and COS cells. However, the AxAxA TCF-4 mutant was similarly inhibitory, despite being expressed at slightly higher levels than wild-type TCF (especially at low doses of transfected plasmid). Furthermore, the mutant was equally responsive to coexpressed CtBP as the wild-type TCF-4. Therefore, although the AxAxA mutation affects the activity of APC(918-1698) in TCF-specific transcription assays, the same mutation in TCF-4 does not affect its activity in these assays. In agreement with this, a comparable double mutation of the CtBP binding motifs in XTcf-3 does not reduce its repressive potential in Xenopus embryos. Note that this double mutation does reduce the in vitro binding of XTcf-3 to CtBP, and so does the AxAxA double mutant of TCF-4. However, the in vitro binding between CtBP and TCF-4 is ~10× less strong than that between TCF-4 and ß-catenin. Thus, the in vitro binding between CtBP and TCF, although apparently specific, is very weak indeed. It may be spurious, given the lack of a detectable association between these proteins in vivo (Hamada, 2004).

In summary, no evidence was obtained for a significant physical or functional interaction between CtBP and TCF. These results thus question the idea that CtBP functions generally as a corepressor of TCF factors. It is agreed that the TCF-4 frameshift mutations observed in microsatellite-unstable colorectal carcinomas are passenger mutations without any functional relevance for TCF-mediated transcription or tumorigenesis (Hamada, 2004).

It was asked whether dCtBP might antagonize Armadillo-mediated transcription during Drosophila development. However, this is not straightforward to test, since dCtBP mutants show highly pleiotropic mutant phenotypes: null mutant embryos are grossly abnormal and do not develop beyond early stages, due to failing interactions between dCtBP and segmentation gene products. This precludes a meaningful analysis of dTCF target gene expression in these mutants. And although dCtBP has been implicated in antagonizing dTCF transcription in the developing midgut, this is an indirect effect mediated by the DNA binding protein Brinker to which CtBP can bind. Likewise, CtBP loss in the mouse causes pleiotropic mutant phenotypes, one of which, unexpectedly, mimics loss of Wnt signaling, but this could also be an indirect effect of CtBP binding to another target protein outside the Wnt pathway (Hamada, 2004).

Thus, to explore the regulatory relationship between dCtBP and Armadillo during development, it was asked whether dCtBP loss would affect the phenotypic consequences of overactive or depleted Armadillo. This is indeed the case: lowering the dose of dCtBP enhances the rough eye phenotype caused by activated Armadillo, but the same condition suppresses the wing nick phenotype due to Armadillo depletion in cells whose stimulation by Wingless is required for normal wing margin formation. These genetic interactions are similar to those of negative components of the Wnt pathway that downregulate Armadillo, such as Drosophila Axin and APC, consistent with dCtBP antagonizing Armadillo. Again, it is emphasized that this antagonism is unlikely to be due to dCtBP being a direct corepressor of dTCF, given that the latter does not contain any CtBP binding motifs. The results suggest that the antagonism between CtBP and Armadillo/ß-catenin is conserved and operates in multiple tissues and cell types (Hamada, 2004).

This study has presented evidence that CtBP binds to APC directly and specifically via the conserved 15Rs of APC and that the association of the two proteins in vivo is functionally relevant since it is required for the full activity of APC in reducing TCF-mediated transcription in colorectal cancer cells. In contrast, no evidence was found for a direct physical or functional interaction between CtBP and TCF in mammalian cells, calling into question whether CtBP acts generally as a transcriptional corepressor of TCF factors (Hamada, 2004).

Instead, the evidence suggests that CtBP antagonizes TCF-mediated transcription by cooperating with APC to sequester nuclear ß-catenin. This sequestration could be a safeguard function of APC, operating in parallel to (and to some extent redundantly with) its other functions in promoting nuclear export and degradation of ß-catenin. It is proposed that APC sequesters ß-catenin by targeting it to CtBP, thus lowering the pool of free nuclear ß-catenin that is available for binding to TCF. The sequestration of the APC/ß-catenin complex by CtBP may be based on spatial segregation within the nucleus (e.g., anchoring of the complex at specific subnuclear bodies). Whatever the precise mechanism, the observed functional cooperation between CtBP and APC in colorectal cancer cells suggests a role of CtBP as a tumor suppressor in the colon (Hamada, 2004).

Interplay between positive and negative activities that influence the role of Bicoid in transcription: Interaction of Bcd with its co-activator CBP

The Drosophila mophogenetic protein Bicoid (Bcd) can activate transcription in a concentration-dependent manner in embryos. It contains a self-inhibitory domain that can interact with the co-repressor Sin3A. A Bcd mutant, Bcd^A57-61, that has a strengthened self-inhibitory function and is unable to activate the hb-CAT reporter in Drosophila cells, has been used to analyze the role of co-factors in regulating Bcd function. Increased concentrations of the co-activator dCBP in cells can switch this protein from its inactive state to an active state on the hb-CAT reporter. The C-terminal portion of Bcd^A57-61 is required to mediate such activity-rescuing function of dCBP. Bcd^A57-61 has a normal ability to bind to a single TAATCC site when analyzed in vitro. Although capable of binding to DNA in vitro, Bcd^A57-61 is unable to access the hb enhancer element in cells, suggesting that its DNA binding defect is only manifested in a cellular context. Increased concentrations of dCBP restore not only the ability of Bcd^A57-61 to access the hb enhancer element in cells but also the occupancy of the general transcription factors TBP and TFIIB at the reporter promoter. These and other results suggest that an activator can undergo switches between its active and inactive states through sensing the opposing actions of positive and negative co-factors (Fu, 2005).

As a molecular morphogen, Bcd can undergo switches, in a concentration-dependent manner, between its active and inactive states in activating transcription of its target genes. The experiments described in this report suggest another mechanism that can facilitate on-off switches of Bcd activity in a Bcd concentration-independent manner. In particular, the mutant Bcd^A57-61 is incapable of activating the hb-CAT reporter gene in S2 cells at all concentrations tested. The inability of this mutant Bcd to activate the hb-CAT reporter reflects a distinct functional state of this protein rather than its defects in protein stability. In fact, this same mutant protein is only modestly weaker than the wt protein on another reporter gene, kni-CAT, which contains the Bcd-responsive kni enhancer element. These and other results suggested that the A57-61 mutation may cause its functionally inactive state on hb-CAT by more efficiently interacting with a co-repressor protein(s), such as Sin3A and its associated complex(es). The experiments described in this report show that increased concentrations of dCBP can restore activity to Bcd^A57-61 on the hb-CAT reporter in cells. These results suggest that the opposing actions of positive and negative co-factors can facilitate Bcd to switch between its active and inactive states in a manner that is Bcd concentration-independent (Fu, 2005).

Although Bcd^A57-61 can bind to both a single site and natural enhancer elements in vitro, it is unable to access the hb enhancer element in cells. These results suggest that the DNA binding defect of this mutant protein is only manifested in a cellular context. This notion is consistent with the finding that the PAH domains of Sin3A do not exhibit any increased ability to reduce DNA binding by Bcd^A57-61 in vitro when compared with wt Bcd. It is proposed that other co-repressors or those that are associated with Sin3A, such as the HDACs, can reduce the ability of Bcd to access a natural enhancer in cells. It is possible that the enzymatic HDAC activity that is more stably associated with Bcd^A57-61 makes it unable to negotiate with histones for accessing DNA. It is also possible that a more stable Bcd-co-repressor complex may sterically hinder the interaction between Bcd^A57-61 molecules and prevent cooperative binding to the enhancer element in cells (Fu, 2005).

The most striking finding of this report is that high levels of dCBP can switch Bcd^A57-61 from its inactive state to an active one on the hb-CAT reporter in cells. ChIP data further show that dCBP increases both the ability of Bcd^A57-61 to access the hb enhancer element in cells and the occupancy of GTFs at the reporter promoter. How does dCBP switch the activity states of Bcd^A57-61 on hb-CAT in cells? Since Bcd and dCBP can physically interact with each other through multiple domains, it is possible that dCBP may increase the DNA binding ability of Bcd in cells by stabilizing the interaction between Bcd molecules and thus enhancing its cooperativity. It is also possible that dCBP may physically compete with co-repressor complexes in interacting with Bcd. Co-IP results suggest that dCBP may negatively affect the interaction between Bcd and Sin3A in cells. dCBP could also play a role in facilitating the interaction between Bcd and the transcription machinery. For all these actions, dCBP may play a structural (rather than enzymatic) role. Finally, the fact that the HAT-defective mutant of dCBP does have a reduced ability to restore activity to Bcd^A57-61 indicates that its enzymatic activity has a positive role, possibly through modifications of histones. It is likely that dCBP can affect the Bcd^A57-61 activity through multiple mechanisms that may be weak individually but, when combined, can lead to a dramatic switch from its inactive state to an active one on the hb-CAT reporter in cells (Fu, 2005).

Currently, it is poorly understood how precisely Bcd activates transcription. Previous studies suggest that much of its activation function is conferred by the C-terminal portion of Bcd. This portion of the protein contains several domains, including the acidic, glutamine-rich and alanine-rich domains, that are characteristic of activation domains capable of interacting with components of the transcription machinery. Interestingly, the alanine-rich domain previously thought to play an activation role was shown recently to exhibit an inhibitory function instead. The C-terminal domain of Bcd can also interact with dCBP, and the results show that this domain is responsible for mediating the activity-switching function of dCBP. Although much of the activation function of Bcd is provided by its C-terminal domain, the N-terminal portion of the protein also contains some activation function. Studies have shown that Bcd(1-246), a derivative lacking the entire C-terminal portion of Bcd, can rescue the bcd^- phenotype when expressed at high levels. These results suggest that Bcd can achieve its activation function through multiple domains presumably by interacting with different proteins, including co-activators and components of the transcription machinery. The results described in this report further support the importance of dCBP in facilitating activation by Bcd (Fu, 2005).

Bcd is a morphogenetic protein whose behavior can be regulated not only by its own concentration but also by the enhancer architecture. On the kni and hb enhancer elements, the N-terminal domain of Bcd is preferentially used for either cooperative DNA binding or self-inhibition, respectively. It is proposed that the interaction between Bcd molecules bound to the kni enhancer element, through its N-terminal domain, can interfere with its interaction with co-repressors, such as Sin3A. Co-activators such as dCBP and co-repressors such as Sin3A can also functionally antagonize each other, possibly by competing for Bcd interaction as part of the mechanisms. Bcd is more sensitive to the self-inhibitory function on the hb enhancer element than on the kni enhancer element: consistent with dCBP's antagonistic role, dCBP increases the activity of Bcd more robustly on the hb enhancer element than on the kni enhancer element. However, the interplay between positive and negative activities that regulate Bcd functions is probably far more complex than the simple physical competition: as already discussed above, dCBP can affect Bcd activity through multiple mechanisms in both HAT-dependent and independent manners. Moreover, in the presence of exogenous dCBP, high levels of Bcd^A57-61 cause a reduction in its activity on the hb-CAT reporter in cells, a reduction that is not observed with wt Bcd, suggesting that the optimal concentration ratio between Bcd and dCBP may vary depending on the strengths of the self-inhibitory function and interaction with co-repressors. In addition, high concentrations of dCBP can rescue the inactive derivative Bcd^A57-61, but not another inactive derivative lacking the C-terminal portion, Bcd(1-246; A57-61), suggesting that the Bcd-dCBP interaction strength can also influence the balance between positive and negative activities that regulate Bcd function (Fu, 2005).

The experiments described in this report suggest that an activator's function is subject to intricate controls by both positive and negative activities in cells. A fine balance between these activities is critical for normal cellular and developmental processes. Transgenic experiments show that both Bcd^A57-61, which has a strengthened self-inhibitory function, and Bcd^A52-56, which has a weakened self-inhibitory function, cause embryonic defects. In addition, embryos with reduced dCBP activity exhibit defects in early expression patterns of a Bcd target gene, even-skipped. Finally, mutations affecting SAP18, a component of the Sin3A-HDAC complex, can alter Bcd function and anterior patterning in embryos. In addition to the co-factors discussed in this study (Sin3A, dCBP and SAP18), Bcd likely has the ability to interact with many other proteins, including not only regulatory proteins but also components of the transcription machinery. Precisely how all these different proteins harmoniously regulate and facilitate the execution of Bcd functions during development remains to be determined. Recent studies have shown that the Bcd gradient in embryos possesses a strikingly sophisticated ability to activate its target genes in a precise manner. These findings further underscore the need of intricate control mechanisms that facilitate Bcd to switch between its active and inactive states in target gene activation. These studies suggest that on-off switches of Bcd activity can be achieved not only in a Bcd concentration-dependent manner but also in a Bcd concentration-independent manner. It remains to be investigated whether and how Bcd interacting proteins, including those yet to be identified, participate in the precision control of target gene activation during development (Fu, 2005).

Hairless induces cell death by downregulation of EGFR signalling activity

Overexpression of the Notch antagonist Hairless (H) during imaginal development in Drosophila is correlated with tissue loss and cell death. Together with the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP), H assembles a repression complex on Notch target genes, thereby downregulating Notch signalling activity. This study investigated the mechanisms underlying H-mediated cell death in S2 cell culture and in vivo during imaginal development in Drosophila. First, the domains within the H protein that are required for apoptosis induction in cell culture were mapped. These include the binding sites for the co-repressors, both of which are essential for H-mediated cell death during fly development. Hence, the underlying cause of H-mediated apoptosis seems to be a transcriptional downregulation of Notch target genes involved in cell survival. In a search for potential targets, transcriptional downregulation of rho-lacZ and EGFR signalling output were noted. Moreover, the EGFR antagonists lozenge, klumpfuss and argos were all activated upon H overexpression. This result conforms to the proapoptotic activity of H, as these factors are known to be involved in apoptosis induction. Together, the results indicate that H induces apoptosis by downregulation of EGFR signalling activity. This highlights the importance of a coordinated interplay of Notch and EGFR signalling pathways for cell survival during Drosophila development (Protzer, 2008).

This work allows two important conclusions: that overexpression of H induces cell-autonomous apoptosis, and that H requires the co-repressors Gro and CtBP for its proapoptotic activity. It is known that H assembles a repression complex together with the two co-repressors, resulting in transcriptional downregulation of Notch target genes. Hence, the ability of H to induce cell death is most likely a consequence of the repression of Notch target genes that are involved in cell survival. It is noted, however, that not every cell that receives an overdose of H dies. One simple explanation for this observation is that the only cells that die are those in which the relevant Notch target genes are normally active, as these cells require a Notch signal for survival. As H results in a repression of Notch activity, these cells would be driven into cell death, whereas those cells that do not depend on higher Notch levels for survival would be resistant to an H overdose. How is this effect of H realised at the molecular level? So far, it has not been possible to narrow down the analyses towards one target gene, the repression of which by the H repressor complex induces apoptosis. The most straightforward idea, repression of the anti-apoptotic protein Diap1, is not supported by the data. Instead, it was found that EGFR signalling activity is downregulated as a consequence of the upregulation of several negative regulators of EGFR (Protzer, 2008).

The existence of a densely woven network of genetic interactions between the EGFR and Notch signalling pathways is well established. This intensive cross-talk harmonises many developmental processes, such as proliferation, differentiation, cell fate specification, morphogenesis and programmed cell death. Still, the molecular basis of this genetic interplay remains largely obscure. So far, few molecular intersections between the Notch and EGFR pathways have been revealed. For example, EGFR signalling causes phosphorylation of the co-repressor Gro, thereby negatively modulating the transcriptional outputs of Notch signalling via the Enhancer of split [E(spl)] genes. Conversely, a myc-Gro complex was shown to inhibit EGFR signalling during neural development in the Drosophila embryo. Although mutual antagonism is probably the most prominent relationship in EGFR-Notch interactions, in some developmental situations both pathways cooperate to potentiate each other's signalling activities. One such example with regard to cell survival has been described in the retina of rugose mutant flies, where cell type-specific cell death could be reversed by an increase in Notch or EGFR signalling activity, indicating that both pathways adopt an anti-apoptotic function in this developmental context. Also, R7 photoreceptor cell specification requires the combined input of both Notch and EGFR signals. Moreover, Notch defines the scope of rho expression in the Drosophila embryo, thereby activating the EGFR pathway required for early ectodermal patterning. Also, during the development of mouse embryonic fibroblast, the Notch receptor-processing γ-secretase presenilin acts as a positive regulator of ERK basal level activity (Protzer, 2008).

A significant decrease was observed in the levels of activated MAPK (diP-ERK), which provides a good assessment of EGFR pathway activation, upon induction of H. Activated MAPK directly phosphorylates two transcription factors, Aop (Yan) and Pointed (Pntp2). Phosphorylation inactivates Aop, which in the unmodified state, represses EGFR targets. At the same time, phosphorylation activates Pointed, which then causes EGFR target gene transcription. As H is a well-defined transcriptional repressor of Notch target genes, it is most unlikely that it impedes EGFR activity at the level of phosphorylation. Moreover, it is not thought that H acts at the level of transcriptional regulation of EGFR target genes, even though combinatorial and antagonistic activities of the nuclear effectors of the EGFR and Notch signalling pathways have been described during eye development. Instead, the hypothesis is favored that H represses the transcription of EGFR activators, or might indirectly provoke the activation of EGFR repressors that affect, for example, the production of EGFR ligands or signal transduction (Protzer, 2008).

