InteractiveFly: GeneBrief

Chip : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Many genes with complex developmental regulation contain multiple enhancers, the binding sites for transcription factors that function at quite a distance from gene coding sequences. It is thought that higher eukaryotes possess factors that facilitate remote enhancer-promoter interactions. Such enhancer-facilitators may be envisioned as helping to form chromatin structures that bring enhancers and promoters closer together; they are different from enhancer-binding activators, coactivators, and basal factors in that they do not participate directly in the activation reaction. Enhancers can interact with proximal promoters from distances of thousands of base pairs. The function of enhancers is disrupted by the Drosophila protein Suppressor of Hairy-wing (Su[Hw]). Su (Hw) binds a DNA sequence in the gypsy retrotransposon and prevents distal enhancers with intervening gypsy insertions from activating target genes without affecting promoter-proximal enhancers. Several observations indicate that su(Hw) does not affect enhancer-binding activators. Instead, su(Hw) may interfere with factors that structurally facilitate interactions between an enhancer and promoter.

To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped (sd), mastermind,(mam) and a previously unknown gene, Chip, have been isolated. A TEA DNA-binding domain in the Scalloped protein binds the cut wing margin enhancer. Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).

The data suggest that sd and mam encode enhancer-binding factors and that Chip may encode an enhancer-facilitator. Both sd and mam mutants display stronger genetic interactions with wing margin enhancer deletions than with gypsy insertions in cut (gypsy insertions block enhancers with the help of su(Hw), a transcription factor that binds the gypsy retrovirus). Chip is also needed for wing margin enhancer activity but appears to play a unique role. Chip is normally required for wing margin enhancer function because Chip mutations enhance the cut wing phenotype of cut mutants. However, in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. In a Chip heterozygote (with the wild-type chromosome able to carry out Chip mediated activation of cut), a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) protein bound to a gypsy insertion in one cut allele acts in a transvection-like manner (one chromosome influencing the activity of the second) to block the wing margin enhancer in the wild-type cut allele on the other chromosome. The simplest interpretation is that Chip protein facilitates enhancer-promoter communication and su(Hw) on one chromosome interferes with Chip mediated enhancer-promoter communication on both chromosomes (Morcillo, 1996 and 1997).

Chip is a LIM protein interactor, as are Chip vertebrate homologs. Chip interacts directly with the LIM domains of Apterous. Chip maternal mutations play a role in segmentation, and evidence supports a role for Chip in regulating the gap gene giant, and possibly the pair-rule gene even-skipped. Moreover, Chip regulates expression of cut and Ultrabithorax during imaginal disc development; these genes are not known to be regulated by LIM domain proteins. Although the role(s) of LIM domain proteins in early Drosophila development is currently unknown, it is possible that LIM domain proteins play broader roles in development than appreciated previously, and that several unknown LIM domain proteins are required for segmentation and imaginal disc development. Another possibile explanation for the broad functions of Chip is that it may interact with other proteins without LIM domains. The two mouse Chip homologs, Nli/Lbd1/Clim-2 and Clim-1 interact directly with P-Otx, a homeodomain protein that lacks LIM domains (Morcillo, 1997).

Does Chip play an novel role in enhancer functions different from that played by transcriptional co-activators? Transcriptional co-activators may be thought of as proteins that serve as a bridge interacting with transcription factors and activating the transcriptional apparatus. Chip and its vertebrate homologs appear to regulate interactions between different transcriptional activator proteins and may function at enhancers to bring together diverse transcriptional factors and form higher order activation complexes; in some cases, to block formation of such complexes. Specific antagonism between Chip and suppressor of Hairy wing suggests a role for Chip in enhancer-promoter communications. The diverse roles suggested for Chip suggest a distinction between Chip and roles for transcriptional co-activators whose targets are thought to be the transcriptional apparatus (Morcillo, 1997).

Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip. Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers (Torigoi, 2000).

Full-length Chip interacts with the HD proteins Bicoid (Bcd) and Ftz, and with a fragment of the Su(Hw) insulator protein. The HD protein Otd binds almost as efficiently as does Bcd and Ftz to Chip, but the Eve HD protein binds poorly, a result possibly attributable to improper folding of the in vitro-translated protein. The domains of Chip involved in homotypic and heterotypic interactions include the LIM interaction domain (LID) and the self-interaction domain (SID). Deletion of the LID reduces interaction with Apterous. That deletion, however, has no effect on interaction with Bcd, Ftz, Su(Hw)DeltaCTD, or Chip. In contrast, two other deletion mutants, ChipDelta404-465 and ChipDelta441-454, reduce binding to Bcd, Ftz, Su(Hw)DeltaCTD, and Chip but have little effect on binding to Apterous. On the basis of this and additional deletion mutants, Chip residues 439-456 are identified as the region that interacts with the HD proteins, Su(Hw), and with Chip itself. This region is termed the other interaction domain (OID) (Torigoi, 2000).

Previous studies have suggested that the SID is sufficient for self-interaction of Chip, but Chip self-interaction is reduced by deletions affecting the OID (ChipDelta404-465 and ChipDelta441-454) but is unaffected by a deletion that removes much of the SID (ChipDelta294-381). An isolated SID fragment (ChipDelta404-519) interacts with itself but does not interact well with intact Chip, whereas a Chip fragment lacking the SID (ChipDelta2-381) interacts both with itself and with intact Chip. Experiments that show interactions between the SID and intact Chip were performed by translating the two interaction partners together in vitro. Evidently, cotranslation permits an interaction not seen by affinity chromatography. It is concluded that Chip interacts with itself through both the SID and the OID (Torigoi, 2000).

The domains of Bcd and Su(Hw) that interact with Chip were mapped to determine if the OID recognizes a common motif in its diverse interaction partners. The N-terminal half of Bcd (residues 1-255) contains the HD and everything needed to rescue bcd mutants in vivo. The N-terminal half of Bcd interacts with Gst-Chip, whereas the C-terminal half (residues 246-489) does not. Smaller Bcd fragments containing the HD (residues 1-190, 1-166 or 57-255) bind more weakly than does the 1-255 fragment, and a fragment (residues 57-166) consisting mostly of the HD (residues 92-151) does not bind. Thus, residues on both sides of the HD are required for strong binding. Similar results were obtained with the Otd HD protein. The region of Su(Hw) that contains 12 zinc fingers (residues 204-672) interacts with Gst-Chip, whereas the N-terminal region (residues 1-190) and the C-terminal region (residues 706-944) do not. Mutation of any one of the 12 zinc fingers does not significantly affect binding to Chip. The regions of Bcd, Su(Hw), and Chip that interact with the Chip OID are not homologous at the primary sequence level (Torigoi, 2000).

The interactions between Chip and HD proteins in vitro raise the question of whether Chip affects the activities of HD proteins in vivo. The effect of Chip on Bcd activity in embryos was tested because both Chip and Bcd are provided maternally and do not regulate each other's expression. Thus, any effect of Chip on Bcd is likely to be direct. The design of the experiment that shows that in embryos reducing Chip activity decreases the activity of a partially defective Bcd protein, was guided by the following considerations. To demonstrate a helping effect of Chip on Bcd activity, maternal Chip could not be simply eliminated because that manipulation results in a more severe segmentation defect than does elimination of Bcd itself. Nor could the dosage of maternal Chip be halved because that change has no effect, even if the maternal Bcd level is also reduced by one-half. Moreover, zygotic Bcd makes no contribution to segmentation; heretofore, no effect has been seen on segmentation by changing the level or nature of zygotically expressed Chip. To detect an effect of Chip on Bcd activity, therefore, the activities of both Bcd and Chip were reduced to less than that provided by a single maternal dose of each. This was accomplished by producing doubly mutant mothers: these mothers were homozygous for the bcd^E3 allele, which encodes a mutant with reduced DNA-binding activity, and were also heterozygous for the Chip^g96.1 allele. This latter mutant allele encodes the SID fragment, which acts as a dominant negative, inhibiting, but not eliminating, maternal Chip activity. It was deduced that the SID fragment inhibits maternal Chip activity from the observations that Chip^g96.1/Chip^g96.1 embryos produced by Chip^g96.1/+ mothers die before reaching the larval stage (some display a mild segmentation defect), whereas all Chip^-/Chip^- embryos produced by Chip^-/+ mothers segment normally and die as larvae. It was further deduced that at least one maternal and two zygotic doses of the SID fragment are required to cause embryonic lethality from the fact that Chip^g96.1/+ embryos from Chip^g96.1/+ mothers segment normally and survive to adulthood. Presumably the SID fragment, produced in this experiment both maternally and zygotically, forms nonfunctional multimers with maternal wild-type Chip. On average, embryos from Chip^g96.1/+; bcd^E3/bcd^E3 mothers (and wild-type fathers) produce nearly one segment less than do embryos from bcd^E3/bcd^E3 mothers (Torigoi, 2000).

These results suggest that Chip increases interactions between Bcd molecules. Thus, in yeast with nonsaturating levels of Bcd, Chip increases activation by Bcd from two strong binding sites separated by a weak site or by a nonbinding spacer, but not from one or three contiguous strong sites. Moreover, Chip does not increase activation above levels that are achieved with high concentrations of Bcd itself. Bcd binds DNA cooperatively, mediated by interactions of regions overlapping those that interact with Chip, and it is suggested that Chip interacts with Bcd to amplify that cooperativity. It is unlikely that Chip itself is a transcriptional activator. Previous experiments have shown that Chip does not activate when tethered upstream of yeast promoters but it can induce activation by recruiting an activation domain fused to LIM domains (Torigoi, 2000).

The idea that Chip increases interactions between certain other proteins agrees with all previous observations on Chip and its homologs. In transient transfection experiments with mammalian cells, Chip homologs increased transcriptional activation by the combination of the P-Otx HD and the Lhx3 LIM-HD proteins from a promoter containing a single binding site for each molecule. The Chip homologs have little effect with P-Otx or Lhx3 alone, indicating that they aid P-Otx-Lhx3 interactions. Furthermore, the nuclear LIM interactor (Nli) homolog of Chip aids formation of different LIM-HD protein dimers in vitro, an effect requiring the Nli SID. Finally, an Apterous-Chip fusion protein, in which the LIM domains of Apterous are replaced by the Chip SID, can replace wild-type Apterous in Drosophila wings, suggesting that Chip aids formation of Apterous dimers in vivo (Torigoi, 2000 and references therein).

Chip potentiates Bcd activity in the Drosophila embryo when the Bcd activity is low. This effect is consistent with previous studies on the expression of segmentation genes in embryos lacking maternal Chip activity. Embryos contain a gradient of Bcd protein, with a high concentration at the anterior end and a low concentration at the posterior end. Loss of maternal Chip strongly reduces all seven blastoderm stripes of Eve protein produced by the eve pair-rule gene. Many, if not all of these stripes are also regulated by Bcd, even though most occur in regions with low to intermediate Bcd concentrations. The eve stripes are activated by several remote enhancers located ~1.5-9 kb from the promoter, and Bcd-binding sites are critical for activation by at least the stripe 2 enhancer. It is likely, therefore, that Chip increases eve expression at least in part by increasing binding of Bcd to the enhancers. Accumulation of the Hb protein is not substantially affected by loss of maternal Chip even though hb expression is dependent on Bcd and several Bcd-binding sites just upstream of the promoter. This lack of an effect of Chip is not unexpected, however, because hb is expressed in the anterior end where the Bcd concentration is the highest (Torigoi, 2000 and references therein).

It is suggested that Chip plays two roles in the regulation of gene expression: (1) Chip is likely to aid binding of proteins to enhancers, and (2) Chip is also likely to function between enhancers and promoters to support enhancer-promoter communication. The in vitro interaction between Chip and the Su(Hw) insulator protein shown here is consistent with the notion that Su(Hw) is directly antagonistic to Chip activity as previously demonstrated genetically at the cut locus. It remains to be seen how, if these speculations are correct, Chip facilitates enhancer-promoter communication and how that communication is disrupted by Su(Hw). It is believed that Su(Hw) blocks activation not by reducing the binding of proteins to enhancers, but rather by hindering enhancer-promoter communication. For instance, an enhancer blocked in its interaction with one promoter by Su(Hw) can nevertheless activate a second promoter located on the opposite side of the enhancer from Su(Hw). Thus, although Su(Hw) is antagonistic to Chip, it is unlikely to affect binding of proteins to enhancers. It is also unlikely that Chip functions merely by preventing binding of Su(Hw) to gypsy because Chip is also important for the expression of several genes, e.g., cut and eve, in the absence of gypsy and Su(Hw) (Torigoi, 2000).

An ancient Pygo-dependent Wnt enhanceosome integrated by Chip/LDB-SSDP

TCF/LEF factors (see Drosophila Pangolin) are ancient context-dependent enhancer-binding proteins that are activated by β-catenin (see Drosophila Armadillo) following Wnt signaling. They control embryonic development and adult stem cell compartments, and their dysregulation often causes cancer. β-catenin-dependent transcription relies on the NPF motif of Pygo proteins. This study used a proteomics approach to discover the Chip/LDB-SSDP (ChiLS) complex as the ligand specifically binding to NPF. ChiLS also recognizes NPF motifs in other nuclear factors including Runt/RUNX2 and Drosophila ARID1, and binds to Groucho/TLE. Studies of Wnt-responsive dTCF enhancers in the Drosophila embryonic midgut indicate how these factors interact to form the Wnt enhanceosome, primed for Wnt responses by Pygo. Together with previous evidence, this study indicates that ChiLS confers context-dependence on TCF/LEF by integrating multiple inputs from lineage and signal-responsive factors, including enhanceosome switch-off by Notch. Its pivotal function in embryos and stem cells explain why its integrity is crucial in the avoidance of cancer (Fiedler, 2015).

TCF/LEF factors (TCFs) were discovered as context-dependent architectural factors without intrinsic transactivation potential that bind to the T cell receptor α (TCRα) enhancer via their high mobility group (HMG) domain. They facilitate complex assemblies with other nearby enhancer-binding proteins, including the signal-responsive CRE-binding factor (CREB) and the lineage-specific RUNX1 (also called Acute Myeloid Leukemia 1, AML1). Their activity further depends on β-catenin, a transcriptional co-factor activated by Wnt signaling, an ancient signaling pathway that controls animal development and stem cell compartments, and whose dysregulation often causes cancer. The context-dependence of TCFs is also apparent in other systems, for example in the embryonic midgut of Drosophila where dTCF integrates multiple signaling inputs with lineage-specific cues during endoderm induction. The molecular basis for this context-dependence remains unexplained (Fiedler, 2015).

In the absence of signaling, T cell factors (TCFs) are bound by the Groucho/Transducin-like Enhancer-of-split (Groucho/TLE) proteins, a family of co-repressors that silence TCF enhancers by recruiting histone deacetylases (HDACs) and by 'blanketing' them with inactive chromatin. TLEs are displaced from TCFs by β-catenin following Wnt signaling, however this is not achieved by competitive binding but depends on other factors. One of these is Pygopus (Pygo), a conserved nuclear Wnt signaling factor that recruits Armadillo (Drosophila β-catenin) via the Legless/BCL9 adaptor to promote TCF-dependent transcription. Intriguingly, Pygo is largely dispensable in the absence of Groucho, which implicates this protein in alleviating Groucho-dependent repression of Wg targets (Fiedler, 2015).

Pygo has a PHD and an N-terminal asparagine proline phenylalanine (NPF) motif, each essential for development and tissue patterning. Much is known about the PHD finger, which binds to Legless/BCL9 and to histone H3 tail methylated at lysine 4 via opposite surfaces that are connected by allosteric communication. By contrast, the NPF ligand is unknown, but two contrasting models have been proposed for its function (1">Figure 1) (Fiedler, 2015).

This study used a proteomics approach to discover that the NPF ligand is an ancient protein complex composed of Chip/LDB (LIM-domain-binding protein) and single-stranded DNA-binding protein (SSDP), also called SSBP. This complex controls remote Wnt- and Notch-responsive enhancers of homeobox genes in flies (Bronstein, 2011), and remote enhancers of globin and other erythroid genes in mammals, integrating lineage-specific inputs from LIM-homeobox (LHX) proteins and other enhancer-binding proteins. Using nuclear magnetic resonance (NMR) spectroscopy, this study demonstrated that Chip/LDB-SSDP (ChiLS) binds directly and specifically to Pygo NPFs, and also to NPF motifs in Runt-related transcription factors (RUNX) proteins and Osa (Drosophila ARID1), whose relevance is shown by functional analysis of Drosophila midgut enhancers. Furthermore, Groucho was identified as another new ligand of ChiLS by mass spectroscopy. This study thus define the core components of a Wnt enhanceosome assembled at TCF enhancers via Groucho/TLE and RUNX, primed for timely Wnt responses by ChiLS-associated Pygo. The pivotal role of ChiLS in integrating the Wnt enhanceosome provides a molecular explanation for the context-dependence of TCFs (Fiedler, 2015).

The discovery of ChiLS as the NPF ligand of Pygo proteins led to the definition of the core components of a multi-protein complex tethered to TCF enhancers via Groucho/TLE and RUNX, and slated for subsequent Wnt responses by Pygo (see Model of the Wnt enhanceosome). ChiLS also contacts additional enhancer-binding proteins via its LID, including lineage-specific and other signal-responsive factors, thereby integrating multiple position-specific inputs into TCF enhancers, which provides a molecular explanation for the context-dependence of TCF/LEF. This complex will be referred to as the Wnt enhanceosome since it shares fundamental features with the paradigmatic interferon β-responsive enhanceosome (Panne, 2007). Its components are conserved in placozoa, arguably the most primitive animals without axis and tissues with only a handful of signaling pathways including Wnt, Notch and TGF-β/SMAD, suggesting that the Wnt enhanceosome emerged as the ur-module integrating signal-responses (Fiedler, 2015).

Other proteins have been reported to interact with the Pygo N-terminus, but none of these recognize NPF. It is noted that this N-terminus is composed of low-complexity (intrinsically disordered) sequences that are prone to non-specific binding (Fiedler, 2015).

NPF is a versatile endocytosis motif that binds to structurally distinct domains, including eps15 homology (EH) domains in epsin15 homology domain (EHD) protein. Indeed, EHDs were consistently identified in lysate-based pull-downs with triple-NPF baits. EHDs are predominantly cytoplasmic, and do not interact with nuclear Pygo upon co-expression, nor are any of the Drosophila EHDs required for Wg signaling in S2 cells. ChiLS is the first nuclear NPF-binding factor (Fiedler, 2015).

NPF binding to ChiLS appears to depend on the same residues as NPF binding to EHD domains, that is, on the aromatic residue at +2, the invariant P at +1, N (or G) at 0 and NPF-adjacent residues, including negative charges at +3 and +4 (whereby a positive charge at +3 abolishes binding to EHD). Indeed, an intramolecular interaction between the +3 side-chain and that of N predisposes NPF to adopt a type 1 β-turn conformation, which increases its affinity to the EHD pocket, while the -1 residue undergoes an intermolecular interaction with this pocket. ChiLS also shows a preference for small residues at -1 and -2, similarly to N-terminal EHDs although RUNX seems to differ at -1 and -2 from Pygo and MACC1 (F/L A/E/D vs S A, respectively) (Fiedler, 2015).

Groucho/TLE is recruited to TCF via its Q domain, which tetramerizes. Intriguingly, the short segment that links two Q domain dimers into a tetramer is deleted in a dTCF-specific groucho allele that abolishes dTCF binding and Wg responses, suggesting that TCF may normally bind to a Groucho/TLE tetramer (Fiedler, 2015).

Groucho/TLE uses its second domain, the WD40 propeller, to bind to other enhancer-binding proteins on Wnt-responsive enhancers, most notably to the C-terminal WRPY motif of RUNX proteins (common partners of TCFs in Wnt-responsive enhancers). This interaction can occur simultaneously with the WD40-dependent binding to ChiLS, given the tetramer structure of Groucho/TLE. In turn, RUNX uses its DNA-binding Runt domain to interact with HMG domains of TCFs, and to recruit ChiLS. RUNX thus appears to be the keystone of the Wnt enhanceosome since it binds to the enhancer directly while undergoing simultaneous interactions with Groucho/TLE (through its C-terminal WRPY motif), TCF and ChiLS (though its Runt domain) (Fiedler, 2015).