Rho activity is required for a timely and spatially regulated release of EGFR ligands. Accordingly, the expression of rho is highly dynamic during Drosophila development, and precedes the appearance of EGFR-induced activated MAPK. Hence, downregulation of rho by H would eventually result in lower levels of activated MAPK (diP-Erk). In contrast to other components of the EGFR signalling pathway, ectopic expression of rho results in EGFR activation in a wide range of tissues, indicating that Rho is an essential and limiting factor. So far, transcriptional control is the only known means of rho regulation. The complex array of enhancers regulating rho expression reflects the dynamic pattern of EGFR activation throughout Drosophila development (Protzer, 2008).

Interestingly, a transcriptional repression of rho-lacZ was observed in H gain-of-function clones that was dependent on the co-repressors Gro and CtBP. This effect might very well be direct, because it was shown previously that rho transcription is regulated by Su(H) in the neuroectoderm as well as in the gut of the Drosophila embryo. As mentioned above, Notch signalling has also been shown to regulate rho expression in the embryonic ectoderm. Moreover, during egg development, a band of Notch activity establishes the boundary between the two dorsal appendage tube cell types, whereby Notch levels are high in rho-expressing cells. In accordance with this, potential Su(H)-binding sites are present in the regulatory regions of rho1 and rho3, making a direct regulation of rho during eye development via the Notch-Su(H)-H complex very likely. It is noted, however, that the downregulation of rho-lacZ and of activated MAPK were focussed at the morphogenetic furrow, where primary photoreceptor cells are specified and ommatidia are founded. Regulation of rho by H would then be expected to interfere with photoreceptor formation rather than with cell survival, which is in agreement with the disturbed cellular architecture of H gain-of-function flies (Protzer, 2008).

Most interestingly, upon H overexpression, ectopic induction of lz, klu and aos was observed. All three genes are known to be involved in cell death induction during pupal eye development. There it was shown that the Runx protein Lz binds to the regulatory regions of klu and aos, resulting in the direct transcriptional activation of these target genes. Therefore, one might speculate that H executes its effect on klu and aos activity via the activation of lz. Moreover, as klu and aos are well-known inhibitors of EGFR signalling activity, this in itself suggests that H impedes EGFR signalling activity via these factors. This interpretation helps to explain why aos expression is induced in H gain-of-function clones, although it is well known that aos is triggered by EGFR signalling, thereby forming an inhibitory loop that acts on EGFR activity. The high levels of Lz still activate aos in H gain-of-function clones, keeping activity of the EGFR pathway low. Alternatively, aos and klu levels might be increased as a consequence of the downregulation, by H, of an as yet unknown repressor. Since H behaves as a kind of 'multi-adaptor protein', which not only recruits the transcriptional silencers Gro and CtBP to Notch targets but also binds other proteins such as Pros26.4, it is also possible that H interacts with positive regulators of lz, klu and aos (Protzer, 2008).

However, a model is favored whereby H influences EGFR signalling activity on two levels. On the one hand, through transcriptional repression of rho, H causes a loss of EGFR signalling output that interferes with cell specification. On the other hand, by interfering with their repressor(s), H relieves the restriction on lz, klu and aos expression, causing their accumulation. In consequence, the survival/death balance is tipped towards apoptosis in those cells that are susceptible to the effects of a lowered EGFR signal. Those cells that do not depend on high Notch and EGFR activity levels for survival would be resistant to an H overdose (Protzer, 2008).

Finally, one can envisage that a downregulation of Notch and EGFR signalling activities, resulting from the overexpression of H, might leave a cell in a state of 'uncertainty' that does not allow any further differentiation towards a certain cell type, but leaves the cell vulnerable to the apoptotic programme (Protzer, 2008).

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor beta ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. This study shows that this repression domain functions through interaction with two corepressors, CtBP and Sin3A, and that either interaction is sufficient for repression. Schnurri contains additional repression domains that function through interaction with CtBP, Groucho, Sin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype evidence is provided that these diverse repression domains are both cooperative and partially redundant. In addition Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor (Cai, 2009).

Drosophila CtBP regulates proliferation and differentiation of eye precursors and complexes with Eyeless, Dachshund, Dan, and Danr during eye and antennal development

Specification factors regulate cell fate in part by interacting with transcriptional co-regulators like CtBP to regulate gene expression. This study demonstrates that CtBP forms a complex or complexes with the Drosophila Pax6 homolog Eyeless (Ey), and with Distal antenna (Dan), Distal antenna related (Danr), and Dachshund to promote eye and antennal specification. Phenotypic analysis together with molecular data indicate that CtBP interacts with Ey to prevent overproliferation of eye precursors. In contrast, CtBP;dan;danr triple mutant adult eyes have significantly fewer ommatidia than CtBP single or dan;danr double mutants, suggesting that the CtBP/Dan/Danr complex functions to recruit ommatidia from the eye precursor pool. Furthermore, CtBP single and to a greater extent CtBP;dan;danr triple mutants affect the establishment and maintenance of the R8 precursor, which is the founding ommatidial cell. Thus, CtBP interacts with different eye specification factors to regulate gene expression appropriate for proliferative vs. differentiative stages of eye development (Hoang, 2010).

The eyes of several CtBP loss-of-function mutant combinations have statistically significantly more ommatidia than wild-type eyes, and CtBP^87De-10 clones ahead of the furrow show a statistically significant increase in the number of cells undergoing mitosis per unit area. These results suggest a role for CtBP in downregulating proliferation of eye precursors, although it cannot be currently exclude that additional processes such as apoptosis that might contribute to the increase in eye size in CtBP- mutants (Hoang, 2010).

The evidence suggests that CtBP is required to downregulate proliferation of eye precursors ahead of the morphogenetic furrow: CtBP- clones are larger and show more mitotic figures than wild type clones in the most anterior regions of the eye field. A number of factors are known to promote proliferation ahead of the furrow, and have been connected to CtBP in some way. Many of these factors can cause massive overgrowth when overexpressed. Thus, the role of CtBP may be to counteract the activity of one or more of these factors, to ensure that cells do not proliferate out of control (Hoang, 2010).

Factors that have previously been linked to CtBP and are known to regulate proliferation of eye precursors ahead of the furrow include the Wingless, Notch and JAK/STAT signaling pathways. This study has not ruled out a possible interaction between CtBP and these signaling pathways in the context of eye precursor proliferation (Hoang, 2010).

However, the combination of Hth, Tsh and Ey also regulates proliferation in ahead of the morphogenetic furrow, and this study has demonstrated that Ey and CtBP are part of a complex in eye-antennal disc cells, and that they interact genetically during eye development. Circumstantial evidence also suggests links between CtBP and Hth and Tsh. For instance, Drosophila and mouse Tsh homologs both contain a PxDLS motif, and have been shown to interact in vitro with Drosophila and mouse CtBP, respectively. In addition, the Drosophila Cdc25 homolog encoded by string, which triggers mitosis, appears to be a target of Hth, although it is not clear if it is a direct target. DamID experiments with CtBP in Kc cells have also identified string as a potential direct CtBP target. Based on current data, it is therefore proposed that CtBP interacts with the Hth/Tsh/Ey complex in eye precursors ahead of the furrow (Hoang, 2010).

CtBP^87De-10 single mutant clones ahead of the furrow in the larval eye disc are larger than wild-type clones and show evidence of eye precursor overproliferation. Accordingly, adult eyes of CtBP transheterozygous combinations or of CtBP^87De-10/M mosaic individuals have more ommatidia than wild-type eyes. In contrast, whereas CtBP^87De-10,dan,danr^ex56 triple mutant clones are similar to CtBP^87De-10 single mutant clones in being larger than wild-type clones ahead of the furrow, CtBP^87De-10,dan,danr^ex56 adults have small rough eyes. This suggests either that the CtBP^87De-10,dan,danr^ex56 triple mutant cells fail to be efficiently recruited into ommatidia and/or eventually undergo apoptosis. In support of the former hypothesis, the phenotypic analysis demonstrates that recruitment of the R8 photoreceptor, which is required to recruit all other ommatidial cells, is affected by loss of CtBP, and is more strongly affected in the CtBP,dan,danr triple mutant (Hoang, 2010).

Given the dynamic and overlapping expression patterns of the retinal determination factors, one intriguing hypothesis that fits the data is that a complex containing CtBP may have different members at different stages of eye development. For instance, a complex containing CtBP, Ey as well as possibly Tsh and Hth anterior to the furrow, might participate in maintaining a 'poised' chromatin structure with respect to eye specific genes, in which genes involved in eye differentiation are not yet expressed and the cells are kept in a proliferative state. It has been suggested that vertebrate Pax6 is a 'pioneering' factor for the lens lineage, and other 'pioneering' factors have been shown to promote a 'bivalent' state in which developmental genes are silenced, but 'poised' for activation. The down-regulation of Ey close to the furrow, and the initiation of Dan, Danr and Dac expression in the same region would be expected to change the composition of the complex containing CtBP and lead to changes in transcription that reflect the transition from proliferation to differentiation (Hoang, 2010).

At present it is not known what genes might be direct targets of the complexes containing CtBP and the eye specification factors. Some possibilities include the cell cycle regulator string, and the pre-proneural gene atonal, which is known to be regulated by Ey, So, Dan and Danr (Ey and So are direct regulators), and which plays a critical role in the transition from proliferation to differentiation of eye precursors. Thus, future work on Drosophila CtBP will shed light on the functions of this important transcriptional regulator, as well as on important transitions during development (Hoang, 2010).

Long- and short-range transcriptional repressors induce distinct chromatin states on repressed genes

Transcriptional repression is essential for establishing precise patterns of gene expression during development. Repressors governing early Drosophila segmentation can be classified as short- or long-range factors based on their ranges of action, acting either locally to quench adjacent activators or broadly to silence an entire locus. Paradoxically, these repressors recruit common corepressors, Groucho and CtBP, despite their different ranges of repression. To reveal the mechanisms underlying these two distinct modes of repression, chromatin analysis was performed using the prototypical long-range repressor Hairy and the short-range repressor Knirps. Chromatin immunoprecipitation and micrococcal nuclease mapping studies reveal that Knirps causes local changes of histone density and acetylation, and the inhibition of activator recruitment, without affecting the recruitment of basal transcriptional machinery. In contrast, Hairy induces widespread histone deacetylation and inhibits the recruitment of basal machinery without inducing chromatin compaction. This study provides detailed mechanistic insight into short- and long-range repression on selected endogenous target genes and suggests that the transcriptional corepressors can be differentially deployed to mediate chromatin changes in a context-dependent manner (Li, 2011).

To directly compare functional aspects of Hairy- and Knirps- mediated repression in the Drosophila embryo, these proteins’ interactions were studied with two segmentally expressed pair-rule genes. Hairy directly represses fushi tarazu (ftz), a secondary pair-rule gene expressed in the blastoderm embryo in a seven-stripe pattern. ftz is regulated by both regionally acting gap genes and the segmentally expressed hairy pair-rule gene. Chromatin immunoprecipitation (ChIP) experiments have revealed dense clusters of peaks around the ftz gene for key transcription factors active in the blastoderm embryo, including Caudal, Hunchback, Knirps, Giant, Huckebein, Krüppel, and Tailless. These transcription factors bind to the promoter-proximal Zebra element, the stripe 1+5 enhancer located 3' of ftz, and a presumptive 5' regulatory region located between 23 kbp and 28 kbp. Hairy has been found to bind in vivo to all of these regions. This repressor is expressed in a striped pattern in the blastoderm embryo; therefore, the ftz gene is active in some nuclei and repressed in others. In order to obtain a homogeneous population of nuclei for chromatin studies, Hairy protein was overexpressed in embryos using a heat-shock driver, which results in complete repression of ftz. This repression requires the recruitment of the Groucho corepressor, because a mutant version of Hairy that does not bind to Groucho fails to repress ftz (Li, 2011).

Interestingly, a titration of heat-shock induction resulted in a nonuniform, progressive loss of specific ftz stripes, with stripe 4 being the most sensitive and stripe 1+5 the least. This result points to the intriguing possibility that Hairy can act locally on specific enhancers, at least very transiently, although the end result of Hairy repression is complete silencing of all enhancer elements. The asynchronous repression of the ftz locus also suggests that Hairy-mediated long-range repression does not act solely by direct targeting the basal promoter, as suggested by a previous model for this class of repressor, because this mechanism should cause uniform inhibition of stripe elements. Similar to ftz, the pair-rule gene even skipped (eve) is also expressed in a seven-stripe pattern and is regulated by multiple modular enhancers. eve is a well-characterized target of the short-range repressor Knirps, which sets posterior boundaries of eve stripe 3 and 4 and anterior borders of eve stripe 6 and 7. After substantial overexpression of Knirps (20 min heat-shock induction), the repressor is able to repress all of the eve stripe enhancers except for the stripe 5 enhancer. When the induction is titrated, Knirps represses individual enhancers in a stepwise manner, with the most sensitive enhancers downregulated earliest, at a low dose of Knirps. Together, these experiments indicate that Hairy can initially act locally but ultimately acts in a globally dominant fashion, whereas Knirps acts in a restricted manner (Li, 2011).

To compare the effects of repression by Hairy and Knirps, chromatin changes associated with repression of ftz and eve were studied via ChIP. No significant change of histone H3 occupancy were detected at regions sampled throughout the ftz locus after Hairy overexpression (although some regions showed modest differences. In contrast, Knirps repression of eve resulted in significantly increased histone H3 density, particularly in two of the three regions corresponding to the Knirps-sensitive enhancers, namely stripe 4+6 and stripe 2. Little change was noted in the promoter region, transcribed region, or the stripe 1 and 5 enhancers, which are not readily repressed by Knirps. An apparent increase in histone H3 density on the repressed stripe 3+7 enhancer, although of low statistical significance, correlates with other alterations common to repressed enhancers, noted below (Li, 2011).

To provide a more detailed picture of chromatin structure, a micrococcal nuclease (MNase) mapping protocol used in yeast and cultured cells was adapted for Drosophila embryos. MNase mapping showed that Hairy repression had little effect on chromatin accessibility throughout the ftz locus, whereas Knirps induced a significant increase in MNase insensitivity specifically at the eve stripe 3+7, 2, and 4+6 enhancers and a minor increase in stripe 1 protection. The promoter and the eve stripe 5 enhancer were little changed, mirroring the patterns noted for overall histone H3 occupancy. The changes noted for the eve locus appear to be specific, because Knirps did not induce any change of a nontargeted intergenic site on the third chromosome. Hairy also had no effect at this locus. The similar results from overall histone H3 density and MNase mapping suggest that Hairy-mediated long-range repression does not involve a general compaction of chromatin on the ftz locus. In contrast, repression by Knirps is associated with an increase in the histone density of targeted enhancer regions, which may result either from Knirps recruitment of factors that mediate chromatin condensation or the blocking of proteins responsible for loosening of chromatin. Recruitment of Groucho by other repressor proteins is also associated with distinct effects: Runt-dependent repression of slp1 does not involve changes in H3 density, but Brinker repression of the vgQ enhancer does. The distance dependence of these repressors has not been established, but in light of the current results, it is apparent that the Groucho corepressor can be involved in distinct effects depending on the context of recruitment (Li, 2011).

Histone acetylation is dynamically regulated on transcribed genes in eukaryotes, with histone acetylation generally correlated with active loci. The histone deacetylase Rpd3 is a component of both Hairy and Knirps corepressor complexes; therefore, histone acetylation levels were assayed across the eve and ftz genes before and after repression. Hairy repression resulted in widespread histone H4 deacetylation throughout the ftz locus. The ectopically expressed Hairy protein itself was not observed to spread but remained restricted to regions of the gene previously observed to bind endogenous Hairy. Using anti-H3-acetylation antibodies, similar widespread H3 deacetylation was also noted. This distributed effect on the ftz locus correlates with prior observations that Hairy-mediated long-range repression might involve a Groucho-mediated 'spreading' mechanism. By this means, Rpd3 may be delivered to extensive areas of a gene. To test whether a spreading of histone deacetylation might correlate with the successive inhibition of ftz enhancers, histone acetylation levels were investigated across ftz after a brief 5 min heat shock followed by immediate fixing, before the entire complement of enhancers can be repressed. In this setting, deacetylation was mostly concentrated around the stripe 1+5 enhancer and the immediate 5' regulatory region, areas that show Hairy occupancy in vivo. More distal 5' regulatory regions and the transcription unit itself showed little initial change, consistent with a spreading action of this repressor during the more extensive repression period (Li, 2011).