In line with this, Runt has pioneering functions in the early Drosophila embryo, shortly after the onset of zygotic transcription, and in the naïve endoderm as soon as this germlayer is formed, in each case prior to the first Wg signaling events. RUNX paralogs also have pioneer-like functions in specifying cell lineages, that is, definitive hematopoiesis in flies and mammals (Fiedler, 2015).

The model predicts that ChiLS, once tethered to the enhanceosome core complex, recruits Pygo via NPF to prime the enhancer for Wnt responses (see Model of the Wnt enhanceosome). Given the dimer-tetramer architecture of ChiLS, its binding to Pygo can occur simultaneously to its NPF-dependent binding to RUNX. In turn, tethering Pygo to the Wnt enhanceosome may require Pygo's binding to methylated histone H3 tail, similarly to Groucho/TLE whose tethering to enhancers depends on binding to hypoacetylated histone H3 and H4 tails. Interestingly, Pygo's histone binding requires at least one methyl group at K4-the hallmark of poised enhancers. Indeed, Drosophila Pygo is highly unorthodox due to an architectural change in its histone-binding surface that allows it to recognize asymmetrically di-methylated arginine 2-a hallmark of silent chromatin. Thus, the rare unorthodox Pygo proteins may recognize silent enhancers even earlier, long before their activation, consistent with the early embryonic function of Pygo, prior to Wg signaling (Fiedler, 2015).

Overcoming the OFF state imposed on the enhancer by Groucho/TLE involves Pygo-dependent capturing of β-catenin/Armadillo, which recruits various transcriptional co-activators to its C-terminus. Although these include CREB-binding protein (CBP), a histone acetyl transferase, its tethering to TCF enhancers is likely to co-depend on CRE-binding factors (CREB, c-Fos) and SMAD which synergize with Armadillo to activate these enhancers-similarly to the interferon-β enhanceosome where CBP recruitment also co-depends on multiple enhancer-binding proteins (Panne, 2007). The ensuing acetylation of the Wnt enhancer chromatin could promote the eviction of Groucho/TLE whose chromatin anchoring is blocked by acetylation of histone H3 and H4 tails, thus initiating the ON state (Fiedler, 2015).

Osa antagonizes Wg responses throughout development, and represses UbxB through its CRE, which also mediates repression in response to high Wg signaling. Osa could therefore terminate enhancer activity, by displacing HAT-recruiting enhancer-binding proteins such as CREB and c-Fos from CREs and by cooperating with repressive enhancer-binding proteins such as Brinker (a Groucho-recruiting repressor that displaces SMAD from UbxB) to re-recruit Groucho/TLE to the enhancer, thereby re-establishing its OFF state. Notably, Osa binds Chip, to repress various Wg and ChiLS targets including achaete-scute and dLMO (Fiedler, 2015).

Therefore, ChiLS is not only a coincidence detector of multiple enhancer-binding proteins and NPF proteins, but also a switch module that exchanges positively- and negatively-acting enhancer-binding proteins (through LID) and NPF factors, to confer signal-induced activation, or re-repression. Its stoichiometry and modularity renders it ideally suited to both tasks. It is noted that the interferon-β enhanceosome does not contain a similar integrating module, perhaps because it is dedicated to a single signaling input (Fiedler, 2015).

ChiLS is essential for activation of master-regulatory genes in the early embryo, similarly to DNA-binding pioneer factors such as Zelda (in the Drosophila embryo) or FoxA (in the mammalian endoderm) which render enhancers accessible to enhancer-binding proteins. Moreover, ChiLS maintains HOX gene expression throughout development, enabling Wg to sustain HOX autoregulation, a mechanism commonly observed to ensure coordinate expression of HOX genes in groups of cells (Fiedler, 2015).

Another hallmark of pioneer factors is that they initiate communication with the basal transcription machinery associated with the promoter. Chip is thought to facilitate enhancer-promoter communication, possibly by bridging enhancers and promoters through self-association. Indeed, Ldb1 occupies both remote enhancers and transcription start sites (e.g., of globin genes and c-Myb), likely looping enhancers to the basal transcription machinery at promoters which requires self-association, but possibly also other factors (such as cohesin, or mediator) (Fiedler, 2015).

It is noted that the chromatin association of Ldb1 has typically been studied in erythroid progenitors or differentiated erythroid cells, following activation of erythoid-specific genes. It would be interesting (if technically challenging) to examine primitive cells, to determine whether ChiLS is associated exclusively with poised enhancers prior to cell specification or signal responses (Fiedler, 2015).

Previous genetic analysis in Drosophila has linked chip predominantly to Notch-regulated processes. Likewise, groucho was initially thought to be dedicated to repression downstream of Notch, before its role in antagonizing TCF and Wnt responses emerged. Moreover, Lozenge facilitates Notch responses in the developing eye, and in hematocytes. Indeed, the first links between Groucho/TLE, RUNX and nuclear Wnt components came from physical interactions, as in the case of ChiLS. The current work indicates that these nuclear Notch signaling components constitute the Wnt enhanceosome. Although the most compelling evidence for this notion is based on physical interactions, the genetic evidence from Drosophila is consistent with a role of ChiLS in Wg responses (Bronstein, 2010). Indeed, mouse Ldb1 has been implicated in Wnt-related processes, based on phenotypic analysis of Ldb1 knock-out embryos and tissues. Notably, Ldb1 has wide-spread roles in various murine stem cell compartments that are controlled by Wnt signaling (Fiedler, 2015).

An interesting corollary is that the Wnt enhanceosome may be switchable to Notch-responsive, by NPF factor exchange and/or LMO-mediated enhancer-binding protein exchange at ChiLS. Hairy/Enhancer-of-split (HES) repressors could be pivotal for this switch: these bHLH factors are universally induced by Notch signaling, and they bind to ChiLS enhancers to re-recruit Groucho/TLE via their WRPW motifs. HES repressors may thus be capable of re-establishing the OFF state on Wnt enhancers in response to Notch (Fiedler, 2015).

Notably, restoring a high histone-binding affinity in Drosophila Pygo by reversing the architectural change in its histone-binding surface towards human renders it hyperactive towards both Wg and Notch targets even though pygo is not normally required for Notch responses in flies. Humanized Pygo may thus resist the Notch-mediated shut-down of the Wnt enhanceosome, owing to its elevated histone affinity that boosts its enhancer tethering, which could delay its eviction from the enhanceosome by repressive NPF factors. The apparent Notch-responsiveness of the Wnt enhanceosome supports the notion that orthodox Pygo proteins (as found in most animals and humans) confer both Wnt and Notch responses (Fiedler, 2015).

Previous genetic studies have shown that the components of the Wnt enhanceosome (e.g., TCF, RUNX, ChiLS and LHX) have pivotal roles in stem cell compartments, as already mentioned, suggesting a universal function of this enhanceosome in stem cells. It is therefore hardly surprising that its dysregulation, that is, by hyperactive β-catenin, is a root cause of cancer, most notably colorectal cancer but also other epithelial cancers. Indeed, genetic evidence implicates almost every one of its components (as inferred from the fly counterparts) in cancer: AML1 and RUNX3 are tumour suppressors whose inactivation is prevalent in myeloid and lymphocytic leukemias, and in a wide range of solid tumors including colorectal cancer, respectively. Likewise, ARID1A is a wide-spread tumor suppressor frequently inactivated in various epithelial cancers. Furthermore, many T-cell acute leukemias can be attributed to inappropriate expression of LMO2. Intriguingly, AML1 and ARID1A behave as haplo-insufficient tumor suppressors, consistent with the notion that these factors compete with activating NPF factors such as Pygo2, RUNX2 and possibly MACC1 (predictive of metastatic colorectal cancer) for binding to ChiLS, which will be interesting to test in future. The case is compelling that the functional integrity of the Wnt enhanceosome is crucial for the avoidance of cancer (Fiedler, 2015).

REGULATION

Targets of Activity

Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. The unsegmented phenotype of Chip mutant germ-line clone embryos resembles that displayed by embryos homozygous for null alleles of the pair-rule gene even-skipped. Eve is expressed in Chip mutants but the pattern is abnormal. There are stripes, but the number is often less than the seven found in wild type embryos. The first stripe to appear is often wider and stronger than in wild type, whereas later-appearing stripes are narrow, weaker, and more uneven than wild type. Pair rule genes are regulated by gap genes. Lack of active Chip affects Giant more severely than the other gap proteins. In wild-type precellular and early cellular blastoderm embryos, Gt is restricted to two broad bands, whereas in Chip germ-line clone embryos, Gt is expressed at low to moderate levels in the entire embryo, including the pole cells. At later stages, Gt expression is similar to wild type. Lack of Giant in embryos lacking active Chip can explain the decreased expression of Kruppel and Knirps because Gt represses Kr and kni. It is conceivable that abnormal Gt expression also weakens Eve stripe 2 (Morcillo, 1997).

Transcriptional regulation by CHIP/LDB complexes

It is increasingly clear that transcription factors play versatile roles in turning genes 'on' or 'off' depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. This study used the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, 189 down-stream targets of CHIP/LDB were identified; these genes are enriched for the binding sites of Apterous (AP) and Pannier (PNR), two well studied transcription factors associated with CHIP/LDB complexes. Extensive genetic screens were performed and target genes were identified that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing, novel roles were uncovered for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, these results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development (Bronstein, 2011).

Drosophila SSDP was identified on the basis of its ability to bind the nuclear adaptor protein CHIP/LDB (van Meyel, 2003; Chen, 2002). Both nuclear localization of SSDP and its ability to modulate the transcription activity of the CHIP-AP complex during wing development depend on its interaction with CHIP/LDB. This study implemented a combination of molecular, bioinformatic and genetic approaches that allowed has led to insight into the effect of SSDP on the transcriptional activity of CHIP/LDB complexes and their role in development. A genome wide screen was conducted for SSDP target genes in Drosophila using expression microarrays with mRNA isolated from larvae bearing hypomorphic alleles of ssdp. Analysis of transcription factor binding site enrichment served as an orthogonal assay that validates and extends the microarray results and thus contributes to understanding of the relation between the CHIP-AP and CHIP-PNR transcription complexes in specific tissues (e.g. wing and thorax) (Bronstein, 2011).

SSDP proteins directly bind DNA and mouse SSDP1 activates the expression of a reporter gene in both yeast and mammalian cells indicating that it is capable of regulating transcription activity. Enrichment was found for SSDP binding sites upstream of the genes identified in the microarray experiments on flies lacking SSDP. Moreover, in agreement with the positive transcriptional role of SSDP, enrichment for SSDP binding sites was restricted to the genes showing decreased expression in mutants. This strongly suggests that a significant number of these genes are bona fide SSDP target genes (Bronstein, 2011).

Consistent with the involvement of SSDP with the CHIP-AP complex, it was found that upstream regulatory regions of the SSDP putative target genes are also enriched for the AP binding site and the SSDP binding site. These sites are likely to be functionally significant, since loss of ssdp enhances the wing notching phenotype of a dominant allele of ap. Additionally, over-expression of Dlmo, whose product negatively regulates the CHIP-AP complex, also interacts with mutants of SSDP target genes, demonstrating that SSDP target genes are involved in the CHIP-AP pathway. The efficiency of finding genetic interactions among the genes differentially expressed in the microarray experiments, demonstrated the power of this approach. Specifically, 72% of the loci tested with Dlmo^Bx2 is more than an order of magnitude higher than an EP insertion screen (1.3% interacting) in a Dlmo^Bx1 sensitized background. Combined microarray and genetic loss of function screen allowed the identification of a similar number of Dlmo-interacting genes by screening a much smaller group of putative target genes (Bejarano, 2008). Of the 35 genes identified by Bejarano only CG1943 was found in the 189 genes identified in the current microarray screen. This study specifically identified down-stream targets of SSDP, while Bejarano searched for any modifiers of the Dlmo wing notching phenotype and thus uncovered genes that function in other regulatory pathways or genes that are upstream of the CHIP-AP complexes. This may explain the limited overlap between the current results and those of Bejarano (Bronstein, 2011).

In contrast to the enrichment of SSDP binding sites in the genes down-regulated in ssdp mutants, the PNR binding site was enriched specifically in the genes up-regulated in the ssdp mutants. A model is therefore presented in which loss of SSDP disrupts the balance between the CHIP-AP and CHIP-PNR complexes. Mammalian SSDP proteins protect LDB, LHX and LMO proteins from ubiquitination and subsequent proteasome-mediated degradation by interfering with the interaction between LDB and the E3 ubiquitin ligase, RLIM. It is therefore possible that in the absence of SSDP proteins, CHIP/LDB and LMO can escape degradation by interacting with GATA and beta-HLH proteins that are not subjected to proteasome-mediated regulation. The N-terminus of CHIP/LDB proteins is responsible for interaction with both PNR and RLIM. Thus, PNR/GATA proteins may partially interfere with the interaction between CHIP/LDB and RLIM making the CHIP/LDB-PNR/GATA complex more resistant to proteasome regulation and less dependant on the levels of SSDP proteins then the CHIP/LDB-LHX/AP complex (Bronstein, 2011).

According to the current model, in cells where both the CHIP-AP and CHIP-PNR complexes are active, loss of SSDP should result in the same phenotype as over-expression of PNR. Indeed, it was found that ssdp^L7/+ flies display duplications of scutellar sensory bristles, similar to gain of function mutations in pnr. In addition, lowered levels of pnr in ssdp^L7/+; pnr^VX6/+ flies suppresses scutellar bristle duplication. This indicates that the duplicated scutellar bristle phenotype of ssdp^L7/+ flies depends on the presence of PNR. As predicted by the model, since both AP and PNR regulate bristle formation, the functional interactions between SSDP target genes and ssdp^L7 and/or Chip^e5.5 resulted in either suppression or enhancement of the duplicated scutellar bristle phenotype (Bronstein, 2011).

These results in flies indicate that SSDP contributes differentially to CHIP/LDB complexes containing AP versus PNR. By contrast, mouse SSDP proteins positively contribute to the transcription activity and assembly of both LDB-GATA and LDB-LHX complexes, but the relative contribution of mammalian SSDP proteins to LDB complexes containing LHX proteins versus GATA proteins has not been specifically examined. It is possible that SSDP alters the balance of LIM-based CHIP/LDB complexes and GATA-containing CHIP/LDB complexes in the development of mice, as occurs in flies (Bronstein, 2011).

The search for enrichment of transcription factor binding sites upstream of the putative SSDP target genes identified additional transcription factors that may warrant future study. Some of these factors are associated with SSDP and CHIP/LDB complexes. For example, the binding sites for PNR and ZESTE (Z) were both enriched in the up-regulated putative SSDP target genes. This is in agreement with previous studies showing that Z can recruit the BRAHMA (BRM, the Drosophila homolog of the yeast SWI2/SNF2 gene) complex via its member OSA, which together negatively regulate the CHIP-PNR complex during sensory bristle formation through direct and simultaneous binding of OSA to both CHIP and PNR (Bronstein, 2011).

Some of the additional regulatory inputs at SSDP target genes may be evolutionarily conserved. For example, enrichment of STAT92E and SSDP binding sites was found in the down-regulated SSDP target genes. This may be significant, as a known role of ssdp is regulation of the JAK/STAT pathway during Drosophila eye development. Interestingly, mammalian STAT1 confers an anti-proliferative response to IFN-γ signaling by inhibition of c-myc expression. Similarly, expression of mammalian SSDP2 in human acute myelogenous leukemia cells and prostate cancer cells leads to cell cycle arrest and inhibits proliferation accompanied by down-regulation of C-MYC. These findings indicate that both in Drosophila and in mammals SSDP and STAT proteins have similar functions and may share common target genes (Bronstein, 2011).

While the transcription factor binding site analysis utilized all of the 189 putative SSDP target genes, genetic screens were conducted on a subset of them due to the availability of mutants. This suggests that more genetic interactions will be found among the untested genes. Even among this more limited subset, there are interesting new stories that suggest future experimental directions. For example, an insertion mutation in the Xbp1 gene suppressed the duplicated scutellar bristle phenotype characteristic of ssdp^L7/+ and Chip^e5.5/+ flies, indicating that XBP1 contributes positively to bristle formation. In contrast, when Xbp1 was silenced in ap-expressing cells both the wings and the scutum displayed a marked excess of sensory bristles while the scutellum was not affected. These results suggest that in the wing and scutum XBP1 acts as a negative regulator of bristle formation. Silencing of Xbp1 in pnr-expressing cells caused a similar excess of bristle on the scutum, accompanied by a reduced number of scutellar bristles, further emphasizing the opposing effects of XBP1 in these two distinct parts of the thorax. Such contrasting phenotypes have been previously documented for several pnr mutants as well. In flies and mammals XBP1 regulates the ER stress response, also termed the unfolded protein response (UPR). Since one of the functions of the ER is the production of secreted proteins, UPR-related pathways are widely utilized during the normal differentiation of many specialized secretory cells. In this respect it would be interesting to examine whether SSDP and CHIP/LDB complexes affect the production of secreted morphogens, such as Wingless (WG), the secreted ligands of the EGFR receptor, Spitz (SPI) and Argos (AOS), or the secreted Notch binding protein Scabrous (SCA) via XBP1 during wing and sensory bristle formation. Alternatively, the transcription factor XBP1 may directly regulate the expression of genes required for differentiation of the wing and sensory bristles. Indeed, carbohydrate ingestion induces XBP1 in the liver of mice, which in turn directly regulates the expression of genes involved in fatty acid synthesis. This role of XBP1 is independent of UPR activation and is not due to altered protein secretory function. Curiously, the two GO function categories 'cellular carbohydrate metabolism' and 'cellular lipid metabolism' which are enriched among Xbp1 target genes in mouse skeletal muscle and secretory cells were also enriched in the list of putative SSDP target genes. Whether this reflects a secondary effect due to the down-regulation of Xbp1 in ssdp mutants or a direct regulation of these processes by SSDP is yet to be determined (Bronstein, 2011).

Additional novel functions for CHIP/LDB complexes are implied by the results regarding the Gs-alpha60A (a.k.a. CG2835) gene. G protein coupled receptors are important regulators of development by for example, signaling via the protein kinase A (PKA) pathway. Activation or inhibition of PKA signaling during pupal wing maturation perturb proper adhesion of dorso-ventral wing surfaces resulting in wing blistering. This phenotype may be due to miss-regulation of wing epithelial cell death in ap-expressing cells. Interestingly, similar wing blisters occur in the wing of Dlmo^Bx2 flies. Moreover, it was found that mutant alleles of Gs-alpha60A enhanced the wing blistering phenotype of Dlmo^Bx2. Silencing of G-salpha60A in ap-expressing cells caused a curled wing phenotype. Such a phenotype can result from differences in the size of the dorsal and ventral wing blade surfaces. In addition, silencing of this gene in pnr-expressing cells caused the posterior pair of scutellar bristles to form in reversed orientation. Bristle orientation have been proposed to be regulated by planar cell polarity genes. Taken together these results point to novel aspects of regulation of wing and sensory bristle development by SSDP and CHIP/LDB complexes mediated by G-alpha proteins (Bronstein, 2011).

This genome-wide expression profiling and bioinformatics analysis of ssdp mutant larvae, combined with genetic screens resulted in gained insight into the intricate context-dependent transcriptional regulation by CHIP/LDB complexes. It was possible to identify 28 putative SSDP target genes that are involved in wing development and 23 putative SSDP target genes that play a role in scutellar bristle formation. Examination of two of these, xbp1 and Gs-alpha60A, suggests novel aspects of developmental regulation such as the involvement of SSDP and CHIP/LDB complexes in ER function and PKA signaling. Furthermore, it was shown that SSDP proteins contribute differentially to transcription activity, and probably to the balance in formation of CHIP-AP and CHIP-PNR complexes. Furthermore potential novel partners of SSDP in regulating transcription of downstream genes during fly development were. It stands to reason that an extension of the genetic analysis to mammals and other vertebrates will reveal a host of additional functions of SSDP and CHIP/LDB during the multifaceted process of transcriptional regulation that underlies the development of multicellular organisms (Bronstein, 2011).