A different picture emerged from studies of Knirps acting on eve. Here, repression led to selective decreases in H3 and H4 acetylation levels, concentrated over the eve stripe 4+6 and stripe 2 enhancers, with lesser decreases noted at stripe 3+7 and stripe 1 enhancers. A local change in acetylation was also noted near the transcriptional initiation site, but not immediately 5' and 3' of this area. The reductions in histone acetylation levels seen on both eve and ftz are consistent with Hairy and Knirps recruiting deacetylases to their target genes. However, it is striking that the broad deacetylation mediated by Hairy on ftz is not associated with dramatic changes in histone density or resistance to nuclease accessibility, whereas increased histone density and resistance to nuclease digestion are associated with Knirps repression on eve. It is possible that in addition to inducing deacetylation, Knirps triggers additional histone modifications or interacts with nucleosome-remodeling complexes to further alter chromatin at the enhancers. H3 lysine 27 methylation is one chromatin signature associated with silenced genes; however, no significant change in this modification was noted at ftz or eve upon repression (Li, 2011).

Previous studies indicated that Hairy can effectively repress a reporter gene without displacing the activators. Attempts were made to test whether this was the case on an endogenous gene, ftz, by examining occupancy by Caudal, a transcription factor that also activates eve. Caudal activates the posterior stripes of both ftz and eve, and it was found that Caudal binds the ftz 5' regulatory region and the promoter-proximal Zebra element. Repression of the locus by Hairy did not affect the Caudal binding pattern, similar to the results obtained with a Hairy-regulated reporter gene. In contrast, Knirps repression decreased Caudal occupancy specifically at the eve 3+7 and 4+6 enhancers, bringing overall protein occupancy down to near baseline levels. This decrease is not an effect of global decrease of Caudal occupancy, because the Caudal binding peak at the eve promoter was not affected. A similar decrease in Caudal occupancy was also observed on a hunchback enhancer after repression by Knirps. Interestingly, Bicoid occupancy of the eve stripe 2 and stripe 1 enhancers was not altered by Knirps, although these enhancers were repressed. Clearly, loss of transcription factor occupancy is not required for short-range repression of a cis-regulatory element. It is possible that different transcriptional activators exhibit differential sensitivity to chromatin changes induced during repression (Li, 2011).

New insights have suggested that many developmental genes, including those regulated by short-range repressors such as Snail, feature RNA polymerase paused in the promoter region even in their inactive state, suggesting postrecruitment levels of regulation. Components of the core machinery were analyzed before and after repression by Hairy and Knirps. Upon Hairy repression, a marked decrease of RNA polymerase II (Pol II) occupancy was observed at the ftz locus. The same trend was observed for the preinitiation, initiation, and elongation forms of Pol II. These results suggest that Hairy directly or indirectly blocks recruitment of Pol II. Similar decreases were noted with levels of TATA box-binding protein (TBP) at the promoter (Li, 2011).

In contrast, induction of Knirps did not change Pol II occupancy at the eve transcription unit, even under condition where most enhancers were repressed. (Under conditions tested in this study, over three-quarters of the embryos had shut down expression of all but stripe 1 and/or 5.) Similarly, TBP occupancy remained at a comparable level before and after Knirps repression. The constant level of RNA polymerase on the eve transcription unit was a surprise in light of the sharp reduction in mRNA production as measured by in situ hybridization. However, there is precedence for this effect: Runt repression of slp1 appears to act through elongation control, which causes no change of the concentration of Pol II on slp1. Knirps may produce a similar effect by inducing a slower transit rate of Pol II on the repressed eve locus. Similar observations have been made at the hsp70 gene upon depletion of elongation factors such as Spt6 or Paf1 (Li, 2011).

The differential distance dependence of short- and long- range repressors such as Hairy and Knirps has been observed in many contexts. However, the mechanisms by which these proteins function have been poorly understood. With the recent demonstration that transcriptional factors considered to be short- and long-range repressors utilize shared cofactors, namely CtBP and Groucho, there has been a question of whether long-range repression is actually functionally distinct from short-range repression (Payankaulam, 2009). The current study provides evidence that the chromatin states associated with long- and short-range repressors are distinct in several ways. It is not yet knowm whether the effects seen on ftz are observed for all Hairy targets, although the similarity of changes observed on the lacZ reporter subject to Hairy repression suggests that they are conserved (Martinez, 2008). Similarly, the reproducibility of Knirps-induced changes at different eve enhancers indicates that this protein can effect related chromatin changes on cis-regulatory modules bound by different activators. Snail, another short-range repressor, also appears to mediate localized deacetylation and activator displacement; thus, this mechanism may be a common feature of this entire class of repressors (Qi, 2009; Y. Nibu, personal communication to Li, 2011). It will be interesting to determine how general are the observations made in this study for long- and short-range repression, a question that can be approached using genome-wide methods. In any event, the highly divergent activities of Knirps and Hairy demonstrated in this study not only underscore the fact that these proteins can mediate biochemically divergent events but also raise interesting questions about how similar cofactors can participate in such distinct effects in a context-dependent manner. It is possible that the corepressors adopt distinct conformations when recruited by different repressors, or the corepressor may form distinct complexes with unique activities. In addition to determining how cis- and trans-acting factors affect repression pathways, these mechanistic insights will provide important contextual information for interpretation of genome-wide transcription factor binding and chromatin modifications and will inform quantitative modeling of cis-regulatory elements for the aim of understanding the activity and evolution of enhancers (Li, 2011).

CoREST acts as a positive regulator of Notch signaling in the follicle cells of Drosophila melanogaster

The Notch signaling pathway plays important roles in a variety of developmental events. The context-dependent activities of positive and negative modulators dramatically increase the diversity of cellular responses to Notch signaling. In a screen for mutations affecting the Drosophila follicular epithelium, a mutation was isolated in CoREST that disrupts the Notch-dependent mitotic-to-endocycle switch of follicle cells at stage 6 of oogenesis. Drosophila CoREST positively regulates Notch signaling, acting downstream of the proteolytic cleavage of Notch but upstream of Hindsight activity; the Hindsight gene is a Notch target that coordinates responses in the follicle cells. CoREST genetically interacts with components of the Notch repressor complex, Hairless, C-terminal Binding Protein and Groucho. In addition, it was demonstrated that levels of H3K27me3 and H4K16 acetylation are dramatically increased in CoREST mutant follicle cells. The data indicate that CoREST acts as a positive modulator of the Notch pathway in the follicular epithelium as well as in wing tissue, and suggests a previously unidentified role for CoREST in the regulation of Notch signaling. Given its high degree of conservation among species, CoREST probably also functions as a regulator of Notch-dependent cellular events in other organisms (Domanitskaya, 2012).

The highly conserved Notch signaling pathway plays a crucial role in a broad array of developmental events, including the maintenance of stem cells, cell fate specification, control of proliferation and apoptosis. Misregulation of the Notch pathway is associated with a number of diseases, including different types of cancer. The binding of the transmembrane ligands DSL (Delta, Serrate, LAG-2) to the extracellular domain of Notch, exposed on a neighboring cell, activates the signaling cascade by triggering a sequence of proteolytic cleavages of Notch protein. Extracellular cleavage (S2) leads to the formation of an intermediate membrane-bound C-terminal fragment of Notch, called NEXT. This event is followed by an intramembranous cleavage (S3) by the γ-secretase complex. The intracellular domain of Notch (NICD) then translocates to the nucleus and binds to a transcription factor of the CSL family [CBF-1, Su(H), LAG-1], converting it from a transcriptional repressor to an activator. In the canonical Notch pathway, Su(H) directly activates Notch target genes in response to signaling. Despite the relative simplicity of the Notch transduction pathway, the presence of a large number of proteins that positively or negatively influence Notch signaling dramatically increases the complexity of the Notch pathway and its cellular responses. For instance, extracellular modulators, such as Fringe, alter ligand-specific Notch activation, whereas cytoplasmic modulators, such as Numb, restrict signal transduction. Nuclear modulators, for instance Mastermind, influence the transcriptional activity of the NICD-containing complex. In addition, there is increasing evidence of the importance of the epigenetic regulation of Notch targets, which can cause differential cellular responses upon Notch activation (Domanitskaya, 2012).

Drosophila serves as an excellent model system to dissect the regulation of the Notch pathway. The Drosophila genome contains only a single Notch protein and two ligands [Delta (Dl) and Serrate (Ser)]. The Notch pathway is involved in several aspects of Drosophila development. The role of Notch in lateral inhibition during neurogenesis has been extensively studied; it restricts neural cell fates in the embryo, and leads to restriction of sensory-organ formation and induction of boundary formation in the wing discs. Notch activity is also required for many aspects of oogenesis, such as the establishment of egg chamber polarity, polar cell formation, control of follicle cell (FC) proliferation, differentiation, cell fate specification and morphogenesis. The Drosophila FCs are somatically derived epithelial cells that form a monolayer covering the germline cells during oogenesis. FCs divide mitotically from stage 2 to stage 6 of oogenesis, followed by the switch from the mitotic cycle to the endocycle (the M/E transition). Endocycles take place from stage 7 to stage 10A of oogenesis and include three rounds of DNA duplication without subsequent cell division. The M/E switch is triggered upon Notch pathway activation. Dl produced in the germline binds to its receptor Notch, expressed in the FCs, and induces activation of the canonical Notch signaling pathway. Removal of Dl from germline cells, or of Notch from FCs, maintains follicle cells in the mitotic cycle throughout oogenesis. NICD complexed with Su(H) activates transcription of downstream target genes required for the M/E switch, such as Hindsight (Hnt). Hnt then mediates the Notch-dependent downregulation of Cut, String (Stg) and Hedgehog (Hh) signaling in the FCs, thus promoting the M/E switch (Domanitskaya, 2012).

This study describes the identification of the transcriptional cofactor Corepressor for element-1-silencing transcription factor (CoREST) as a positive modulator of Notch signaling in the FCs and during wing development. CoREST is required for the promotion of the M/E switch during oogenesis. CoREST acts downstream of NICD release but upstream of Hnt activity, and it is a previously unidentified modulator of the Notch pathway. The genetic interactions between CoREST and Hairless (H), CtBP and Groucho (Gro), members of the Notch repressor complex, suggest that CoREST might influence the activity of either Notch transcriptional repressor or activator complexes. In addition, CoREST specifically affects tri-methylation of lysine 27 of histone 3 (H3K27) and acetylation of H4K16 in FCs, because these chromatin modifications show elevated levels in the CoREST mutant cells. These findings point to a possible role of CoREST in regulation of the activity of the Notch repressor-activator complexes and/or epigenetic regulation of the components of the repressor-activator complexes or of factors involved in the transduction of the signaling or directly of target genes of the Notch signaling pathway (Domanitskaya, 2012).

Initially, CoREST was identified in humans as a corepressor with REST (RE1 silencing transcription factor) in mediating repression of the proneuronal genes, and thus as an important factor in the establishment of non-neural cell specificity. Subsequently, CoREST was identified in a variety of vertebrate and invertebrate species, and was shown to play a functionally conserved role in neurogenesis. Recent studies show that CoREST regulates a very broad range of genes by both REST-dependent and REST-independent means, including genes encoding members of key neural developmental signaling pathways, such as BMP, SHH, Notch, RA, FGF, EGF and WNT. Analysis of CoREST downstream target genes and their developmental expression profiles suggested that the liberation of CoREST from gene promoters is associated with both gene repression and activation depending on the cell context. In the work reported in this study, a lethal allele of Drosophila CoREST was isolated, and the contribution of CoREST to the development of FCs, a process that involves cell proliferation and differentiation, was analyzed. This study has implicated CoRESTin the regulation of Notch signaling, and acts as a positive modulator of the Notch pathway in Drosophila FCs (Domanitskaya, 2012).

This study has identified a role for CoREST in the Notch-mediated regulation of the M/E switch during stage 6 of oogenesis. Loss of CoREST activity in FCs primarily disrupts the Notch signaling pathway. We further demonstrated that CoREST regulates the Notch pathway downstream of NICD release and upstream of Hnt. The misexpression of Hnt in the CoREST mutant clones rescues the failure in the M/E switch. Furthermore, the role of CoREST in Notch pathway regulation is not restricted to FCs: CoREST also interacts with Notch during wing development. Interestingly, CoREST was identified as a negative modulator of Notch signaling in Caenorhabditis elegans in a genetic screen for suppressors of the developmental defects in sel-12 presenilin mutants. Presenilin is a component of the γ-secretase complex that performs the S3 cleavage of Notch. Mutations in spr-1, the C. elegans homolog of CoREST, suppress the developmental defects observed in sel-12 animals by derepressing the transcription of the other functionally redundant presenilin gene, hop-1. Therefore, CoREST acts as a negative regulator of the γ-secretase complex in C. elegans, and hence proteolytic cleavage of Notch and release of NICD. By contrast, Drosophila CoREST does not affect the processing of the Notch receptor in the follicle cells, and instead acts as a positive modulator of the Notch pathway functioning downstream of NICD release (Domanitskaya, 2012).

CoREST plays transcriptional and epigenetic regulatory roles: it can promote gene activation in addition to repression, as well as being able to modify the epigenetic status of target gene loci distinct from its effects on transcription. Several possible scenarios of how CoREST could be involved in the regulation of Notch signaling are discussed, based on the previous knowledge about CoREST and considering the current data (Domanitskaya, 2012).

hnt, the downstream target gene of Notch signaling in FCs, fails to be properly upregulated upon Notch activation in the CoREST mutant cells. CoREST might therefore act as a transcriptional repressor for an unknown factor, which is in turn involved in the transcriptional repression of hnt. Alternatively, CoREST could be directly involved in the transcriptional regulation of hnt and act as an activator. hnt was shown to be a putative direct target of Notch signaling in DmD8 cells from the analysis of genes for which mRNA levels increase within 30 minutes of Notch activation, and which contain regions occupied by Su(H). If hnt is a direct target of Notch in FCs, its transcription would be regulated by the balance between Notch repressor and activator complexes, and CoREST might be involved in the regulation of stability or activity of either of these. Interestingly, CoREST was shown to interact with CtBP1 in mammals (Kuppuswamy, 2008), and to bind to the SIRT1-LSD1-CtBP1 complex, which is required for the repression of certain Notch target genes (Mulligan, 2011). Thus, Drosophila CoREST might similarly directly bind to the repressor complex containing CtBP and modify its activity or destabilize it. However, CoREST could be involved in the transcriptional regulation of the components of Notch repressor or activator complexes. In this scenario, in CoREST mutant FCs, upregulation of negative regulator(s) would lead to greater activity of negative than positive regulators, resulting in disruption of Notch signaling. Both suggested models of the direct and indirect transcriptional role of CoREST are consistent with the current results, given that the CoREST mutant phenotype could be suppressed by removal of one copy of H, CtBP or Gro, components of the Notch repressor complex (Domanitskaya, 2012).

More recently, epigenetic mechanisms have emerged as an important interface regulating context-dependent and stage-specific gene regulation. Mammalian CoREST acts as a scaffold for recruitment of transcriptional regulators such as REST, and epigenetic factors such as the enzymes HDAC1, HDAC2 and LSD1. In Drosophila, using two-hybrid interaction, CoREST was also shown to interact with Su(VAR)3-3 (Drosophila homolog of LSD1) and Rpd3 (HDAC1). This study has shown that the levels of H3K27me3 and H4K16 acetylation are significantly and specifically increased in the CoREST mutant FCs. Recently, the H3K27me3 demethylase UTX was shown to act as a suppressor of Notch- and Rb-dependent tumors in Drosophila eyes, and in addition to increased level of H3K27me3 staining, an excessive activation of Notch was detected in Utx mutant eye discs. The observation of increased levels of H3K27me3 coupled to cell overproliferation and modified Notch signaling in both of these cases suggests that the increased H3K27me3 results in epigenetic regulation of genes involved in Notch signaling and/or of Notch target genes. However, in the eye tumor system, this increase in H3K27me3 promotes Notch signaling, whereas in the follicle cells, it reduces Notch signaling. This indicates a strong context-dependent effect on Notch signaling by certain chromatin modifications. Thus, these chromatin modifications might be involved in cell-context-dependent Notch target gene silencing and/or activation. Interestingly, many Notch-regulated genes are highly enriched in a characteristic chromatin modification pattern, termed a bivalent domain, consisting of regions of H3K4me3, a marker for actively expressed genes, and H3K27me3, a marker for stably repressed genes; and Notch signaling could be involved in resolving these domains, leading to gene expression (Schwanbeck, 2011). Therefore, the increased level of H3K27me3 in CoREST mutant FCs might lead to a repression of certain Notch target genes, for instance hnt (Domanitskaya, 2012).