Protein Interactions

Chip was cloned and found to encode a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins, which bind nuclear LIM domain proteins. Chip protein physically interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins (Morcillo, 1997).

Formation of the dorsal-ventral axis of the Drosophila wing depends on activity of the LIM-homeodomain protein Apterous (Ap). Ap activity levels are modulated by dLMO, the protein encoded by the Beadex (Bx) gene. Overexpression of dLMO in Bx mutants interferes with Apterous function. Conversely, Bx loss-of-function mutants fail to down-regulate Apterous activity at late stages of wing development. Biochemical analysis shows that dLMO protein binds to Chip, thus competing with Apterous binding to Chip. These data suggest that Apterous activity depends on formation of a functional complex with Chip and that the relative levels of dLMO, Apterous, and Chip determine the level of Apterous activity. The dominant interference mechanism of dLMO action may serve as a model for the mechanism by which LMO oncogenes cause cancer when misexpressed in T cells (Milan, 1998).

LIM domains are thought to mediate protein interactions and are found in a variety of different types of proteins, often in combination with other recognized protein domains, as in the LIM-homeodomain proteins. dLMO belongs to a class of LIM domain proteins that have two LIM domains and no other recognizable motifs (hence, the designation LMO, for LIM only). In view of the effects of dLMO on Ap function, experiments were designed to discover whether dLMO can interact with Ap, a LIM homeodomain protein and Chip, an LDB (LIM domain binding) protein. LDB proteins bind to the LIM domains of nuclear LMO proteins of the type encoded by Bx. Genetic interactions between chip and ap suggest that as with Ldb1 and XLim1, Chip binding might activate Ap function. When overexpressed, Bx appears to interfere with Ap function without affecting either Chip or Ap protein expression. This raises the possibility that dLMO might interfere with binding between Ap and Chip. This was tested using a coimmunoprecipitation assay in which the binding between constant amounts of Chip and Ap proteins was challenged by increasing concentrations of Bx protein. Chip protein can be immunoprecipitated with T7-epitope-tagged Ap protein and anti-T7 antibody, showing that Ap and Chip proteins bind in vitro. Binding between Chip and Ap was challenged by adding increasing amounts of in vitro-translated dLMO protein. A dose-dependent decrease in the amount of Chip immunoprecipitating with Ap is observed as the amount of dLMO protein is increased and a corresponding increase in the amount of Chip immunoprecipitating with dLMO is also observed. These observations indicate that dLMO can bind to Chip in vitro and can compete for binding between Chip and Ap in a concentration-dependent manner. As a further test, the LIM domains of Ap were expressed as a GST fusion protein and tested for binding to full-length dLMO, Chip, and Ap proteins. Ap binds to itself and to Chip but not to dLMO in the GST-pull-down assay. This suggests that dLMO interferes with formation of the active Ap-Chip complex by competing with Ap for binding to Chip (Milan, 1998).

Two distinct functional domains have been identifed within NLI, the vertebrate homolog of Chip; the amino-terminal 200 amino acids (aa) mediate homodimerization, and 38 aa near the carboxyl terminus are required for high-affinity binding to LIM domains of LIM-HD proteins. These two domains within Chip are predicted to be present by primary sequence similarities between NLI, Chip, and orthologs from other species. To perform biochemical assays in vitro and misexpression studies in vivo, recombinant mutant Chip proteins were prepared in which either the predicted dimerization domain (DD) or the predicted LIM interaction domain (LID) was deleted. In addition, a control full-length Chip with no mutations was used. Five copies of the c-myc epitope were fused to the carboxyl terminus of each protein to monitor each protein's expression (van Meyel, 1999).

To determine whether the predicted DD and LID of Chip are indeed required for self-dimerization and LIM-HD interaction, respectively, double-immunoprecipitation assays were performed to biochemically detect high-affinity protein interactions. A 32-amino acid LID has been shown to be required for Chip to interact with Apterous, Islet, and Lim3. Deletion of 156 amino acids of the putative DD severely impairs the ability of Chip to dimerize. The DD is dispensable for LIM-HD interactions, and the LID is not required for Chip self-dimerization, indicating that these two domains of Chip function independently. Because deletion of the DD (aa 221-376) does not completely abolish the ability of Chip to self-dimerize, it is suspected that in conservative efforts to avoid disturbing nearby motifs, not all components of the dimerization domain were removed. Chip contains nearly 200 unique N-terminal amino acids not present in NLI that could participate in Chip dimerization (van Meyel, 1999).

Ap is the only known LIM-HD family member expressed in the developing wing disc. Chip interacts genetically with ap to cause disruptions of the wing margin, suggesting that these two genes act in the same pathway (Morcillo, 1997). In vitro analysis of Chip interactions with LIM-HD factors further suggests that the functionally relevant complexes required for transcriptional regulation in the wing disc are comprised of two Ap molecules bridged by two dimerized molecules of Chip. Alternatively, it is possible that Ap function is independent of Chip and that Chip, although expressed coincidentally with Ap, participates in an independent pathway and modulates Ap function indirectly by sequestering it from alternative complexes (van Meyel, 1999).

To distinguish whether Ap and Chip form functional complexes in vivo, or whether Ap works independently of Chip, the GAL4-UAS system was used to express Chip (ChipFL) and mutant Chip proteins (ChipdeltaDD and ChipdeltaLID). ap^GAL4, a P[GAL4] insertion in the ap locus, was used to direct reproducibly high levels of UAS transgene expression in the ap cells of the wing disc. As assayed by staining for the c-myc epitope, all Chip variants were expressed and were localized to the nucleus. Control ap^GAL4/+ heterozygotes display no wing defects, nor do they in the presence of a UAS-ap transgene. The latter observation is consistent with the hypothesis that a Chip/Ap tetrameric complex is functionally relevant, since it would not be compromised by Ap overexpression. In contrast, overexpression of ChipFL in the wing discs of ap^GAL4/+ heterozygotes results in defects resembling hypomorphic ap mutants. Wings were severely compromised in size and structure, and the wing margin was poorly demarcated. These wing defects are suppressed by simultaneous overexpression of Ap using a UAS-ap transgene. This indicates that the stoichiometry between Ap and Chip is an important factor in wing development and further suggests that overexpression of ChipFL titrates endogenous Ap to form incomplete complexes in which LID domains of Chip molecules remain vacant. The suppression of wing defects caused by co-overexpression of Ap is consistent with the idea that these incomplete complexes can be rendered fully functional by providing additional Ap to occupy vacant LID sites (van Meyel, 1999).

Overexpression of ChipdeltaDD results in severe wing defects that mimic those of extreme ap mutants. Like ChipFL, ChipdeltaDD is predicted to bind and sequester endogenous Ap but is unable to dimerize. The relative severity of ChipdeltaDD suggests that it renders Ap completely nonfunctional and blocks it from forming further Chip-bridged multimolecular interactions. Simultaneous overexpression of Ap suppresses the wing defects induced by ChipdeltaDD, suggesting that a pool of endogenous, dimerized Chip molecules exists to which ectopic Ap molecules bind and ''fill in'' unoccupied LID sites to form functional complexes. The suppression was not to the same extent as that achieved by Ap coexpressed with ChipFL and suggests that the poisoned ChipdeltaDD/Ap complexes can compete with fully functional complexes for binding to control elements in target genes (van Meyel, 1999).

Overexpression of ChipdeltaLID causes wing defects resembling those induced by overexpression of ChipFL. This experiment demonstrates a key role for Chip in wing development, since ChipdeltaLID is unable to interact with Ap and thus cannot simply sequester it from biological function. Importantly, these defects are not suppressed by simultaneous overexpression of Ap, as are those induced by ChipFL or ChipdeltaDD. Therefore, Ap cannot reconstitute function of complexes containing ChipdeltaLID, strongly supporting a role for direct physical interaction between Ap and Chip for function in vivo (van Meyel, 1999).

These results suggest that a tetrameric Chip/Ap complex mediates Chip and Ap function in the wing. In a simple model, the Ap LIM domains and the Chip LID domain bind one another and permit the homeodomains of two Ap molecules to be bridged by Chip dimerization. To test this model directly, the domains responsible for the interaction were removed and the remainder of each protein was tethered by fusing ChipdeltaLID directly to a LIM domain deleted version of Ap. This chimeric molecule should allow reconstitution of the Chip/Ap complex independently of the LID and LIM domains and rescue ap mutant wing defects if Chip and Ap are indeed required to bind one another. ap^GAL4 has been shown to act as a strong hypomorphic allele of ap. Using ap^GAL4 as a driver in an ap mutant background, it was found that ApdeltaLIM and ChipdeltaLID are incapable of rescuing any ap mutant wing defects. In contrast, the ChipdeltaLID:ApdeltaLIM chimera does rescue ap mutants. While the extent of rescue is not as complete as for Ap, flies expressing ChipdeltaLID:ApdeltaLIM exhibit significant wing outgrowth and a clearly demarcated margin with a triple row of sensory bristles. It is concluded from this result that physical interactions between Ap and Chip are required for appropriate margin formation and wing outgrowth (van Meyel, 1999).

In experiments to be reported elsewhere, it has been found that loss-of-function Chip mutations reveal ap-like defects in axon pathfinding and neurotransmitter production by Ap interneurons. Having implicated a role for Chip in three distinct functions of Ap, and given the widespread expression of Chip, it is speculated that Chip is an obligate cofactor for other LIM-HD activities. In fact, it would appear that the only way to exclude Chip from participating in complexes with LIM-HD factors is to selectively render it ineffective, perhaps by sequestration or by the formation of more favorable interactions between LIM-HDs and other factors. Indeed, both of these processes may act to limit the range of activity of Chip. (1) Chip may be sequestered in cells that coexpress dLMO, a member of the LIM-only subclass of nuclear LIM-containing proteins that have no homeodomain. dLMO can selectively inhibit Chip interactions with Ap in vitro and can modulate Ap function in the wing. (2) LIM-HD proteins may directly interact with proteins other than Chip. For example, in the absence of NLI, the LIM domains of Lhx-3 mediate direct heterodimerization with Isl-1 and Isl-2 but not with other LIM-HD family members. Drosophila Lim3 and Isl are likewise capable of forming heterodimers in the absence of Chip, suggesting that they can participate in both Chip-dependent and Chip-independent heterodimeric complexes in cells in which they are coexpressed. In fact, the data show that direct Islet/Lim3 interactions may be of higher affinity than Islet/Chip interactions, raising the possibility that under certain conditions Islet/Lim3 interactions may be favored over interactions with Chip. Recent studies have shown that Islet and Lim3 function in a combinatorial manner to specify motor axon pathway selection in flies (Thor, 1999), and analyses of Lhx-3/4 mutant mice indicate that a similar code operates in vertebrates. The implementation of any LIM-HD combinatorial code relies not only on the availability, concentration, and relative affinities of LIM-HDs; in addition, Chip/NLI and possibly LMO cofactors are also involved (van Meyel, 1999).

To test whether the active form of Ap is a complex involving two molecules of Ap and two molecules of Chip, the effects of expressing dominant-negative forms of Chip that differ in their ability to bind Ap were compared. Overexpression of wild-type Chip has dominant-negative activity in vivo. It has been suggested that this could be due to formation of trimeric complexes (Ap:Chip:Chip) that lack a second Ap molecule because the dominant-negative activity of Chip can be suppressed by overexpression of Ap. It was reasoned that a truncated form of Chip lacking the LIM interaction domain (ChipdeltaLID) would also serve as a dominant negative but that its activity should not be suppressed by overexpression of Ap. Before testing the activity of the ChipdeltaLID construct in vivo, it was verified that the molecular interactions between Ap and Chip in vitro are consistent with the expectations based on analysis of the human LDB protein, NLI (Milan, 1999).

Complex formation between Ap, Chip, and ChipdeltaLID was assayed using in vitro translated proteins. Ap was expressed with a T7-epitope tag, incubated with 35S-labeled Chip or ChipdeltaLID, and immunoprecipitated with anti-T7. Full-length Chip coprecipitates with T7-Ap. ChipdeltaLID is not recovered above background levels when incubated with T7-Ap, confirming that Chip needs the LID to bind effectively to Ap. ChipdeltaLID coprecipitates when incubated with T7-Ap and full-length Chip, demonstrating formation of a three part Ap:Chip:ChipdeltaLID complex. To verify that a Chip dimer can bridge two Ap molecules, a tagged version of Ap (Ap-TAP) was used. The biological activity of Ap-TAP is comparable to that of wild-type Ap when ectopically expressed in vivo under GAL4 control. Ap-TAP was in vitro translated and bound to IgG beads. The beads were washed and incubated with labeled Ap with or without unlabeled Chip. Without Chip, only background levels of 35S-Ap are recovered. When Chip is present, Ap-TAP beads bind 35S-Ap, indicating formation of the tetrameric complex in vitro (Milan, 1999).

Overexpression of Chip represses the Ap targets fringe-lacZ and dLMO (Beadex). Overexpression of ChipdeltaLID using patched GAL4 also represses fringe-lacZ and dLMO, indicating that both forms of Chip suppress Apterous activity when overexpressed. Overexpression of Chip under control of ap-GAL4 interferes with wing formation, producing a phenotype resembling the lack of ap function. This can be suppressed by coexpression of UAS-Chip and UAS-Ap. Overexpression of ChipdeltaLID using ap-GAL4 causes a mild apterous phenotype: distal notching of the wing margin and dorsal-to-ventral transformation of the alula. Although the phenotype suggests only partial reduction of Ap activity, the defects caused by overexpression of ChipdeltaLID cannot be suppressed by coexpression of Ap. These results suggest that ChipdeltaLID acts as a dominant negative for Ap activity in vivo by promoting the formation of a trimeric Ap:Chip:ChipdeltaLID complex that cannot bind another Ap molecule (Milan, 1999).

The LIM domain protein dLMO can compete with Ap for binding to its cofactor Chip. If the active form of Ap in vivo is a LIM-HD dimer bridged by a dimer of cofactor, dLMO might compete for Ap activity by displacing an Ap molecule from the Ap:Chip complex to form an Ap:dLMO heterodimer bridged by the cofactor. This model was tested by preparing a form of Ap that could dimerize but that could not be displaced by dLMO. To do so, a fusion protein consisting of the N-terminal dimerization domain of Chip linked to a C-terminal fragment of Ap containing the homeodomain (aa 270-469) was expressed. The new protein, called ChAp, lacks the LIM interaction domain of Chip and the LIM domains of Ap. Its structure should allow it to form an Ap dimer (Milan, 1999).

A test was performed to see whether ChAp has activity comparable to Ap in vivo. When expressed along the anteroposterior compartment boundary under control of dpp-GAL4, UAS-ap and UAS-ChAp produce essentially identical phenotypes. In both cases, ectopic wing margins are induced on both sides of the dpp-GAL4 stripe in the ventral compartment. The ectopic wing margin is due to the ectopic expression of Wingless in the ventral compartment. This correlates with ectopic induction of the dorsally expressed Ap targets fringe-lacZ and dLMO in ventral cells. These observations show that ChAp can mimic the effects of Ap in ectopic expression assays. A rescue assay was used to ask whether ChAp can functionally substitute for Ap in vivo. The wing defect in ap^GAL4/ap^rk568 flies is completely suppressed when wild-type Ap is expressed in dorsal cells using ap-GAL4. Dorsal expression of ChAp produces a comparable rescue. These results show that ChAp behaves like wild-type Ap when ectopically expressed and that ChAp can substitute for Ap in vivo (Milan, 1999).

According to the dimer model, ChAp should be sensitive to the dominant-negative activity of ChipdeltaLID but should not be subject to regulation by dLMO in vivo. dLMO is induced by Ap in the wing disc, and loss-of-function dLMO mutants produce defects that are thought to result from overactivation of Ap. ChAp overexpression in the dorsal compartment of an otherwise wild-type wing disc gives a phenotype that closely resembles the dLMO loss-of-function phenotype. The wings are smaller than wild type and are held in an abnormal position. The dorsal compartment is typically smaller than the ventral compartment, giving the wing a slightly curled appearance. The pattern of veins is also abnormal. Coexpression of the dominant-negative form of the cofactor, ChipdeltaLID, suppresses the ChAp overexpression phenotype. This indicates that dimerization is required for ChAp activity in vivo (Milan, 1999).

To ask whether ChAp is subject to regulation by dLMO, the ability of Ap and ChAp to suppress the effects of dLMO overexpression in vivo were compared. apGAL4/+; UAS-dLMO/+ wings show loss of the normal wing margin and sporadic patches of ectopic wing margin, thus promoting local overgrowth. Overexpression of wild-type Ap does not suppress the dLMO overexpression phenotype. Antibody staining shows that Wg is not expressed at the DV boundary in these discs. The wing pouch is very small, and the normally straight boundary between cells expressing Ap and adjacent nonexpressing cells is uneven. These observations suggest that Ap is nonfunctional in these discs in spite of being overexpressed. In contrast, coexpression of ChAp and dLMO restores Wg expression at the DV boundary even though dLMO is expressed at high levels. The resulting wings have a normal wing margin and resemble the dLMO mutant wing. These results suggest that ChAp overexpression phenocopies the dLMO loss-of-function mutant because ChAp is not sensitive to downregulation by dLMO. Consequently, ChAp remains active in the presence of excess dLMO (Milan, 1999).

The bridged dimer model suggests that removing dLMO activity would result in excess Ap activity. To test this, the properties of Chip and Ap interaction were exploited to regulate Ap activity in a dLMO mutant background. dLMO loss-of-function mutants were generated by excision of a GAL4-P element insertion in the second intron of the dLMO gene. Fortuitously, excision line hdp^R590 strongly reduces dLMO expression but leaves GAL4 and the cis-regulatory region unaffected, so that the mutant expresses GAL4 in the normal pattern of dLMO. hdp^R590 causes aberrant Serrate expression and a reduced dorsal wing pouch. The dLMO loss-of-function phenotype in this mutant can be suppressed by expression of ChipdeltaLID. The small wing size of hdp^R590 is fully rescued, and the abnormal venation is partially suppressed. Likewise, expression of a mutant form of Ap lacking only the homeodomain completely suppresses the hdp^R590 phenotype. Both of these constructs have mild dominant-negative effects that reduce Ap activity in vivo. These results confirm that the dLMO loss-of-function phenotype results principally from excess Ap activity at later stages of wing development. Further, they support the proposal that the normal function of dLMO is to regulate Ap activity levels by interfering with formation of an active complex consisting of two Ap molecules bridged by a dimer of Chip molecules (Milan, 1999).

The finding that ChAp can completely replace Apterous function in vivo suggests that the relevant feature of this tetrameric complex is the formation of a dimer of Ap. This molecule is not subject to negative regulation by dLMO and so remains constitutively active. The consequence is a failure to downregulate Ap activity as development proceeds. The phenotypic consequences of excess Apterous activity are comparable to those of the dLMO lack-of-function mutant. These findings support the view that the tetrameric complex between Ap and its cofactor Chip provides a means to generate the requisite bridged dimer of Ap, while allowing the activity of the complex to be regulated by the competitive inhibitor dLMO. It is suggested that this may provide a general model for regulation of LIM-HD activity. LMO family proteins may be generic antagonists of LIM homeodomain proteins through binding to their common LDB cofactors. The active complexes may be cofactor-bridged homodimers (as is the case for Ap in wing development) or heterodimers with other LIM-HD transcription factors or other types of LDB-binding transcription factors. Combinations of different transcription factors bridged by a cofactor dimer might broaden the range of possible target genes (Milan, 1999).