To further understand the function of the Drosophila CoREST in Notch pathway regulation, identification of other CoREST essential and specific binding partners would be useful. One previously identified partner for CoREST is Chn (Tsuda, 2006). Given that wild-type expression of Hnt and Cut was observed in chn mutant cells, this factor does not appear to partner CoREST in regulation of Notch signaling in FCs. Using yeast two-hybrid analyses and an embryonic cDNA fusion protein library, it was shown that all three splice variants of Drosophila CoREST interact with the unique C-terminus of Tramtrack88 (Ttk88), a known repressor without homology to REST. In addition, a Ttk69 splice variant can form a complex with CoREST and Ttk88. However, Ttk88 was not detected in the ovary by immunofluorescence or western blot analysis, and disruption of Ttk88 does not have any impact on oogenesis. Conversely, Ttk69 is steadily expressed in FCs before stage 10 and it is required for the M/E transition. However, in contrast to CoREST, which acts upstream of Hnt, Hnt expression is not affected in ttk^1e11 mutant FCs, indicating a role of Ttk69 downstream of Hnt in the control of the M/E switch. Additionally, Ttk69 is not required for cell differentiation, as expression of FasIII, a cell fate marker for immature follicle cells, is normal in ttk^1e11 mutant FCs. From these important phenotypic differences between Ttk69, Ttk88 and CoREST, it appears that CoREST plays a Ttk-independent role in Notch pathway regulation in the FCs. Future work to identify transcription regulators that act as binding partners of CoREST will help in determining the precise biochemical role of CoREST in modulating Notch signaling (Domanitskaya, 2012).

These results demonstrate an unexpected role for CoREST in positively regulating Notch signaling. The effect of the loss of CoREST is particularly strong in the PFCs and relatively mild in the lateral and anterior follicle cells. This implies that CoREST is crucially required in cells that are more sensitive to loss of Notch signaling. The difference between the PFCs and the other follicle cells is established at approximately stages 6-7 of oogenesis by EGF receptor activation in response to Gurken produced by the oocyte. EGF signaling, therefore, is active around the same time as the Notch pathway and hence it is probable that downstream effector(s) of EGFR signaling result in the increased sensitivity of PFCs to the loss of CoREST. In the model of CoREST negatively affecting a repressor of Notch signaling, EGFR signaling would be expected to act positively to enhance expression and/or activity of a Notch repressor. Thus, loss of CoREST from the PFCs would occur in a cell type where repressor activity is already augmented, which would explain the observation of differential loss of Notch signaling in the PFCs (Domanitskaya, 2012).

In summary this study has shown that CoREST, a component of transcriptional repressor complexes, acts positively in Notch signaling in the ovarian follicle cells of Drosophila. The results also show that different cell types are differentially sensitive to loss of this repressor. Future identification of partners and targets of CoREST in the follicle cells should further elucidate how activity of EGFR and other signaling pathways are integrated in this process (Domanitskaya, 2012).

Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types

Transcriptional repressors function primarily by recruiting co-repressors, which are accessory proteins that antagonize transcription by modifying chromatin structure. Although a repressor could function by recruiting just a single co-repressor, many can recruit more than one, with Drosophila Brinker (Brk) recruiting the co-repressors CtBP and Groucho (Gro), in addition to possessing a third repression domain, 3R. Previous studies indicated that Gro is sufficient for Brk to repress targets in the wing, questioning why it should need to recruit CtBP, a short-range co-repressor, when Gro is known to be able to function over longer distances. To resolve this, genomic engineering was used to generate a series of brk mutants that are unable to recruit Gro, CtBP and/or have 3R deleted. These reveal that although the recruitment of Gro is necessary and can be sufficient for Brk to make an almost morphologically wild-type fly, it is insufficient during oogenesis, where Brk must utilize CtBP and 3R to pattern the egg shell appropriately. Gro insufficiency during oogenesis can be explained by its downregulation in Brk-expressing cells through phosphorylation downstream of EGFR signaling (Upadhyai, 2013).

A structure/function analysis of the transcriptional repressor Brk has been performed by replacing the endogenous brk gene with a ΦC31 bacteriophage attP site into which mutant forms of brk were introduced by integrase-mediated transgenesis. The goal was to generate mutations that disrupted the ability of Brk to recruit the CoRs Gro and CtBP and/or that deleted the less well characterized 3R repression domain and to test their activity in different tissues at different times of development to determine if and why they are required by Brk to repress transcription. Previous studies with Brk and other TFs that can recruit both CoRs indicated that Gro recruitment is essential for at least some of the activities of these TFs, but the reason for recruiting CtBP has proven more elusive. This study has confirmed that Gro recruitment is essential for Brk activity, but have also showed that Brk needs to recruit CtBP and to possess the 3R domain for full activity in some tissues, in particular during oogenesis (Upadhyai, 2013).

Lethality of the brk^GM mutant reveals Gro recruitment is necessary for Brk activity. The brk^Δ3RCM mutant, which utilizes Gro as its sole repressive activity, can progress from fertilization to an almost morphologically wild-type adult, indicating that Gro is close to sufficiency in this regard. However, brk^Δ3RCM mutants often die as embryos and show defective oogenesis, with eggs having aberrant egg shell pattering, a characteristic of brk null mutants. The single mutants, brk^Δ3R and brk^CM, show less severe egg shell defects and reduced fertility, the latter probably relating to a defective micropyle, the structure through which sperm normally enter. The apparent inactivity of Brk^Δ3RCM protein in follicle cells appears to be explained by active, unphosphorylated Gro being reduced there. The egg shell is patterned by the surrounding follicle cells, where Brk is expressed at high levels in the dorsal anterior. This coincides with high levels of EGFR signaling and previous studies have shown that Gro activity is attenuated following phosphorylation by MAPK downstream of EGFR signaling. As expected, lower levels of unphosphorylated or active Gro were found in the dorsal-anterior follicle cells. Consistent with the activity of Brk^Δ3RCM being compromised by EGFR-dependent downregulation of Gro activity, upregulation of EGFR signaling in the wing disc of brk^CM mutants results in derepression of the targets salE1 and ombZ (Upadhyai, 2013).

EGFR signaling also probably reduces the levels of active Gro available for Brk in other tissues, including the ventral ectoderm where Brk activity is required to ensure proper patterning of the denticle belts and where EGFR signaling is known play a key role. Many brk^Δ3RCM mutants do not survive embryogenesis and demonstrate defects in denticle patterning similar to, but weaker than, those of null mutants. In addition, the VDB phenotype of brk^GM mutants is less severe than in brk^KO or brk^3M mutants. Thus, CtBP and 3R appear to provide repressive activity in the ventral ectoderm (Upadhyai, 2013).

No Brk targets have been characterized in the follicle cells, but these would be expected to be partially derepressed in both brk^CM and brk^Δ3R mutants and possibly completely derepressed in brk^Δ3RCM mutants based on the egg shell phenotypes, although there might be some differences between brk^CM and brk^Δ3R given the differences between CtBP and 3R just discussed. However, again, this would not imply that these targets are CtBP/3R specific, because the inability of Gro to participate in their repression is presumed to be due to its unavailability. Thus, although studies have indicated that TFs that have the ability to recruit both Gro and CtBP may only recruit one or other at specific targets, this might not reflect a CoR specificity for individual targets, but rather a cell-specific availability of CoRs (Upadhyai, 2013).

It is possible that if Gro were available in all cells then the CiM and 3R domain would be dispensable and so, at least for Brk, downregulation of Gro by MAPK phosphorylation could be considered inconvenient. This might be true for other TFs, including Hairy, Hairless and Knirps, which also function in multiple tissues, many of which are exposed to RTK signaling, and might explain why these TFs need to resort to recruiting CtBP as well as Gro. It should also be noted that Gro activity can be downregulated in other ways, including phosphorylation by Homeodomain-interacting protein kinase. This downregulation of Gro activity has been explained in terms of reducing the activity of specific repressors in specific tissues, such as E(Spl) factors during wing vein formation. This appears to be a somewhat illogical way to downregulate the activity of specific repressors, as there are almost certainly many other TFs utilizing Gro in the same cells and in other tissues exposed to RTK signaling and their activity might be compromised. There are no data indicating whether the downregulation of Gro activity in follicle cells serves any purpose and could simply be a consequence of the decision to downregulate Gro activity by this means in other tissues. However, this has serious implications for Brk and has required Brk to be versatile in its mechanisms of repression. Of course, the possibility has not been ruled out that downregulation of Gro activity does serve a purpose for Brk in follicle cells; for example, if Gro were available here it might provide Brk with too much activity or allow it to inappropriately repress a target that CtBP or 3R cannot. This might be tested by assessing egg shell phenotypes after driving unphosphorylatable Gro at physiological levels in a brk^Δ3RCM mutant, but currently this is technically challenging (Upadhyai, 2013).

The idea that repressors need to be versatile in their repressive mechanisms because of variable CoR availability presumably extends beyond Brk and Hairless, Hairy and Knirps. In fact, other repressors in Drosophila possess both CtBP- and Gro-interaction motifs, including Snail. This might not be simply related to downregulation of Gro activity, as CtBP activity can also be modulated; for example, SUMOylation and acetylation of mammalian CtBPs is implicated in regulating their nuclear localization. In addition, other CoRs might similarly be available only in some cells; MAPK activity has been shown to phosphorylate and lead to the nuclear export and inactivation of the SMRT CoR complex. Finally, a further consideration raised by the present study is that care should be taken in assuming that a TF requires and can use a specific CoR to repress its targets in a particular tissue simply because it possesses an interaction motif for that CoR (Upadhyai, 2013).

Escargot restricts niche cell to stem cell conversion in the Drosophila testis

Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. Esg-interacting proteins were identified, and an interaction was demonstrated between Esg and the corepressor C-terminal binding protein (CtBP), which is also required for maintenance of hub cell fate. These results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo (Voog, 2014)

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing

Trithorax (Trx) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these Trx functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. This study report that the SET domains of Trx and Trx-related (Trr) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of Trx and Tyr2404 of Trr prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3 H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by Trx-dependent H3K27 acetylation by CREB-binding protein (CBP). Perturbation of Polycomb silencing by Trx overexpression requires CBP. It was also shown that Trx and Trr are each physically associated with CBP in vivo, that Trx binds directly to the CBP KIX domain, and that the chromatin binding patterns of Trx and Trr are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of Trx and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory (Tie, 2014).

The major findings presented in this study are: (1) TRX and TRR are monomethyltransferases and together account for the bulk of the H3K4me1 in vivo; (2) the catalytic activities of both TRX and CBP are required to antagonize PcG silencing; (3) TRX and TRR are physically associated with CBP in vivo and TRX binds directly to the CBP KIX domain via a region that contains multiple KIX-binding motifs; (4) TRX and TRR colocalize genome-wide with H3K4me1 and CBP at PREs and enhancers; and (5) H3K4me1 enhances histone acetylation by CBP. Together, these data suggest that the primary target of TRX monomethyltransferase activity is not promoters but PREs and neighboring enhancers. They suggest a new model for how TRX antagonizes Polycomb silencing, stimulates active enhancers, and establishes a cellular memory of active transcription. This differs significantly from the previous view that TRX trimethylates H3K4 (Tie, 2014).

The evidence presented in this study indicates that the SET domains of TRX, TRR and their human orthologs possess intrinsic H3K4 monomethyltransferase activities and are prevented from being trimethyltransferases by the presence of the bulkier Tyr residue at their respective F/Y switch positions, as previously shown for MLL1. Although these data do not rule out the possibility that TRX and TRR complexes might have some H3K4 trimethylation activity in vivo in some chromatin contexts, the reduced H3K4me1 and apparently normal H3K4me3 levels in the catalytically inactive trx^Z11 and trr³ mutants strongly suggest that H3K4 monomethylation is the predominant activity of TRX and TRR in vivo. Moreover, the absence of detectable H3K4me1 in trr³; trx^Z11 double-mutant embryos suggests that they are the principal H3K4 monomethyltransferases in vivo, consistent with their genome-wide colocalization with H3K4me1 at PREs and enhancers. Suppression of Pc³ mutant phenotypes by trx^Z11 further suggests that the monomethyltransferase activity of TRX plays a role in antagonizing Polycomb silencing (Tie, 2014).

This study found that TRX and TRR are physically associated with CBP in embryo extracts, confirming a previous report for TRX. The direct binding of TRX to the CBP KIX domain and the genome-wide correlation of H3K27ac with H3K4me1 on active genes suggests that their activities are coupled in vivo. Consistent with this, TRX-CBP complexes pulled down from embryo extracts have both H3K4 monomethyltransferase and H3K27 acetyltransferase activities. Moreover, the impaired Polycomb silencing caused by TRX overexpression in vivo (which elevates both H3K4me1 and H3K27ac levels) requires CBP and presumably the TRX-CBP interaction. Mutating the CID will be required to show this conclusively. No direct interaction between CBP and the TRR C-terminus was found, but it has been previously reported that CBP interacts directly with the H3K27 demethylase UTX, which is another subunit of the TRR complex. Together, these data suggest that these direct interactions are required for TRX- and TRR-dependent H3K27 acetylation and further suggest that TRX and TRR complexes function by fundamentally similar mechanisms (Tie, 2014).

The enhanced in vitro acetylation of H3K27 on K4me1-containing recombinant H3 substrates suggests that H3K4me1 might be a preferred CBP substrate in vivo. Consistent with this, altering the H3K4me1 level in vivo by manipulating LSD1 causes concordant changes in H3K27ac in adults. Moreover, a genome-wide analysis of hundreds of bona fide enhancers in purified mesodermal cells from Drosophila embryos revealed that H3K27ac is not present on enhancers without H3K4me1, whereas H3K4me1 is present without H3K27ac prior to enhancer 'activation'. This suggests that the presence of H3K4me1 might be a prerequisite for the deposition of H3K27ac at enhancers. Interestingly, some of the catalytically inactive trx^Z11 mutants survive until the late pupal period and exhibit strong homeotic transformations. This suggests that TRX catalytic activity might be more important for stimulating enhancers that drive robust homeotic gene expression, whereas the physical association of TRX with CBP, which is intact in trx^Z11, is more important for preventing silencing of normally active PcG-regulated genes in the embryo (Tie, 2014).

H3K4me1 and CBP are part of a conserved chromatin 'signature' of enhancers and H3K27ac marks 'active' enhancers. The data strongly suggest that TRX and TRR are responsible both for the H3K4me1 on enhancers and, via their physical association with CBP, for the H3K27ac on active enhancers. Determining which H3K4me1 is TRX dependent will require ChIP-seq analysis of trx^Z11 mutant cells (Tie, 2014).

Like TRX, H3K4me1 and CBP are also present at PRE/TREs of both active and inactive genes, suggesting that PRE/TREs have a functional connection to enhancers. Functional analyses of the strong bxd PRE/TRE in vivo suggest that PRE/TREs are distinct from enhancers, do not possess enhancer activity, but can boost enhancer-dependent transcription in a TRX-dependent manner. A GAL4-TRX fusion protein tethered to a transgene reporter exhibits these same properties (Tie, 2014).

TRR was recently shown to occupy many presumed enhancers. This study has found that TRR binds more sites than TRX and also co-occupies most TRX binding sites genome-wide, including PRE/TREs. This raises the possibility that both TRX and TRR regulate many PcG-regulated genes, perhaps in different contexts or in response to different signals. The presence of UTX in the TRR complex suggests that TRR can facilitate switching of PcG-regulated genes from silent to active, whereas TRX might only be capable of maintaining the expression of genes activated prior to the onset of Polycomb silencing in the early embryo, or genes subsequently derepressed by the UTX activity associated with the TRR complex. This might explain the previously reported critical requirement for TRX in early embryogenesis (0-4 hours; i.e. prior to the onset of Polycomb silencing) for later robust expression of the homeotic genes in imaginal discs. Absence of TRX in 0- to 4-hour embryos cannot be compensated by its subsequent restoration. Further investigation will be required to determine whether and in what contexts there is functional collaboration or division of labor between TRX and TRR (Tie, 2014).

Although it is required continuously, the critical early requirement for TRX might provide an important clue to its function. This suggests that TRX and CBP, bound to PRE/TREs, might be required for de novo 'priming' of surrounding enhancers with H3K4me1 and H3K27ac in the early embryo, prior to the onset of Polycomb silencing and perhaps even prior to transcriptional activation of the zygotic genome (Tie, 2014).

There is little detectable H3K27me3 in 0- to 4-hour embryos, whereas the H3K27ac level is already high relative to later embryonic stages. H3K4me1 is already present during syncytial stages. It is speculated that before zygotic genome activation, TRX and CBP are constitutively bound to PRE/TREs and deposition of H3K4me1 and H3K27ac might initially be restricted to nucleosomes adjacent to PRE/TREs. Binding of activators to early-acting enhancers promotes spreading of H3K4me1 and H3K27ac from PRE/TREs across adjacent cis-regulatory regions to form broad domains, perhaps facilitated by interactions between activators and TRX/CBP complexes. Spreading of H3K27ac initially proceeds unchecked by H3K27me3, encompassing all surrounding enhancers, including those that will be 'activated' later (e.g. the imaginal disc enhancers) and protects them from subsequent deposition of H3K27me3 by PRC2 at the onset of Polycomb silencing. PcG-regulated genes that are not activated in the early embryo become subject to deposition/spreading of H3K27me3 in similar broad domains, blocking subsequent H3K27 acetylation. There might also be some active removal of pre-existing H3K27ac by PRC1/PRC2-associated RPD3. Subsequent activation requires removal of H3K27me3 by UTX, and thus might require TRR, which is also present at PRE/TREs and so is poised to respond to the binding of TRR-dependent activators, such as EcR (Tie, 2014).