At the sequence level, Drosophila Lim1 is highly related to its vertebrate homologs. Given their conservation, it was of interest to see if this sequence homology translates into functional similarities at a molecular level. In Xenopus, Xlim-1 and the LIM-domain-binding protein (Xldb-1/NLI/CLIM- 2) interact in vitro, and cooperate in vivo to induce secondary axis structures (Agulnick, 1996). As the name implies, this association takes place through the LIM domains. More recently, the Drosophila homolog of Xldb1/NLI/CLIM-2, Chip has been cloned and shown to interact with the Ap protein (Morcillo, 1997; Fernandez-Funez, 1998). To determine if Lim1 and Chip interact in vitro, co-immunoprecipitation experiments were carried out. Using the Lim1 antibody, the ability of Chip to be immunoprecipitated by full-length and truncated Lim1 proteins was carried out. The results show that Chip can be immunoprecipitated in the presence of full-length Lim1, and a truncated Lim1 protein that contains the LIM domains (LIM-Lim1). HD-Lim1, which lacks the LIM domains and includes the homeodomain fails to coimmunoprecipitate Chip. Additionally, Chip by itself is not immunopreciptated by the Lim1 antibody. These results show that Lim1 has the capacity to interact with the LIM-domain-binding protein, Chip. This interaction requires the LIM domains of Lim1 and is independent of the Lim1 homeodomain. Similar to its vertebrate counterparts, and Ap in Drosophila, Lim1 and Chip may cooperate in vivo to modulate the transcriptional activity of its downstream target genes. Chip is ubiquitously expressed and therefore is present in all Lim1-expressing cells suggesting that an in vivo interaction is possible (Lilly, 1999).

The LIM homeodomain (LIM-HD) protein Apterous (Ap) and its cofactor DLDB/CHIP control dorso-ventral (D/V) patterning and growth of the Drosophila wing. To investigate the molecular mechanisms of Ap/CHIP function, their relative levels of expression were altered and mutants were generated in the LIM1, LIM2 and HD domains of Ap, as well as in the LIM-interacting and self-association domains of CHIP. Using in vitro and in vivo assays it was found that: (1) the levels of CHIP relative to Ap control D/V patterning; (2) the LIM1 and LIM2 domains differ in their contributions to Ap function; (3) Ap HD mutations cause weak dominant negative effects; (4) overexpression of ChipDeltaSAD mutants mimics Ap lack-of-function, and this dominant negative phenotype is caused by titration of Ap because it can be rescued by adding extra Ap; and (5) overexpression of ChipDeltaLID mutants also causes an Ap lack-of-function phenotype, but it cannot be rescued by extra Ap. These results support the model that the Ap-CHIP active complex in vivo is a tetramer (Rincon-Limas, 2000).

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip acts as a cofactor of the dorsal selector Apterous, and both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax (Ramain, 2000).

Pnr is a member of the GATA-1 family of transcription factors and activates proneural function by binding to the dorsocentral (DC) enhancer located 4 kb and 30 kb upstream of ac and sc, respectively. Reported in this study is the characterization of Chip^E, a viable allele of Chip, that interacts with pnr genetically. Chip^E mutants show reduced ac-sc expression in the DC, associated with loss of DC bristles, and produce a phenotype similar to that of loss-of-function pnr alleles. This genetic interaction correlates with a physical interaction between Chip, Pnr, and the bHLH heterodimers (Ac/Sc-Da). Pnr interacts with the N terminus of Chip through its COOH terminus encompassing two helices that are conserved between D. melanogaster and D. virilis and that are probably both involved in protein-protein interactions. Chip dimerizes with the bHLH heterodimers through its C-terminal LID, known to mediate heterodimerization with LIM-containing proteins (Ramain, 2000).

In vertebrates, the Ldb1/NLI protein associates with GATA-1, Lmo2, and the bHLH E47, Tal1/SCL in an erythroid complex whose function is poorly understood. A similar Drosophila complex functions in vivo to regulate the ac/sc genes directly during establishment of the proneural prepattern.

Accurate coexpression of ac/sc is mediated by Pnr binding to the DC. The ac and sc promoters include E boxes that are targets for the Ac/Sc-Da heterodimers and support autoregulation during development. In cultured chicken embryonic fibroblast (CEF) cells, Pnr and the Ac/Sc-Da heterodimer activate expression of a CAT reporter linked respectively to the DC enhancer and to the ac promoter. Physical interactions between Pnr and the bHLHs lead to synergistic activation of the reporter when the regulatory sequences are associated. Pnr and the Ac/Sc-Da heterodimers are both required in flies for expression of a LacZ reporter linked to the promoter associated with the enhancer, but the analysis of Chip^E shows that Chip is also required for full activation in vivo. The interactions between Pnr and the bHLH mediated by Chip suggest that Pnr might also be involved in autoregulation. Interestingly, Chip interacts with Ac/Sc through the Ac/Sc bHLH domains, and it has been shown that the overexpression of a homologous bHLH domain is sufficient to mediate the proneural function of Ac/Sc (Ramain, 2000).

Chip has been identified in a genetic screen for mutations that reduce activity of the wing margin enhancer of the cut locus, and it has been proposed that Chip may act as a bridge allowing enhancer-promoter communications. Thus, if the flies have a unique functional Chip allele, they display a cut margin phenotype, and this effect is observed exclusively when they carry a gypsy insertion between the enhancer and the promoter on one chromosome. It has been proposed that binding of the Su(Hw) insulator protein to the gypsy insertion blocks communication on the mutant chromosome, thereby interfering with the functioning of the wild-type homolog. The interchromosomal insulation is detectable when Chip activity is reduced, and Chip and Su(Hw) are antagonistic to each other, suggesting that Chip may be a facilitator target of Su(Hw) (Ramain, 2000).

The Chip^E mutation specifically disrupts interactions with the bHLH and strongly affects the expression of a LacZ reporter linked to the ac promoter/DC enhancer in flies, suggesting that Chip also mediates enhancer-promoter communication in the ASC. Further evidence is provided by the Hw¹ mutant. Hw¹ carries a gypsy insertion within ac such that sc, which is located further downstream from the DC enhancer, is no longer expressed. In addition, the removal of the gypsy insulator largely restores sc expression in the DC proneural cluster (Ramain, 2000).

Thus, a complex containing Pnr, Chip, and the Ac/Sc-Da heterodimer activates ac-sc expression, and its function is antagonized by Ush, Ap, and Emc. Ush and Emc dimerize respectively with Pnr and the HLH Ac/Sc. The repressing effect of Ap may reflect its ability to interact with Chip, thereby depriving Pnr of its essential cofactor. Alternatively, Ap may weaken the enhancer activity of Pnr. Thus, Ap may compete directly with Chip for binding to Pnr (Ramain, 2000).

Within the domain of Pnr expression, Ap and Pnr compete for binding to their common Chip cofactor. Ap activity is mediated by a Chip dimer, whereas activation of ac-sc by Pnr requires a Chip monomer. Chip acts as a bridge between the Ac-Da heterodimer bound to the E boxes of the ac promoter and Pnr bound to the GATA sites of the DC enhancer. The activity of the resulting complex is antagonized by Ush and Emc, which negatively regulate Pnr and Ac/Sc functions during development. The repressing effect of Ap is mediated either by dimerization of Ap with Chip and/or Pnr or by Chip-assisted binding of Ap to sites located between the DC enhancer and the ac promoter. Additional cofactors, such as dLMO, may participate in this complex (Ramain, 2000).

Chip is required in flies for ASC activation, whereas it appears dispensable in CEF cells. This observation may reflect the nature of the reporter used in the transfection experiments where the DC enhancer is close to the ac promoter and poorly mimics the genomic organization of the ASC, where the DC enhancer has to regulate ac and sc simultaneously. Furthermore, the chromatin structure and its modifications associated with gene expression are probably not reproduced in the transient expression assay. Thus, ASC expression in flies may require additional coactivators recruited by Chip, including chromatin remodeling factors. Moreover, the activation of ac/sc probably requires the assembly of a higher-order nucleoprotein complex containing multiple transcription factors (enhanceosome), and Chip may allow the correct positioning of Pnr and the Ac/Sc-Da heterodimer in this structure (Ramain, 2000).

Chip^E mutants affect the scutellar and dorsocentral bristles in opposite fashions. It will be of interest to compare the regulation of the activity of the corresponding enhancers by Chip and Pnr (Ramain, 2000).

It has been proposed that appropriate combinations of proteins represent the positional cues that activate a given enhancer of the ASC complex. The disc is divided in large territories, but almost nothing is known concerning how these territories are further subdivided or how the positional information revealed by the accurate ac-sc expression is created. The present study provides a link between the spatial regulation of the proneural genes and the compartmentalization of the disc. ac-sc expression is stimulated by a complex containing the prepattern factor Pnr, Chip, and the bHLH proteins Ac/Sc and Da. Chip is an essential cofactor of the dorsal selector Ap, and these interactions coordinate the spatial transcription of the proneural genes. Ap is expressed specifically in the dorsal compartment of the wing pouch, and the juxtaposition of Ap-expressing with Ap-nonexpressing cells defines the dorsal/ventral organizing boundary where wingless (wg) expression is induced. On the thorax, ap and Chip are ubiquitously expressed, whereas wg expression occurs in a stripe straddling the lateral border of the domain of pnr expression. Moreover, Pnr activates wg. Pnr associates with Chip, and the domain of pnr expression appears devoid of Ap activity. As a consequence, this domain may define a boundary between a region devoid of Ap activity and a region where Ap is active. Alternatively, Pnr may associate with Ap, and the resulting heterodimer may regulate wg. Further studies will help to resolve this issue (Ramain, 2000).

The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or is a facilitator of activation that is inhibited by Su(Hw). Evidence is provided that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. The C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. It is inferred that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. It is theorized that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein (Gause, 2001).

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. A difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo (Weihe, 2001).

Since dLMO competes with Ap for binding to Chip, the possibility that Ap protein may be protected when it is in a complex with Chip was examined. To test this, Chip^e5.5 mutant clones, which lack Chip protein and therefore lack Ap activity, were created. Ap protein levels were reduced in Chip mutant clones, and increased in the wild-type twin spots which contain a higher level of Chip protein. To verify that reduced Chip activity does not affect ap mRNA levels ap-lacZ reporter gene expression was examined in discs expressing the dominant negative form of Chip, ChipDeltaLID. Ap protein levels were reduced in cells expressing ChipDeltaLID but ap-lacZ levels were unaffected. Thus, loss of Chip leads to reduced levels of Ap protein. It was noted that Chip mutant clones also lack dLMO expression. Thus, loss of Ap protein in Chip mutant clones does not correlate with expression of dLMO, as in wild-type cells. Rather, reduction of Ap levels correlates with the availability of Chip as a binding partner. This suggested that binding to Chip contributes to stabilization of Ap (Weihe, 2001).