Other functions of H3K4me1 and H3K27ac at PREs and enhancers are not yet understood, but they might (1) recruit H3K4me1 and H3K27ac 'readers' that further stimulate/maintain the active transcriptional state, (2) facilitate the targeting of enhancers to promoters and (3) perpetuate the broad domains of H3K4me1 and H3K27ac by enhancing their own deposition by TRX and CBP, as suggested by the enhancing effect of H3K4me1 on H3K27 acetylation in vitro. Perpetuation of the broad domains of H3K4me1 and possibly H3K27ac through replication and mitosis could also constitute the elusive cellular memory of past transcriptional activity (Tie, 2014).

CtBP represses Dpp signaling as a dimer>

C-terminal binding protein (CtBP) is a highly conserved transcriptional co-repressor in animal development and human diseases. In Drosophila, CtBP is critical for fly development and is thought to exert its repressive roles in many signaling pathways including Dpp/BMP pathway. This study provides evidence that although wild type CtBP negatively and dominantly influences Dpp signaling in fly presumptive wings, mutant CtBP unable to form dimer does not, indicating that dimerization is required for the repression role of CtBP in Dpp signaling in vivo (Bi, 2018).

DEVELOPMENTAL BIOLOGY

Developmental Northern analysis of wild-type flies using the CtBP two-hybrid insert as a probe shows that three major transcripts (2.5, 2.7 and 4.0 kb) are expressed dynamically throughout all stages of development, whereas an additional 3.5 kb transcript is detected predominantly in adult females and embryos stages. CtBP transcript levels increase both early, during oogenesis and embryogenesis, and later in pre-pupae stages. CtBP expression is detected ubiquitously in the germarium and early oogenic stages and is highly expressed in nurse cells by stage 10. This transcript is dumped into the oocyte and is detected ubiquitously as a maternal transcript in early and cellular blastoderm stage embryos (Poortinga, 1998).

EFFECTS OF MUTATION

It has been demonstrated that CtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal CtBP activity. While hairy is probably not the only segmentation gene interacting with CtBP, dose-sensitive genetic interactions exist between CtBP and hairy mutations (Poortinga, 1998).

The P1590 strain carries a homozygous lethal insertion, with the homozygotes dying as pharate adults. When dissected from their pupal cases, CtBP/1590 homozygotes exhibit duplicated and ectopic bristles (macrochaetes) on the notum and scutellum. The P1590 strain also exhibits a strong maternal effect phenotype. It was on the basis of its maternal requirement that the P-1590 allele was identified in a screen for maternal-effect lethals. In this screen, a change of function mutation in an RNA polymerase II subunit (wimp) was used to reduce, but not eliminate, P-1590 maternal contribution. Embryos derived from mothers trans-heterozygous for wimp and the P1590 allele die, and cuticle preparations of these embryos show segmentation defects, ranging from pair-wise fusions of adjacent denticle bands to more widespread denticle fusions (Poortinga, 1998).

Since wimp reduces, but does not eliminate maternal function, loss of CtBP/P1590 function in germline clones was examined using the FLP-DFS technique. The FLP-DFS system incorporates the presence of a dominant female sterile (DFS) mutation, ovo^D1, and the FLP-FRT yeast site-specific recombination system to create germline-specific mosaics. A P1590 FRT82B chromosome had been generated previously as part of a screen using the FLP-DFS technique to look for maternal phenotypes in zygotic single P-element-induced mutations. Embryos derived from germline clones generated with this chromosome were reported to have segmentation defects resulting in pair-wise fusions, as well as large holes in the ventral cuticle (Perrimon, 1996). Using this P1590 FRT82B stock, more severe cuticle disruptions have been obtained than previously reported: embryos were consistently observed that are significantly shorter than wild-type, with either 'lawns' of denticles on the ventral cuticle or severely fused or missing denticle bands (Poortinga, 1998).

If the segmentation defects observed in embryos lacking maternal CtBP are due to its interaction with Hairy, disruptions in patterning similar to those found in hairy mutations or loss of maternal Groucho are expected. In particular, the expression of the other primary pair-rule genes are expected to be disrupted and fushi tarazu expression to be derepressed. Consistent with this, Ftz stripes are found to be expanded in embryos lacking maternal CtBP. However, this broad band of expression later resolves into stripe-specific ftz repression, with stripes 2, 4, 5 and 6 predominantly affected. Aberrant expression of the primary pair-rule gene proteins, Eve and Runt, as well as of Hairy itself are also observed. Since the primary pair-rule genes respond directly to gap gene cues, gap gene expression was examined in embryos lacking maternal CtBP. Expression of the three gap genes examined, Hunchback, Krüppel and Knirps, appears normal in these embryos. In addition to its effects on anterior-posterior patterning, embryos lacking maternal CtBP also show disruptions of dorsoventral patterning. Beginning with the expression of the pair-rule genes, a lack of segmentation gene expression is detected on the ventral surface (Poortinga, 1998).

In addition to the disruption of patterning in P1590 germline clones, CtBP/P1590 was examined for genetic interaction with hairy. h mutations result in a range of cuticle phenotypes from loss or fusion of adjacent denticle bands to a fusion of most of the segments ('lawn' phenotype), with the most common phenotype called the classic pair-rule phenotype that results from the loss of alternating segment-wide regions. Larvae homozygous for a strong h allele, h^7H, display the extreme 'lawn' phenotype, whereas larvae trans-heterozygous for the h^7H allele and a weaker h allele, h^12C, display the classic pair-rule phenotype. This h^7H/h^12C allelic combination was initially used to examine if reducing the CtBP dose maternally would suppress or enhance the intermediate pair-rule phenotype. P1590 was genetically recombined onto a chromosome containing the h^7H allele. Reducing the dose of CtBP maternally results in the suppression of the h^7H/h^12C mutant cuticle phenotype. Likewise, reducing the dose of CtBP maternally in the severe h^7H background suppresses the extreme lawn phenotype. No alterations in viability or phenotype of any progeny classes are observed when P1590 is trans-heterozygous with h^7H, or when the h^7H P1590 recombinant chromosome is crossed to wild-type (Poortinga, 1998).

CtBP is required for proper development of peripheral nervous system in Drosophila

C-terminal binding protein (CtBP) is an evolutionarily and functionally conserved transcriptional corepressor known to integrate diverse signals to regulate transcription. Drosophila CtBP (dCtBP) regulates tissue specification and segmentation during early embryogenesis. This study investigated the roles of dCtBP during development of the peripheral nervous system (PNS). This study includes a detailed quantitative analysis of how altered dCtBP activity affects the formation of adult mechanosensory bristles. dCtBP loss-of-function was shown to result in a series of phenotypes with the most prevalent being supernumerary bristles. These dCtBP phenotypes are more complex than those caused by Hairless, a known dCtBP-interacting factor that regulates bristle formation. The emergence of additional bristles correlated with the appearance of extra sensory organ precursor (SOP) cells in earlier stages, suggesting that dCtBP may directly or indirectly inhibit SOP cell fates. It was also found that development of a subset of bristles was regulated by dCtBP associated with U-shaped through the PxDLS dCtBP-interacting motif. Furthermore, the double bristle with sockets phenotype induced by dCtBP mutations suggests the involvement of this corepressor in additional molecular pathways independent of both Hairless and U-shaped. It is therefore proposed that dCtBP is part of a gene circuitry that controls the patterning and differentiation of the fly PNS via multiple mechanisms (Stern, 2009).

This study provides evidence that dCtBP is required for different aspects of PNS development. In addition, extensive genetic characterization demonstrates how altered dCtBP activity can influence the formation of the adult dorsal thoracic mechanosensory organs. The data show that overexpression of dCtBP impairs mechanosensory formation. In contrast, reduction of dCtBP activity leads to variable bristle phenotypes, suggesting that dCtBP is likely operating in different molecular complexes. Namely, the mechanisms by which dCtBP regulates cell fate specification within the PNS may involve protein–protein interactions between dCtBP and at least two factors: Ush and possibly H (Stern, 2009).

The data strongly suggest that dCtBP associates with the Ush-Pnr repressor complex through the Ush PxDLS motif to inhibit the expression of achaete and scute in particular PNCs. This model is supported by the following evidence. First, the ush loss-of-function and gain-of-function phenotypes were phenocopied by the corresponding genetic alterations to dCtBP activity. Second, Ush interacts with Pnr and the Ush-Pnr complex inhibits expression of the achaete and scute genes through GATA sites located within the DC enhancer. Third, the additional SOP cells were formed in both the dCtBP and ush mutant imaginal discs. Fourth, both ush and pnr alleles exhibited dominant genetic interactions with dCtBP. Finally, disruption of the PxDLS motif of Ush partially mitigated the effects of ush overexpression on particular bristles (Stern, 2009).

The evolutionarily conserved physical interaction of dCtBP with Ush is essential for the propagation of certain cell lineages, such as blood cells (crystal cells) of the fruit fly, but not for heart development, processes known to be regulated by Ush and the GATA factors, Pnr and Serpent. Surprisingly, the interaction between CtBP and FOG-1 is not required for erythroid development in mice, despite the fact that this interaction was found to be important in tissue culture experiments and in frog embryos. The current results from the ush overexpression assay suggest that Ush may utilize both the PxDLS motif and another repression domain(s) to fully function, since particular bristles are affected by disruption of the PxDLS motif of Ush. A putative corepressor that interacts with the additional repression domain may act additively or cooperatively with dCtBP or function in different tissue/cell-type contexts. In fact, recently other repression domains in Ush, required for repression of the D-mef2 cardiac gene, were identified and these seemed to cooperatively work with the dCtBP-dependent motif. Consistent with this hypothesis, some dCtBP-interacting factors contain multiple repression domains. Knirps (nuclear receptor), Snail (zinc-finger protein), and H all have two repression domains, dCtBP-dependent and -independent, which can function additively in transgenic flies and/or in tissue culture. It has been also demonstrated that H has an additional repression activity independent of Groucho and dCtBP-binding. Krüppel (zinc-finger protein) has two evolutionarily conserved repression domains. The dCtBP-dependent domain is functional in tissue culture and in transgenic embryos, while the other repression domain is only active in tissue culture but not in transgenic embryos, suggesting a cell-type specific effect. Finally, Brinker (a helix-turn-helix protein) contains at least three repression domains (dCtBP-dependent, Groucho-dependent, and the third repression domain) that are important for repression of different target genes (Stern, 2009).

The physical interaction of dCtBP with H is implicated in sensory organ formation, wing formation, and embryonic patterning. H acts as an adaptor protein to bridge the Groucho and dCtBP corepressors to the DNA-binding factor Su(H), to ultimately inhibit Notch target genes. Vertebrate Notch target genes are similarly repressed by a complex consisting of CtBP with RBP-Jkappa (the mammalian counterpart to Su(H)) and the SHARP/CtIP corepressors. This study demonstrates that the bristles that are affected in dCtBP mutants also show defects in H loss-of-function mutants, although the effect of H is stronger than that of dCtBP. H mutations induce two distinct phenotypes associated with loss of bristles; one is the bald phenotype (a complete loss of both sockets and bristles) due to lack of SOP cells, and the other is the double-socket phenotype (also lack of bristles). A similar bald phenotype was observed in dCtBP mutant backgrounds, such as dCtBP RNAi, the dCtBP^87De-10/dCtBP⁰³⁴⁶³ transheterozygote, the dCtBP^87De-10 clonal backgrounds. Although compared to what is seen in dCtBP mutants, reduction of H activity interferes more uniformly with the formation of all 11 bristles that were analyzed, the bald phenotype further supports previous observations that dCtBP is involved in H-mediated repression. The double-socket phenotype seen in H loss-of-function mutants was never observed in dCtBP mutants. This distinct phenotype suggests that H may play a role independent of dCtBP, possibly by interacting with another corepressor Groucho. Interestingly, the bald phenotype was also induced by overexpression of dCtBP. The mechanism by which overexpression causes the bald phenotype in all regions except the DC region remains unclear, although one simple explanation could be that overproduction of dCtBP may disrupt the stoichiometric balance of the H/dCtBP/Groucho repression complex (Stern, 2009).

The double bristle phenotype observed in dCtBP mutants suggests that dCtBP may be required to execute cell fate decisions within the SOP lineage. A similar phenotype seen in the H gain-of-function background was the result of a socket-to-bristle cell fate transformation. Of note, this phenotype is clearly distinct from the double bristle phenotype observed in dCtBP mutants, which is always associated with a socket(s). This dCtBP phenotype implies that cousin-to-cousin cell fate conversions may be occurring within the sensory organ lineage. This type of cell fate switch could be similar to the conversion of sheath to bristle observed in hamlet mutants. Hamlet is a zinc-finger transcription factor and interestingly contains a PLDLS peptide sequence located between amino acid 747 and 751, identical to the CtBP-interacting motif. Future experiments will address whether dCtBP and Hamlet can physically interact and function together within the same biological process (Stern, 2009).

Based on the results, it is concluded that dCtBP regulates the development of the mechanosensory organs likely via multiple mechanisms. This highlights the centrality of this transcriptional corepressor in integrating multiple inputs to define boundaries and thereby control pattern formation during development (Stern, 2009).

Conserved catalytic and C-terminal regulatory domains of the C-terminal binding protein corepressor fine-tune the transcriptional response in development

Transcriptional corepressors play complex roles in developmental gene regulation. These proteins control transcription by recruiting diverse chromatin-modifying enzymes, but it is not known whether corepressor activities are finely regulated in different developmental settings or whether their basic activities are identical in most contexts. The evolutionarily conserved C-terminal binding protein (CtBP) is recruited by a variety of transcription factors that play crucial roles in development and disease. CtBP contains a central NAD(H) binding core domain that is homologous to D2 hydroxy acid dehydrogenase enzymes, as well as an unstructured C-terminal domain. NAD(H) binding is important for CtBP function, but the significance of its intrinsic dehydrogenase activity, as well as that of the unstructured C terminus, is poorly understood. To clarify the biological relevance of these features, genetic rescue assays were establised to determine how different forms of CtBP function in the context of Drosophila development. The mutant phenotypes and specific gene regulatory effects indicate that both the catalytic site of CtBP and the C-terminal extension play important, if nonessential roles in development. The results indicate that the structural and enzymatic features of CtBP, previously thought to be dispensable for overall transcriptional control, are critical for modulating this protein's activity in diverse developmental settings (Zhang, 2011).

CtBP is recruited by diverse transcription factors to effect repression of target genes in numerous metazoan regulatory pathways. Reflecting its evolutionary relatedness to dehydrogenases, CtBP has a functional NAD(H) binding cleft that is integral for overall protein structure, as well as for interaction with transcription factors and cofactors. In addition, CtBP contains active-site residues that are both evolutionarily conserved and confer in vitro dehydrogenase activity. In this study, an in vivo developmental assay was used to address key questions about the function of CtBP. In a genomic rescue assay that comprehensively tests biological function, it was shown that the residues required for enzymatic activity, as well as the C-terminal regulatory domain, are essential for normal function in development. Interestingly, animals programmed solely with the catalytic mutant protein or the form containing the C-terminal regulatory extension had significantly impaired viability and strong wing phenotypes. Specific transcriptional defects were also evident; embryonic eve expression was severely disrupted in these mutant embryos and the expression levels of particular targets in adults were derepressed. The overall phenotypes of these mutants were for the most part enhanced manifestations of effects noted with less penetrant alleles, suggesting that loss of enzymatic function or too-extensive provision of the C-terminal extension reduced CtBP activity. The effects noted in this stdy are likely to represent a significant loss of activity, because CtBP function exhibits a considerable degree of robustness: the CtBP gene is recessive, showing no phenotype with ~50% of normal dosage, and wild-type flies carrying extra copies of these transgenes (up to ~200% of normal CtBP levels) are unaffected. Despite the lack of overt phenotypes for catalytic or C-terminal variants when expressed in cell-based assays, this analysis clearly indicates that the catalytic activity and C-terminal extension are important features that regulate CtBP function (Zhang, 2011).

The results illuminate the overall functional understanding of CtBP, leading to a model that explains the roles of NAD(H) binding and catalysis. Previous results demonstrated that NAD(H) binding is important for folding of CtBP and influences dimerization, binding of cofactors, and interaction with transcription factors. NAD(H) has been suggested to do more than influence the structure of CtBP complexes, however. The observation that the CtBP-NAD mutant largely localizes in the cytoplasm suggests that NAD(H) binding might also regulate the protein's subcellular localization. In agreement with this hypothesis, mammalian CtBP1-NAD mutants also showed improper nuclear localization in MEF90 (CtBP^−/−) cells. In addition, because of a reported higher affinity of the corepressor for the reduced NADH dinucleotide over NAD⁺, CtBP has been suggested to serve as a mediator that links cellular redox status to transcriptional output, and treatment of cells with agents that affect NADH levels can influence CtBP-mediated repression and occupancy of promoters (Zhang, 2011).

Building on these insights, an essential additional question is how a putative dehydrogenase activity may function in transcriptional control. Here, there is little in the way of precedent to go on; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in transcriptional regulation as an essential component of the OCA-S/Oct-1 coactivator complex, where it plays a critical role in S-phase activation of histone H2B expression. This dehydrogenase is also proposed to function as a redox sensor, monitoring changes during S phase, but how dehydrogenase activity itself affects transcription in this case remains a mystery. With respect to CtBP, numerous cell-based studies have shown that the dehydrogenase-defective mutant remains active, suggesting that the enzymatic activity is dispensable for CtBP function in these settings. These previous studies were not designed to test whether CtBP dehydrogenase activity is important only under certain physiological conditions or on specific target genes, however. The current genomic rescue data strongly suggest that the dehydrogenase activity is important for proper development and is involved in gene regulation. Two models would explain this function: possibly in response to metabolic signals, the enzymatic activity may be important as a mechanism to interconvert the reduced and oxidized forms of NAD, altering CtBP's structure and, thus, interaction with cofactors, or the CtBP dehydrogenase activity may be important for the reduction or oxidation of specific as-yet-unknown substrates important for gene regulation. It cannot be rule out that the mutation in the catalytic site also affect CtBP interactions with cofactors, but the results of structural and mutational studies indicate that the catalytic site is located in a buried cleft, separate from the cofactor binding domain (Zhang, 2011).

An additional area of CtBP biology concerns possible regulation through the C-terminal region. Diverse metazoan CtBP proteins feature evolutionarily divergent C-terminal extensions that are dispensable for corepressor activity. Indeed, although Drosophila genes and those of other arthropods encode a conserved C-terminal region as an alternatively spliced exon, the major isoform lacks this domain. Previous studies have indicated that the C-terminal domain is subject to posttranslational modifications through sumoylation and phosphorylation that may regulate CtBP through alternative cellular localization and degradation. This study has shown that mutant animals that only express the CtBP_L isoform with the C-terminal extension are viable but exhibit impaired viability, defective wings, and gene expression defects, clearly indicating the functional importance of this domain. It appears that equipping all CtBP molecules with this extension reduces CtBP activity, suggesting that this domain serves a negative regulatory function, possibly as a recipient of posttranslational modifications that may reflect signal transduction activity. In total, these results indicate that CtBP may function as a nexus of signal integration, responding to metabolic status through NAD(H) binding and to signaling pathways through the C terminus to affect transcriptional levels. An important question is whether this signaling would affect CtBP function generally or in a gene-specific manner. The differential effects of the CtBP-CAT mutant and CtBP_L on eve, mmp1, Rel, prd, and stv provide some support for the latter possibility. A genome-wide comparison of expression profiles between CtBP-WT and CtBP-CAT mutants, as well as CtBP_L mutants, will provide important insights on this question (Zhang, 2011).

In light of the involvement of CtBP in numerous regulatory events in Drosophila, including gap gene repression function in the blastoderm embryo, development of the peripheral nervous system, and recruitment in Notch, wingless, and transforming growth factor β (TGF-β) signaling pathways, it was initially quite surprising that the prevalent phenotypes observed in different CtBP rescues were specific for wings. However, further characterization of impaired embryo viability and disrupted eve expression in embryos, as well as bristle phenotypes and altered expression of specific genes in adult flies, clearly shows that the effects are not limited to wings. It is likely that changes in CtBP activity produce various effects in different tissues and that those individuals with the strongest effects in nonwing tissues do not survive (Zhang, 2011).

In summary, this study has demonstrated that the dehydrogenase activity of CtBP and the C-terminal domain are important for fine-tuning CtBP function in the context of development. A detailed biochemical characterization of how these activities are integrated remains to be elucidated; important clues will likely come from a comprehensive picture of how possible modifications and metabolic signals are focused through CtBP to affect gene expression at a genomic level. (Zhang, 2011).

EVOLUTIONARY HOMOLOGS

CtBP is an NAD+-regulated dehydrogenase

Transcriptional repression is based on the selective actions of recruited corepressor complexes, including those with enzymatic activities. One well-characterized developmentally important corepressor is the C-terminal binding protein (CtBP). Although intriguingly related in sequence to D2 hydroxyacid dehydrogenases, the mechanism by which CtBP functions remains unclear. Biochemical and crystallographic studies reveal that CtBP is a functional dehydrogenase. In addition, both a cofactor-dependent conformational change, with NAD+ and NADH being equivalently effective, and the active site residues are linked to the binding of the PXDLS consensus recognition motif on repressors, such as E1A and nuclear receptor interacting protein RIP140. Together, these data suggest that CtBP is an NAD+-regulated component of critical complexes for specific repression events in cells (Kumar, 2002).

The dehydrogenase domain alone is sufficient to bind the PXDLS motif. This domain is highly conserved within CtBP family members from C. elegans to vertebrates. However the C terminus extension (C') is highly variable with no predicted secondary structure. It is possible that C' is a regulatory region, likely to mediate CtBP function after recruitment to a PXDLS motif. In Drosophila, there are three splice variants of CtBP differing only in the C', and the shortest of these splice variants is essentially only composed of the dehydrogenase domain. It was initially speculated, based on the crystal structure, that the PXDLS motif might bind in a cavity near the entrance to the active site cleft. However, mutations in this cavity do not disrupt E1A interactions in vitro or the repression function in vivo. Unexpectedly, mutation of the active site residues do affect E1A binding, suggesting that the PXDLS motif interacts with these residues at the periphery of the active site cleft. The cleft is walled off by a loop extending from the 2-fold related subunit that may provide additional interactions with the PXDLS sequence. Indeed, CtBP may be a simple dehydrogenase that has evolved or gained an extra ability to bind a PXDLS recognition motif (Kumar, 2002).

Nuclear receptor interaction protein p140 is highly recruited to ligand receptors on cognate DNA sites. An interaction between CtBP and RIP140 has also been reported, which is intriguing because RIP140 is recruited to nuclear receptors in response to ligand based on RIP140 LXXLL motifs; it competes with other coactivators. While at ambient levels of CtBP, liganded retinoic acid receptor induces recruitment of coactivators, including CBP and p160 co-activator factors; it is hown here that increased expression of CtBP completely blocks RAR activation, an effect entirely dependent on RIP140. Again, this effect requires specific CtBP catalytic residues, consistent with the expected role for these residues in stabilizing binding to interacting cofactors. This also implies that, in the presence of ligand, regulation of CtBP can be a key component to the nature of the transcriptional response (Kumar, 2002).

The finding that E1A-CtBP requires NAD+ has interesting implications. In particular, it raises the possibility that alterations in NAD+ levels might modulate the binding of CtBP to specific repressor complexes, as well as regulating its own enzymatic activity. Alterations in NAD+ level has been documented in response to DNA damage, and the reported associations between CtBP and p130/Rb complex, BRCA1, and KU70 may be critical regulatory components of cellular homeostasis. The ratio of NAD+/NADH can vary in response to activation of metabolic dehydrogenases during day-night periods of food intake and starvation, and rhythmic cycles in the cellular redox state have been shown to regulate DNA binding of Clock and NPAS2 heterodimeric transcription factors (Kumar, 2002).

Using CtBP prepared and expressed in a mammalian system, no evidence was found that E1A-CtBP interaction is regulated differently by NADH versus NAD+. Intriguingly, these results differ from those reported for a bacterially expressed CtBP, where NADH is two to three orders of magnitude more effective than NAD+ in stimulating CtBP-E1A interaction. This discrepancy may reflect different sources of CtBP; since in vitro transcribed and translated (TnT) CtBP was used in this study. The rabbit reticulocyte lysate used for the TnT reaction is known to posttranslationally modify proteins, including phosphorylation, acetylation, and isoprenylation. It is possible that one or more of these modifications dampen the differential effect observed with bacterial CtBP. In all, it is not easy to see from the structure how NADH could be up to three orders of magnitude more effective than NAD+ in stimulating E1A-CtBP interaction, considering that the two cofactors differ chemically by only a hydrogen atom on the nicotinamide ring. This may be further clarified by comparing the structure described in this study to a complex of CtBP with NADH (Kumar, 2002).

CtBP is not the only transcription corepressor to bind NAD+. The Sir2 family of transcriptional corepressors also binds NAD+ as a cofactor for histone deacetylation reactions, and furthermore, there is direct evidence that activity of NAD+-dependent Sir2 repressors can regulate life span in C. elegans. Whether levels of nuclear NAD+ vary during development, viral infection, or transcriptional silencing remains to be determined, but it marks an intriguing new direction for future research (Kumar, 2002).

The C-terminal binding protein 2 (CtBP2) is a 48 kDa phosphoprotein reported to function as a co-repressor for a growing list of transcriptional repressors. CtBP is a dimeric NAD+-regulated D-isomer-specific 2-hydroxy acid dehydrogenase. However, the specific substrate(s) of CtBP enzymatic activity and the relationship of this activity to its co-repression function remain unknown. The ability of a human CtBP to bind and serve as a co-repressor of E1A has been shown to be regulated by nuclear NADH levels. This study extends the functional characterization of CtBP by demonstrating that amino acid substitutions at Gly189 in the conserved NAD+-binding fold both abrogate the ability of CtBP2 to homodimerize and are associated with a dramatic loss of co-repressor activity. Consistent with the known enzymatic activity of CtBP2, mutations at Arg272 in the substrate-binding domain and at His321 in the catalytic domain result in significant loss of CtBP2 transcriptional co-repressor activity. High resolution serial C-terminal deletion analysis of CtBP2 also reveals a novel N-terminal repression domain that is distinct from its dehydrogenase domain. These results suggest a model in which CtBP2 co-repressor function is regulated, at least in part, through the effect of NADH on CtBP2 homodimerization (Thio, 2004).

It is proposed that the CtBP2 homodimerization function serves as a cellular redox sensor for its co-repressor function. In such a role, CtBPs can detect the readiness of a cell to proceed with various cellular processes by the levels of cellular NAD+/NADH present. The ability of CtBP co-repressors to sense the cellular redox state is likely to be specific to NAD+/NADH. It has been reported that the binding of hCtBP1 to E1A was insensitive to NADP+, NADPH and FAD+. This is not surprising since the NAD+-binding domain of CtBPs contain the conserved GXGXXG motif (where X is any amino acid), characteristic of enzymes that use NADH as a cofactor. The nucleotide-binding domains of enzymes that use NADPH as a cofactor contain a distinct GXGXXA motif. The nucleotide-binding domain in the 2HAD [PDB] family of bacterial enzymes moderates enzyme activity by determining the availability of the active site. The active site is found in a crevice between the nucleotide-binding and substrate-binding domains, which are connected by a flexible hinge region. Cofactor binding causes a conformational change of the dimer, thus closing the active site cleft. This effectively brings the NAD+ cofactor into a favorable position relative to the substrate. Once the hydride transfer from substrate to NAD+ is completed, the cleft opens again to allow the release of the enzymatic product. The inability to bind NAD+/NADH, as in the case of the Gly189 mutation in mCtBP2, would leave the active site closed once the substrate is bound, thus rendering the enzyme incapable of carrying out any further catalytic reactions (Thio, 2004).

This proposal is supported by a well-established paradigm of redox-regulated transcriptional regulation. The silent information regulator 2 protein (Sir2) in yeast mediates transcriptional silencing by promoting the deacetylation of histones in a reaction that absolutely depends on the presence of NAD+. A detailed mechanism for the reaction catalyzed by Sir2 has been proposed. The proto-oncogenes c-fos and c-jun are known to be regulated by the cellular redox state. Their protein products, Fos and Jun, function cooperatively by forming a heterodimeric complex known as activator protein-1 (AP-1). AP-1 binds to the DNA regulatory element known as the AP-1-binding site. The ability of AP-1 to bind DNA is regulated by the reduction-oxidation of a single conserved cysteine residue of the DNA-binding domain of these two proteins. The reduction of this residue is carried out by redox factor-1 (Ref-1), which is itself subject to redox-mediated regulation. The oxidized or reduced state of cells would thus trigger a redox cascade involving Ref-1, AP-1 and possibly many other proteins, which would eventually lead to the transcriptional repression or activation of downstream target genes. NADPH has been reported to reverse the oxidation of the yeast homolog of AP-1 (YAP-1). The demonstration that CtBP is an NAD+-regulated dehydrogenase that mediates transcriptional repression, and the finding that the level of NADH controls and regulates CtBP2 co-repressor function through its effect on CtBP2 homodimerization, provides further evidence for a broader functional role of enzymatic activity in general, and NAD+-dependent dehydrogenase activity in particular, in gene transcriptional regulation (Thio, 2004).

CtBp interacts with viral oncoproteins

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, termed C-terminal binding protein (CtBP). The cDNA for CtBP was cloned via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain reveals that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation (Schaeper, 1995).

A region of the C-terminus of adenovirus type 2/5 E1A protein has been associated with negative modulation of tumorigenicity, as well as the extent of oncogenic transformation. In contrast with the N-terminus of the E1A protein, which has been extensively characterized and shown to associate with a number of cellular proteins, the function of the C-terminus is poorly understood. To date, a single 48-kDa protein, CTBP1, has been shown to associate with this region. Human (CTBP2) and mouse (Ctbp2), both highly related to CTBP1, have been identification and sequenced and these also are likely to bind to the E1A protein. CTBP2 is expressed in all tissues tested, with a higher level of expression in the heart, skeletal muscle, and pancreas. CTBP1 and CTBP2 map to human chromosomes 4p16 and 21q21.3, respectively (Katsanis, 1998).

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, cellular proteins were sought that complex with CtBP. A 125-kDa protein, CtIP, has been cloned and characterized that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. These results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif (Schaeper, 1998).

The adenovirus E1A-243R protein has the ability to force a resting cell into uncontrolled proliferation by modulating the activity of key targets in cell cycle control. Most of these regulatory mechanisms are dependent on activities mapping to conserved region 1 (CR1) and the non-conserved N-terminal region of E1A. CR1 functions as a very patent transactivator when it is tethered to a promoter through a heterologous DNA binding domain. However, artificial DNA binding is not sufficient to convert full-length E1A-243R to a transactivator. Thus, an additional function(s) of the E1A-243R protein modulates the effect of CR1 in transcription regulation. A 44 amino acid region at the extreme C-terminus of ElA inhibits transactivation by a Gal4-CR1 fusion protein. Inhibition correlates with binding of the nuclear 48 kDa C-terminal binding protein (CtBP), which has been implicated in E1A-mediated suppression of the metastatic potential of tumou cells. This suggests that CtBP binding can regulate E1A-mediated transformation by modulating CR1-dependent control of transcription (Sollerbrant, 1998).

Because of the high sequence conservation from human to Drosophila CtBP, it was expected that Drosophila CtBP would also interact with adenovirus E1a. dCtBP indeed interacts strongly with the C-terminus of the Ad2 E1a protein in a directed two-hybrid assay. Full-length E1a fused to LexA is lethal to yeast cells and could not be tested. Point mutations within a six amino acid motif in the E1a C-terminus, PXDLSX, eliminate or attenuate CtBP binding. A similar sequence was sought within the sufficient 25 amino acid interaction region of Hairy; the five amino acid PLSLV sequence identified from full-length Hairy abolishes interaction with dCtBP while still retaining Hairy's ability to interact with other proteins, including Groucho, which binds to the adjacent WRPW sequence. The dCtBP interaction domain for E(spl)mdelta was also mapped. 16 amino acids (143-158) are found to be sufficient for this interaction. Deletion of five amino acids similar to the Hairy consensus from full-length E(spl)mdelta abolishes interaction with dCtBP, while deletion of an adjacent five amino acids has no effect (Sollerbrant, 1998).

Adenovirus E1A mediates its effects on cellular transformation and transcription by interacting with critical cellular proteins involved in cell growth and differentiation. The amino terminus of E1A binds to CBP/p300 and associated histone acetyltransferases such as P/CAF. The carboxyl terminus binds to the carboxyl-terminal binding protein (CtBP), which associates with histone deacetylases. 12S E1A can be acetylated by p300 and P/CAF, and one of the acetylation sites maps to Lys-239. This Lys residue is adjacent to the consensus CtBP binding motif, PXDLS. Mutation of Lys-239 to Gln or Ala blocks CtBP binding in vitro and disrupts the E1A-CtBP interaction in vivo. Peptide competition assays demonstrate that the interaction of E1A with CtBP is also blocked by Lys-239 acetylation. Supporting a functional role for Lys-239 in CtBP binding, mutation of this residue to Ala decreases the ability of E1A to block cAMP-regulated enhancer (CRE)-binding protein (CREB)-stimulated gene expression. Lys-239 is acetylated in cells as detected by using an antibody directed against an acetyl-Lys-239 E1A peptide. CtBP interacts with a wide variety of other transcriptional repressors through the PXDLS motif, and, in many instances, this motif is followed by a Lys residue. It is suggested that acetylation of this residue by histone acetyltransferases, and the consequent disruption of repressor complexes, might be a general mechanism for gene activation (Zhang, 2000).

CtBP has been shown to be a highly conserved corepressor of transcription. E1A and all the various transcription factors to which CtBP binds contain a conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif (amino acids [aa] 728 to 732). EBNA3C binds to CtBP both in vitro and in vivo and the interaction requires an intact PLDLS. The C terminus of EBNA3C (aa 580 to 992) has modest trans-repressor activity when it is fused to the DNA-binding domain of Gal4, and deletion or mutation of the PLDLS sequence ablates this and unmasks a transactivation function within the fragment. However, loss of the CtBP interaction motif has little effect on the ability of full-length EBNA3C to repress transcription. A striking correlation between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in the immortalization and transformation of primary rat embryo fibroblasts has also been revealed (Touitou, 2001).

SUMOylation of CtBP and CtBP binding to NOS

PDZ domains function in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, affinity chromatography was performed of brain extract with the nNOS PDZ domain. The carboxyl-terminal-binding protein (CtBP) was identified as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, these data suggest a new function for nNOS as a regulator of CtBP nuclear localization (Riefler, 2001).

The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. CtBP1 is SUMOylated and its SUMOylation (see Drosophila SUMO) profoundly affects its subcellular localization. SUMOylation occurs at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacks the SUMOylation site and is not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifts CtBP1 from the nucleus to the cytoplasm, while it has little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolishes the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 is inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression (Lin, 2003).

CtBP functions in invertebrates

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of many devastating human diseases. An important adaptation for anautogenicity is the previtellogenic arrest (the state of arrest) preventing the activation of the yolk protein precursor (YPP) genes Vg and VCP prior to blood feeding. A novel GATA factor (AaGATAr) that recognizes GATA binding motifs (WGATAR) in the upstream region of the YPP genes serves as a transcriptional repressor at the state of arrest. Importantly, AaGATAr can override the 20-hydroxyecdysone transactivation of YPP genes, and its transcriptional repression involves the recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is present only in the adult female fat body. Furthermore, in nuclear extracts of previtellogenic fat bodies with transcriptionally repressed YPP genes, there is a GATA binding protein forming a band with mobility similar to that of AaGATAr. The specific repression of YPP genes by AaGATAr in the fat body of the female mosquito during the state of arrest represents an important molecular adaptation for anautogenicity (Martin, 2001).

CtBP interacts with mammalian transcription factors

Basic Kruppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. BKLF is capable of repressing transcription, and its repression domain has been mapped to the N-terminus. A two-hybrid screen against BKLF was carried out, and a novel clone was isolated encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein that binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG (Drosophila homolog: U-shaped). These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators (Turner, 1998).

GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 a functional analysis of the FOG-2 protein was performed. The results demonstrate that (1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins; (2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and (3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, a novel N-terminal domain of FOG-2 (amino acids 1-247) has been identifed that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development (Svensson, 2000).

CACCC-boxes are recognized by transcription factors of the Sp/Kruppel-like Factor (Sp1/KLF) family. Described here is one member of this family, KLF8/ZNF741/BKLF3 (KLF8). KLF8 contains a characteristic C-terminal DNA-binding domain comprised of three Kruppel-like zinc fingers, but it also has limited homology to another family member, KLF3/Basic Kruppel-like Factor (KLF3/BKLF), in its N-terminus. Most significantly, it shares with KLF3/BKLF a Pro-Val-Asp-Leu-Ser/Thr motif. In KLF3/BKLF this motif mediates contact with the co-repressor protein C-terminal Binding Protein (CtBP). The KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP. The N-terminus of KLF8 functions as a repression domain and its activity relies on the integrity of the CtBP recognition motif. The zinc fingers of KLF8 recognize CACCC elements in DNA, and full-length KLF8 can repress a CACCC-dependent promoter. KLF8 is broadly expressed in human tissues. These results establish KLF8 as a CACCC-box binding protein that associates with CtBP and represses transcription (van Vliet, 2000).

deltaEF1, a representative of the zinc finger-homeodomain protein family, is a transcriptional repressor that binds E2-box (CACCTG) and related sequences and counteracts the activators through transrepression mechanisms. It has been shown that the N-proximal region of the protein is involved in the transrepression. deltaEF1 has a second mechanism of transrepression, recruiting CtBP1 or CtBP2 as its corepressor. A two-hybrid screen of mouse cDNAs with various portions of deltaEF1 has identified these proteins, which bind to deltaEF1 in a manner dependent on the PLDLSL sequence located in the short medial (MS) portion of deltaEF1. CtBP1 is the mouse ortholog of human CtBP, known as the C-terminal binding protein of adenovirus E1A, while CtBP2 is the second homolog. Fusion of mouse CtBP1 or CtBP2 to Gal4DBD (Gal4 DNA binding domain) makes these proteins Gal4 binding site-dependent transcriptional repressors in transfected 10T1/2 cells, indicating their involvement in a transcriptional repression mechanism. When the MS portion of deltaEF1 is fused to Gal4DBD and used to transfect cells, a strong transrepression activity is generated, but this activity is totally dependent on the PLDLSL sequence that serves as the site for interaction with endogenous CtBP proteins, indicating that CtBP-1 and -2 can act as corepressors. Exogenous CtBP1/2 significantly enhances transcriptional repression by deltaEF1, and this enhancement is lost if the PLDLSL sequence is altered, demonstrating that CtBP1 and -2 act as corepressors of deltaEF1. In the mouse, CtBP1 is expressed from embryo to adult, but CtBP2 is mainly expressed during embryogenesis. In developing embryos, CtBP1 and CtBP2 are expressed broadly with different tissue preferences. Remarkably, their high expression occurs in subsets of deltaEF1-expressing tissues, e.g., cephalic and dorsal root ganglia, spinal cord, posterior-distal halves of the limb bud mesenchyme, and perichondrium of forming digits, supporting the conclusion that CtBP1 and -2 play crucial roles in the repressor action of deltaEF1 in these tissues (Furusawa, 1999).

Previous work has demonstrated the critical role for transcription repression in quiescent cells through the action of E2F-Rb or E2F-p130 complexes. Recent studies have shown that at least one mechanism for this repression involves the recruitment of histone deacetylase. Nevertheless, these studies also suggest that other events likely contribute to E2F/Rb-mediated repression. Using a yeast two-hybrid screen to identify proteins that specifically interact with the Rb-related p130 protein, it has been demonstrated that p130, as well as Rb, interacts with a protein known as CtIP. This interaction depends on the p130 pocket domain, which is important for repression activity, as well as an LXCXE sequence within CtIP, a motif previously shown to mediate interactions of viral proteins with Rb. CtIP interacts with CtBP, a protein named for its ability to interact with the C-terminal sequences of adenovirus E1A. Recent work has demonstrated that the Drosophila homolog of CtBP is a transcriptional corepressor for Hairy, Knirps, and Snail. Both CtIP and CtBP can efficiently repress transcription when recruited to a promoter by the Gal4 DNA binding domain, thereby identifying them as corepressor proteins. Moreover, the full repression activity of CtIP requires a PLDLS domain that is also necessary for the interaction with CtBP. It is proposed that E2F-mediated repression involves at least two events, either the recruitment of a histone deacetylase or the recruitment of the CtIP/CtBP corepressor complex (Meloni, 1999).

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF (Melhuish, 2000).

Ectopic production of the EVI1 transcriptional repressor zinc finger protein (Drosophila homolog: CG10568) is seen in 4%-6% of human acute myeloid leukemias. Overexpression also transforms Rat1 fibroblasts by an unknown mechanism, which is likely to be related to its role in leukemia and which depends upon its repressor activity. Mutant murine Evi-1 proteins, lacking either the N-terminal zinc finger DNA binding domain or both DNA binding zinc finger clusters, function as dominant negative mutants by reverting the transformed phenotype of Evi-1 transformed Rat1 fibroblasts. The dominant negative activity of the non-DNA binding mutants suggests sequestration of transformation-specific cofactors and recruitment of these cellular factors might mediate Evi-1 transforming activity. C-terminal binding protein (CtBP) co-repressor family proteins bind PLDLS-like motifs. The murine Evi-1 repressor domain has two such sites, PFDLT (site a, amino acids 553-559) and PLDLS (site b, amino acids 584-590), which independently can bind CtBP family co-repressor proteins, with site b binding with higher affinity than site a. Functional analysis of specific CtBP binding mutants show site b is absolutely required to mediate both transformation of Rat1 fibroblasts and transcriptional repressor activity. This is the first demonstration that the biological activity of a mammalian cellular transcriptional repressor protein is mediated by CtBPs. Furthermore, it suggests that CtBP proteins are involved in the development of some acute leukemias and that blocking their ability to specifically interact with EVI1 might provide a target for the development of pharmacological therapeutic agents (Palmer, 2001).

Evi-1 is a zinc finger nuclear protein whose inappropriate expression leads to leukemic transformation of hematopoietic cells in mice and humans. This expression blocks the antiproliferative effect of transforming growth factor beta (TGF-beta). Evi-1 represses TGF-beta signaling by direct interaction with Smad3 through its first zinc finger motif. Evi-1 represses Smad-induced transcription by recruiting C-terminal binding protein (CtBP) as a corepressor. Evi-1 associates with CtBP1 through one of the consensus binding motifs, and this association is required for efficient inhibition of TGF-beta signaling. A specific inhibitor for histone deacetylase (HDAc) alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting that HDAc is involved in the transcriptional repression by Evi-1. This identifies a novel function of Evi-1 as a member of corepressor complexes and suggests that aberrant recruitment of corepressors is one of the mechanisms for Evi-1-induced leukemogenesis (Izutsu, 2001).

ZEB is an active transcriptional repressor that regulates lymphocyte and muscle differentiation in vertebrates. Its homolog in Drosophila (Zfh-1) is also essential for differentiation of somatic and cardiac muscle. ZEB and Zfh-1 are shown to interact with the corepressor CtBP to repress transcription. ZEB and Zfh-1, both contain the sequence PLDLS in the same region of the repressor domain, and this sequence is shown to bind CtBP-1 and -2. In vertebrate species, ZEB contains two additional CtBP-like binding sites (variations of the PLDLS sequence) that also bind CtBP proteins and are required for full repressor activity. The three sites have an additive effect, and mutation of all three sites is necessary to abolish both binding to CtBP and repressor activity. Finally, the interaction of CtBP with ZEB at the promoter is shown to be necessary for repressor activity (Postigo, 1999).

Balancing signals derived from the TGFbeta family are crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFbeta/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB-1/deltaEF1 and ZEB-2/SIP1) regulate TGFbeta/BMP signaling in opposite ways: ZEB-1/deltaEF1 synergizes with Smad-mediated transcriptional activation, while ZEB-2/SIP1 represses it. These antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB-1/deltaEF1 binds to p300 and promotes the formation of a p300-Smad transcriptional complex, ZEB-2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFbeta family-dependent genes during Xenopus development (Postigo, 2003).

CtBP (carboxyl-terminal binding protein) participates in regulating cellular development and differentiation by associating with a diverse array of transcriptional repressors. Most of these interactions occur through a consensus CtBP-binding motif, PXDLS, in the repressor proteins. The CtBP-binding motif in E1A is flanked by a Lys residue and it has been suggested that acetylation of this residue by the p300/CBP-associated factor P/CAF disrupts the CtBP interaction. The interaction between CtBP and the nuclear hormone receptor corepressor RIP140 has been shown to be regulated similarly, in this case by p300/CBP itself. CtBP interacts with RIP140 in vitro and in vivo through a sequence, PIDLSCK, in the amino-terminal third of the RIP140 protein. Acetylation of the Lys residue in this motif, demonstrated in vivo by using an acetylated RIP140-specific antibody, dramatically reduces CtBP binding. Mutation of the Lys residue to Gln results in a decrease in CtBP binding in vivo and a loss of transcriptional repression. It is suggested that p300/CBP-mediated acetylation disrupts the RIP140-CtBP complex and derepresses nuclear hormone receptor-regulated genes. Disruption of repressor-CtBP interactions by acetylation may be a general mode of gene activation (Vo, 2001).

The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress (Mirnezami, 2003).

The recruitment of hCtBP1 by proteins containing a PXDLS motif is regulated by changes in cellular redox potential. The central dehydrogenase domain of hCtBP1 contains a high-affinity binding site for NADH (GXGXXG), occupation of which induces a conformational change in the hCtBP1 molecule and an increase in binding to proteins such as E1A and ZEB. A mutation in hCtBP1 in the GXGXXG motif (G183A) abolishes NADH responsiveness. This site in hCtBP2 is conserved (amino acids 187-192): it was asked whether NADH could regulate the Hdm2:hCtBP2 interaction. NADH concentrations (0.01 to 1 mM) known to promote the interaction of hCtBP1 with PXDLS motif proteins inhibit binding of full-length GST-hCtBP2 to Hdm2. This inhibition did not occur when either GST-hCtBP2(1-110), lacking the dehydrogenase domain, or hCtBP2(G189A), containing a mutation in the NADH binding site, were used in the assays. Therefore, in contrast to interactions with PXDLS motif proteins, the conformational changes induced by NADH binding to the CtBP dehydrogenase domain result in a reduced affinity of hCtBP2 for Hdm2. Exposure of cells in culture to CoCl₂ can be used as a model for the induction of a hypoxia-like stress response. CoCl₂ treatment (200 μM) induces an increase in the cellular NADH/NAD⁺ ratio sufficient to promote binding of CtBP proteins to PXDLS motif proteins in the cell. 200 μM CoCl₂ reduces the formation of Hdm2:hCtBP2 complexes in MCF-7 cells. Hypoxia, which has a greater effect on the cellular NADH/NAD⁺ ratio than CoCl₂, is more effective than CoCl₂ in reducing the Hdm2:hCtBP2 interaction. These data demonstrate, therefore, that the NADH-induced regulation of the Hdm2:hCtBP2 interaction also occurs in vivo (Mirnezami, 2003).

The distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, binds two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppresses binding of both SRF and YY1 complexes and abolishes DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppress MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and beta-galactosidase is induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter is no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration (L'honore, 2003).

The transcriptional repressor Tel plays an evolutionarily conserved role in angiogenesis: it is indispensable for the sprouting of human endothelial cells and for normal development of the Danio rerio blood circulatory system. Tel orchestrates endothelial sprouting by binding to the generic co-repressor, CtBP. The Tel-CtBP complex temporally restricts a VEGF (vascular endothelial growth factor)-mediated pulse of dll4 expression and thereby directly links VEGF receptor intracellular signalling and intercellular Notch-Dll4 signalling. It further controls branching by regulating expression of other factors that constrain angiogenesis such as sprouty family members and ve-cadherin. Thus, the Tel-CtBP complex conditions endothelial cells for angiogenesis by controlling the balance between stimulatory and antagonistic sprouting cues. Tel control of branching seems to be a refinement of invertebrate tracheae morphogenesis that requires Yan, the invertebrate orthologue of Tel. This work highlights Tel and its associated networks as potential targets for the development of therapeutic strategies to inhibit pathological angiogenesis (Roukens, 2010).

Homeodomain-interacting protein kinase-2 mediates CtBP phosphorylation and degradation in UV-triggered apoptosis

Homeodomain-interacting protein kinase-2 (HIPK2) is a serine/threonine kinase involved in transcriptional regulation and apoptosis. The transcriptional corepressor CtBP (carboxyl-terminal binding protein) also plays a fundamental role in these processes. HIPK2 has been shown to participate in a pathway of UV-triggered CtBP clearance that results in cell death. HIPK2 phosphorylates CtBP at Ser-422 in vitro. A Ser-422 phospho-specific antibody was developed to demonstrate that CtBP is phosphorylated on this residue in response to UV irradiation. HIPK2 knock-down blocks the UV-induced Ser-422 phosphorylation and degradation. The proteasomal inhibitor MG-132 treatment increases levels of ubiquitinated CtBP induced by UV. Interference with HIPK2 function via the kinase-dead mutant decreases CtBP ubiquitination. Furthermore, a phosphopeptide spanning Ser-422 blocks UV-triggered CtBP degradation, confirming that Ser-422 phosphorylation marks CtBP for clearance. Consequently, interference with HIPK2 action in H1299 cells rescues UV-triggered apoptosis (Zhang, 2004).

HIPK2 is a Ser/Thr kinase that has been shown previously to be activated by UV irradiation, TGF- ß treatment, Wnt signaling, and TAK1 action. It plays important roles in transcriptional regulation and promotes cell growth arrest and apoptosis. HIPK2 activation triggers Ser-46 phosphorylation on p53 and prevents MDM2-mediated ubiquitination and degradation of p53, resulting in the apoptotic response. A recent report also demonstrated that HIPK2 controls sensory neuron survival by suppressing the Brn3a-dependent transcription of Brn3a, TrkA, and Bcl-xL. Trigeminal sensory neurons, which are especially susceptible to HIPK2-induced apoptosis, express the highest levels of HIPK2 during the peak of apoptosis in vivo, supporting the idea that HIPK2 participates in programmed cell death in the developing peripheral nervous system. This study confirmed that the transcriptional corepressor CtBP is a HIPK2 target. In addition to stabilizing p53, HIPK2 phosphorylates Ser-422 on the antiapoptotic factor CtBP. This action destabilizes CtBP, thus providing an alternative pathway for programmed cell death. Of note, this pathway should be functional in p53-deficient cells, which includes most human tumors (Zhang, 2004).

Precisely how Ser-422 phosphorylation marks CtBP for clearance is uncertain. This modification on Ser-422 must be read and interpreted by the cellular machinery. It is likely that CtBP phosphorylation causes the recruitment of a particular E3 ligase that triggers ubiquitination. Alternatively, novel enzyme(s) and bridging factor(s) might play critical roles in CtBP clearance. Elucidating the mechanism of the phosphorylation-dependent degradation of CtBP is essential for understanding CtBP regulation (Zhang, 2004).

It was interesting that Ser-422 phosphorylation and ubiquitination was observed even in the absence of UV stimulation, suggesting that these modifications are a part of CtBP homeostasis. This finding is in agreement with the finding that the Ser-422 mutant of CtBP is detected at a higher level than the wild-type protein when both were expressed in a CtBP-null background. Whether basal activity of HIPK2 or other kinases are responsible for this action is unknown (Zhang, 2004)

C-terminal binding proteins are essential pro-survival factors that undergo caspase-dependent downregulation during neuronal apoptosis

C-terminal binding proteins (CtBPs) are transcriptional co-repressors that are subject to proteasome-dependent downregulation during apoptosis. Alternative mechanisms that regulate CtBP expression are currently under investigation and the role of CtBPs in neuronal survival is largely unexplored. This study shows that CtBPs are downregulated in cerebellar granule neurons (CGNs) induced to undergo apoptosis by a variety of stressors. Moreover, antisense-mediated downregulation of CtBP1 is sufficient to cause CGN apoptosis. Similarly, the CtBP inhibitor, 4-methylthio-2-oxobutyric acid, induces expression of the CtBP target Noxa and causes actinomycin-sensitive CGN apoptosis. Unexpectedly, it was found that the mechanism of CtBP downregulation in CGNs undergoing apoptosis varies in a stimulus-specific manner involving either the proteasome or caspases. In the case of CGNs deprived of depolarizing potassium (5K apoptotic condition), caspases appear to play a dominant role in CtBP downregulation. However, incubation in 5K does not enhance the kinetics of CtBP1 degradation and recombinant CtBP1 is not cleaved in vitro by caspase-3. In addition, 5K has no significant effect on CtBP transcript expression. Finally, mouse embryonic stem cells display caspase-dependent downregulation of CtBP1 following exposure to staurosporine, an effect that is not observed in DGCR8 knockout cells which are deficient in miRNA processing. These data identify caspase-dependent downregulation of CtBPs as an alternative mechanism to the proteasome for regulation of these transcriptional co-repressors in neurons undergoing apoptosis. Moreover, caspases appear to regulate CtBP expression indirectly, at a post-transcriptional level, and via a mechanism that is dependent upon miRNA processing. It is concluded that CtBPs are essential pro-survival proteins in neurons and their downregulation contributes significantly to neuronal apoptosis via the de-repression of pro-apoptotic genes (Stankiewicz, 2013).

Effects of CtBP mutation

Mice harboring mutations in both Ctbp1 and Ctbp2 were generated to address the in vivo function of CtBPs during vertebrate development. Ctbp1 mutant mice are small but viable and fertile, whereas Ctbp2-null mice show defects in axial patterning and die by E10.5 due to aberrant extraembryonic development. Mice harboring various combinations of Ctbp1 and Ctbp2 mutant alleles exhibit dosage-sensitive defects in a wide range of developmental processes. The strong genetic interaction, as well as transcription assays with CtBP-deficient cells, indicates that CtBPs have overlapping roles in regulating gene expression. It is suggested that the observed phenotypes reflect the large number of transcription factors whose activities are compromised in the absence of CtBP (Hildebrand, 2002).

Aberrant Wnt/beta-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, it was shown that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear beta-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing beta-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of beta-catenin require the additional activation of KRAS (K-ras). These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and beta-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human familial adenomatous polyposis adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear beta-catenin, whereas nuclear beta-catenin was detected in carcinomas (Phelps, 2009).

CtBP and the modification of chromatin

Binding of the C-terminal binding protein, CtBP, to the adenovirus E1A moiety of a Gal4-E1A fusion protein abolishes conserved region (CR) 1-dependent transcription activation. In contrast, a non-promoter targeted E1A peptide, capable of binding CtBP, can induce transcription from the proliferating cell nuclear antigen (PCNA) promoter (See Drosophila PCNA). CtBP is shown here to bind the histone deacetylase HDAC1, suggesting that a promoter targeted CtBP-HDAC1 complex can silence transcription from the PCNA promoter through a deacetylation mechanism. Expression of the CtBP binding domain of E1A is sufficient to alleviate repression, possibly due to the displacement of the CtBP-HDAC1 complex from the promoter (Sundqvist, 1998).

Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs, two-hybrid screens were performed with Xenopus Pc (XPc) and human Pc2 (HPC2). C-terminal binding protein (CtBP) interacts with XPc and HPC2; CtBP and HPC2 coimmunoprecipitate, and CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, it is found that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, CtBP is shown to be a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP (Sewalt, 1999).

Ikaros can repress transcription through the recruitment of histone deacetylase complexes. Ikaros can also repress transcription through its interactions with the co-repressor, C-terminal binding protein (CtBP). CtBP interacts with Ikaros isoforms through a PEDLS motif present at the N terminus of these proteins but not with homologs like Aiolos, which lack this motif. Mutations in Ikaros that prevent CtBP interactions reduce its ability to repress transcription. CtBP interacts with Sin3A (see Drosophila Sin3A) but not with the Mi-2 co-repressor and it represses transcription in a manner that is independent of histone deacetylase activity. These data strongly suggest that CtBP contributes to a histone deacetylase activity independent mechanism of repression by Ikaros. The viral oncoprotein E1A, which also binds to CtBP, shows a strong association with Ikaros. This Ikaros-E1A interaction may underlie Ikaros's decreased ability to repress transcription in E1A transformed cells (Koipally, 2000).

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. The amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs (Zhang, 2001).

The transcriptional co-repressor CtBP (C-terminal binding protein) is implicated in tumorigenesis because it is targeted by the adenovirus E1A protein during oncogenic transformation. Genetic studies have also identified a crucial function for CtBP in animal development. CtBP is recruited to DNA by transcription factors that contain a PXDLS motif, but the detailed molecular events after the recruitment of CtBP to DNA and the mechanism of CtBP function in tumorigenesis are largely unknown. A CtBP complex has been identified that contains the essential components for both gene targeting and coordinated histone modifications, allowing for the effective repression of genes targeted by CtBP. Inhibiting the expression of CtBP and its associated histone-modifying activities by RNA-mediated interference results in alterations of histone modifications at the promoter of the tumor invasion suppressor gene E-cadherin and increased promoter activity in a reporter assay. These findings identify a molecular mechanism by which CtBP mediates transcriptional repression and provide insight into CtBP participation in oncogenesis (Shi, 2003).

To understand how CtBP represses transcription, the biochemical purification of CtBP was undertaken. Stable HeLa cells were generated expressing human CtBP1 tagged with both the Flag and hemagglutinin (HA) epitopes at its amino terminus. Nuclear extracts from these cells were subjected to sequential purification with anti-Flag and anti-HA antibody columns. About 20 polypeptides were found to be specifically associated with the tagged CtBP1, whose identities were determined by mass spectrometry. These CtBP1- associated proteins can be grouped into at least four classes based on their functions, which include the following: the DNA-binding proteins such as ZEB 1, which have been shown to interact with CtBP and to repress transcription in a CtBP-dependent manner; the histone-modifying enzymes represented by the histone deacetylases HDAC1 and 2 and the related histone methyltransferases (HMTs) G9a and Eu-HMTase1 (EuHMT), and two chromodomain-containing proteins HPC2 and CDYL. The last group includes CoREST (a protein found as a co-repressor to the transcription factor REST; see Drosophila CoRest), a CoREST-related protein (KIAA1343) and a protein (KIAA0601) that shares sequence homology with polyamine oxidases, which is referred to as NPAO (nuclear polyamine oxidase). The presence of CoREST and its associated proteins in the CtBP complex suggests a possible interplay between these two co-repressors (Shi, 2003).

It is proposed that the action of the CtBP complex is initiated by the DNA-binding repressors that anchor the CtBP complex to its target promoters. This is followed by the action of HDAC1/2, which removes the acetyl group from the histone tails of transcriptionally active chromatin, allowing Lys 9 of histone H3 to be methylated by G9a and EuHMT. The methylated Lys 9 (and perhaps Lys 27) are then recognized by HPC2 and/or CDYL, which might contribute to the formation of a local repressive chromatin structure. These coordinated biochemical and enzymatic events help convert an active chromatin environment to one that is conducive to transcriptional repression. Biologically, the identification of the proteins involved in tumorigenesis in the CtBP complex, and the finding that CtBP and the associated HMTs repress transcription of the tumor invasion suppressor E-cad, shed significant light on the role of CtBP in tumorigenesis. Last, it is noted that the CtBP complex might contain additional enzymes. Both CtBP complex and recombinant CtBP possess dehydrogenase activity. In addition, NPAO is related to polyamine oxidases, and CDYL shares homology with enoyl CoA isomerases/hydratases. It is speculated that, whereas HDACs and HMTs are involved in histone modifications, the other putative enzymes might also have a role in CtBP-regulated transcription, perhaps by targeting non-histone components of the chromatin. Future studies aimed at delineating their functions and possible relationships with histone-modifying enzymes in the CtBP complex are likely to provide new insight into eukaryotic gene regulation (Shi, 2003).

Regulation of the brown and white fat gene programs through a PRDM16/CtBP transcriptional complex

Brown fat is a specialized tissue that can dissipate energy and counteract obesity through a pattern of gene expression that greatly increases mitochondrial content and uncoupled respiration. PRDM16 is a zinc-finger protein that controls brown fat determination by stimulating brown fat-selective gene expression, while suppressing the expression of genes selective for white fat cells. To determine the mechanisms regulating this switching of gene programs, native PRDM16 protein complexes were purified from fat cells. The PRDM16 transcriptional holocompex contains C-terminal-binding protein-1 (CtBP-1) and CtBP-2, and this direct interaction selectively mediates the repression of white fat genes. This repression occurs through recruiting a PRDM16/CtBP complex onto the promoters of white fat-specific genes such as resistin, and is abolished in the genetic absence of CtBP-1 and CtBP-2. In turn, recruitment of PPAR-γ-coactivator-1α (PGC-1α) and PGC-1β to the PRDM16 complex displaces CtBP, allowing this complex to powerfully activate brown fat genes, such as PGC-1α itself. These data show that the regulated docking of the CtBP proteins on PRDM16 controls the brown and white fat-selective gene programs (Kajimura, 2008).

Despite sharing the ability to accumulate triglycerides, the physiological roles of WAT and BAT are almost diametrically opposite. WAT stores most of the excess energy in mammals and also contributes to systemic energy balance through releasing a variety of adipokines in response to changes in energy status. In contrast, BAT plays an important role in energy expenditure by dissipating chemical energy in response to cold or excess feeding. BAT is not thought to play a substantial role in systemic signaling both because the mass of this tissue is much smaller than WAT and because not all the adipokines are expressed in BAT. This study shows that PRDM16 coordinately represses the expression of a wide range of WAT-selective genes in a CtBP-dependent manner, while simultaneously activating BAT-selective genes responsible for mitochondrial biogenesis and oxidative metabolism (Kajimura, 2008).

Notably, PRDM16 directly suppresses at least two important WAT-selective adipokines: resistin and angiotensinogen. Resistin is increased in diet-induced and genetic obesity, and inhibits the action of insulin on hepatic glucose production. Angiotensinogen, a unique substrate of rennin, is a precursor of the vasoactive peptide angiotensin II. WAT is a major extrahepatic synthesis site of angiotensinogen, which shows positive correlation with body mass index and can cause high blood pressure. In addition to these two well-known WAT-selective molecules, PRDM16 also represses serpina3k, a WAT-selective secreted protein that belongs to serpin (serine protease inhibitors) family. Although the function of serpina3k is unknown, two adipokines, PAI-1 (plasimogen inhibitor-1) and vaspin (visceral adipose tissue-derived serpin), are also serpin members, and are associated with obesity-linked cardiovascular disease and insulin resistance, respectively (Kajimura, 2008).

This study shows a nearly absolute requirement for the CtBPs in the PRDM16-mediated repression of white fat genes by two independent lines of experimental evidence: (1) A mutant PRDM16 that no longer binds CtBPs loses the ability to repress nearly the entire set of white fat-selective genes regulated by wild-type PRDM16. (2) Genetic ablation of the CtBPs causes a loss of wild-type PRDM16’s suppressive effects on white fat-selective gene expression (Kajimura, 2008).

CtBP was originally identified as a phosphoprotein that bound the C-terminal region of the human adenovirus EIA protein. It has been shown that CtBP functions predominantly as a transcriptional repressor by associating with other corepressors and histone modifying enzymes, including class I histone deacetylases HDAC1/2, histone methyltransferases (HMTs, G9a, and GLP/Eu-HMT1), histone lysine-specific demethylase (LSD1), and CoREST. The repressive effects are thought to occur through coordinate histone modifications by HDACs and G9a/GLP actions on deacetylation and methylation of H3K9, and also through additive demethylation on H3K4 by LSD1. Notably, PRDM16 also contains a SET domain, a structural hallmark of histone lysine methyltransferase shared by G9a, GLP, SETDB1, and Suv39h1/2. An aberrant form of PRDM16 lacking PR-domain (SET domain) is expressed exclusively in adult T-cell leukemia cells. This short form of PRDM16, but not the regular form of PRDM16 possesses 'oncogenic' potential. The functional importance of the SET domain in PRDM16 for silencing gene expression in the context of brown fat determination remains unanswered (Kajimura, 2008).

PGC-1α and PGC-1β are critical molecules in the brown fat phenotype. PGC-1α is a major coactivator of the thermogenic program of BAT in response to cAMP signaling, but is not required for the identity of brown fat cells. Double deficiency in PGC-1α and PGC-1β, however, causes a near total loss of the molecular identity of brown fat cells, suggesting an absolute requirement for PGC-1 coactivators in the brown fat program. A previous study has shown that PRDM16 robustly induced brown fat gene expression at least in part by directly binding to and stimulating both PGC-1α and PGC-1β. In this study, it was also shown that PGC-1α and/or β directly compete with CtBP for binding to the PRDM16 complex. Although PGC-1s and CtBP bind to the distinct domains of the PRDM16 molecule in the primary sequence, they presumably compete at the level of three-dimensional structure of PRDM16. These results suggest a model in which PRDM16 represses white fat-selective gene expression including resistin and angiotensinogen by recruiting CtBP onto their promoters. Recruitment of PGC-1α/β to PRDM16 complex displaces CtBP, leading to the activation of the brown fat-selective genes such as PGC-1α itself. The binding of PGC-1s or CtBP to PRDM16 is mutually exclusive. Obviously, there must be at least some other components, most likely a transcription factor, onto which PRMD16 is recruited and determines whether PRDM16 will bind either to CtBP or PGC-1 coactivators (Kajimura, 2008).

It also seems likely that this switching mechanism might be affected by signal transduction pathways. Since BAT is highly innervated by the sympathetic nerve system, activation of β-adrenergic receptors by catecholamines is essential for many aspects of brown fat development and thermogenic program, including UCP-1 gene expression. Upon stimulation, an increase in intracellular cAMP levels activates cAMP-dependent protein kinase A (PKA) and its downstream p38MAPK (mitogen-activated protein kinase), which leads to the phosphorylations of PGC-1α and ATF-2/CRE. This phosphorylation event activates the recruitment of PGC-1α and ATF-2/CRE onto the regulatory regions of the PGC-1α gene itself and the UCP-1 gene, inducing their transcription. cAMP signaling augments the activity of PRDM16 to induce both PGC-1α and UCP-1 gene expression. PKA directly phosphorylates CtBP-1 at T144 and modulates the association with GCN5 and SF-1. Thus, it is conceivable that modulations of PRDM16 by cAMP signaling and other pathways may influence the ability of PRDM16 to form complexes with PGC-1α/β and/or other coregulator proteins, to fine tune the activation of the brown fat gene program. Investigating this aspect will be necessary for fully understanding the transcriptional mechanisms of brown fat determination (Kajimura, 2008).

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Search PubMed for articles about Drosophila C-terminal binding protein

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