ChipDeltaLID is capable of binding to full-length Chip through its dimerization domain, but cannot bind to Ap. Consequently, ChipDeltaLID leads to formation of trimeric complexes and thereby blocks Ap activity in vivo. The observation that ChipDeltaLID leads to reduced Ap stability without affecting ap-lacZ expression suggests that stabilization might require formation of tetrameric complexes between Chip and Ap. The tetrameric form of Chip and Ap is thought to be the active DNA-binding complex. Overexpression of ChipDeltaLID does not decrease the availability of LIM-binding sites in wild-type Chip, but does compete for tetramer formation. This raises the possibility that Ap stability might depend on whether it is able to form a DNA-binding complex with Chip (Weihe, 2001).

To test the effect of Ap-binding sites on Ap protein stability, a constant amount of a plasmid directing expression of a myc-tagged Ap protein was co-transfected with varying amounts of a plasmid carrying nine tandem repeats of a binding-site for the mammalian Ap-homolog Lhx2. This DNA sequence binds Drosophila Ap in S2 cells. The total amount of DNA in the transfection assay was held constant by varying the ratio of plasmid containing the binding sites and empty vector. Increasing the ratio of the plasmid containing the binding sites results in dose-dependent stabilization of Ap-myc protein. This observation supports the idea that availability of binding sites limits the amount of Ap protein that is stable in the cell when mRNA levels are held constant (Weihe, 2001).

Another means to test this possibility is by competition between Ap and a related protein for a fixed number of binding sites. For these experiments use was made of a fusion protein between Chip and Ap (called ChAp). In this protein the dimerization domain of Chip mediates dimerization of the DNA-binding domains of Ap. Thus, ChAp dimers should compete with endogenous Chip:Ap tetramers for DNA-binding sites. Use of the Myc tag versions of both proteins allowed direct comparison of their relative levels in co-transfected cells. Using this assay it was verified that increasing the level of ChAp-myc decreases the level of co-transfected Ap-myc in a dose-dependent manner. Expression of Chip-myc as a control has little effect on Ap-myc levels. Note that the level of Ap-myc construct was held constant in all samples. ChAp-myc and Chip-myc expression levels were controlled by varying the ratio of the expression constructs to the empty expression vector in the transfections (Weihe, 2001).

It was next asked whether competition for DNA-binding sites would affect Ap stability in the wing disc. Fortuitously, the antibody raised against Ap does not recognize ChAp. This allows the level of the endogenous Ap protein to be measured in cells expressing ChAp. ChAp expression under dpp^Gal4 control leads to a decrease in the level of endogenous Ap protein. Together, these observations suggest that Ap protein is unstable in vivo unless bound to DNA as part of an active complex with its co-factor Chip (Weihe, 2001).

This report addresses the problem of asymmetry in the competition between dLMO and Ap. The simplest model for competitive inhibition by dLMO would suggest that Ap should compete effectively with dLMO for binding to Chip when overexpressed. However, overexpression of Ap does not produce an excess of Ap activity. dLMO competes effectively for Ap activity, but the reverse is not true. Swapping the LIM domains of Ap for those of dLMO produces a functional Ap protein that is able to compete effectively with dLMO. This finding may provide an explanation for the non-reciprocal properties of Ap and dLMO. The effectiveness of dLMO as an inhibitor of Ap activity is attributed to an intrinsic difference in the ability of the LIM domains of these two proteins to bind to Chip. It is considered likely that the LIM domains of dLMO bind the LID of Chip with higher affinity than the LIM domains of Ap. However, these proteins have not been produced in soluble form at adequate concentrations and so the affinities of these binding interactions have not been determined directly (Weihe, 2001).

Other proteins might also contribute to stabilization of Chip-dLMO complexes or to destabilization of Chip-Ap complexes in vivo. Interactions involving Ap, Chip and other proteins have been reported. For example, Pannier interacts with Chip and competes with Apterous for patterning of the thorax. In this model, Chip is found in a complex with Pannier and dLMO, which promotes dorsal thorax formation. Chip is also found in a complex with Ap. The level of Chip is not in great excess, so competition occurs between Ap and Pannier for formation of Chip complexes, despite the fact that Pannier and Ap do not bind to Chip in the same way. It was noted that overexpression of dLAp-flag appears to interfere with Pannier complex formation, because it causes the formation of a cleft in the thorax, resembling a pannier loss-of-function mutant phenotype. Comparable overexpression of Ap does not do so. This suggests that dLAp competes more effectively than Ap for binding to Chip and so is more effective at sequestering Chip from Pannier-containing complexes. The relative affinity of these proteins appears to play an important role in maintaining the proper balance of complex formation in vivo. Numerous LIM-HD proteins have been found to play important roles in the development of a number of species. It seems likely that other LIM-homeodomain transcription factors will be regulated in similarly complex ways (Weihe, 2001).

Lim1 expression becomes discernible slightly later than expression al, clawless (cll/C15) and Bar, and maximal al expression in late third instar depends on Lim1 function. Cll expression is also significantly reduced in Lim1^7B2 (a null allele) clones in late third instar discs, indicating that not only al but also cll is positively regulated by Lim1 in the late third instar. Chi encodes a LIM domain binding protein and has been suggested to act as a co-factor for Lim1. Cll and Al signals are significantly reduced in clones of Chi^e5.5, a null allele of Chi. The concerted action of Lim1 and Chi is thus shown to be required for the maximal expression of cll and al in the late-third-instar pretarsus region (Kojima, 2005).

When Lim1 is misexpressed using blk-GAL4, the expression of al but not cll is induced. Thus, unlike al, cll may require an additional component for its maximal expression. Alternatively, cll may be less sensitive to activation by Lim1 than al (Kojima, 2005).

Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development

The LIM-domain-binding protein Ldb1 is a key factor in the assembly of transcriptional complexes involving LIM-homeodomain proteins and other transcription factors that regulate animal development. Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) have been identified as components of Ldb1-associated nuclear complexes in HeLa cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development was identified. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 during invertebrate and vertebrate development (Chen, 2002).

To search for new interaction partners of Ldb1, a HeLa cell line was generated that expresses Ldb1 proteins carrying an N-terminal FLAG/HA epitope tag. Nuclear extracts prepared from these cells (and from nontransduced control cells) were incubated with immobilized anti-FLAG antibodies and the specifically bound materials were eluted by competition with excess amounts of FLAG peptide. Thereafter, immobilized anti-HA antibodies were used in a second round of purification. SDS/PAGE separation and silver staining of the final eluate revealed at least six polypeptides that were specific for the epitope-tagged Ldb1 sample and were not observed in the mock control (Chen, 2002).

All specific bands were analyzed by mass spectrometry. As expected, the 56-kDa band corresponded to epitope-tagged Ldb1. The 50-kDa doublet contained closely related proteins. Two peptides, SAQTFLSEIR and NSPNNISGISNPPGTPR, present in tryptic digests of the lower band of this doublet, correspond to human Ssdp1 (gi:13449489). The upper band contained several tryptic peptides (LALYVYEYLLHIGAQK, SAQTFLSEIR, and SSPGAVAGLSNAPGTPR) that correspond to Ssdp3 (gi:13400104), a structural relative of Ssdp1. Database searches revealed four human and three mouse Ssdp sequences that are closely related. In addition, a unique Drosophila Ssdp sequence was identified. Mouse Ssdp1 and Drosophila Ssdp were selected for functional studies reported below (Chen, 2002).

The function of Lim1 in axis formation in Xenopus depends on cooperation with Ldb cofactors. It was asked whether Ssdp might synergize with Xenopus Lim1 and Ldb1 in this system. Xenopus embryos were injected in the prospective ventral marginal zone with different combinations of synthetic mRNAs encoding Xlim1, Ldb1, and mouse or Drosophila Ssdp. Neither of the Ssdp proteins alone had any axis-inducing activity at the levels tested . High levels of Xlim1 and Ldb1 mRNAs induced incomplete secondary axes, whereas lower levels were ineffective. The lower levels of Xlim1 and Ldb1 mRNAs became highly effective, however, when coinjected with either mouse Ssdp1or Drosophila Ssdp mRNAs. Injection of low levels of Ldb1 plus Ssdp, or of Xlim1 plus Ssdp RNAs did not induce secondary axes. All secondary axes generated in this manner were incomplete. It seems that injection of high levels of Xlim1 plus Ldb1 mRNAs or of the triple combination of mRNAs caused both secondary axis induction and an inhibition of gastrulation, because those embryos that did not display a secondary axis were abnormal, mostly because of an open blastopore. This finding may explain the fact that injection of 80 pg of Xlim1, 80 pg of Ldb1, and 100 pg of mouse Ssdp1 mRNAs led to a lower proportion of secondary axis induction than 40 pg of Xlim1, 40 pg of Ldb1, and 100 pg of mouse Ssdp1 mRNAs. It is believed that the higher Xlim1/Ldb1 levels more effectively interfered with gastrulation, leading to a higher proportion of abnormal embryos rather than axis-duplicated embryos. Drosophila Ssdp mRNA was almost as effective as mouse Ssdp1 mRNA in inducing secondary axes when coinjected with Xlim1 and Ldb1 mRNAs. Thus, mouse Ssdp1 and Drosophila Ssdp proteins are sufficiently similar in their functions as to be interchangeable in ectopic expression experiments. A deletion of amino acid residues 1 to 121 from mouse Ssdp1 yielded a protein that did not bind Ldb1 after cotransfection into cultured cells and was also unable to synergize with Xlim1 and Ldb1 in axis induction. It is concluded from these results that Ssdp synergizes with Xlim1 and Ldb1 in vivo during gastrulation in Xenopus, and that this synergy is likely to require interaction between Ssdp and Ldb1 (Chen, 2002).

The single D. melanogaster Ssdp protein is encoded by CG7187 in polytene chromosome bands 90F1–2 in the right arm of the third chromosome. Searches of the EST databases identified 29 Ssdp ESTs, as well as ESTs for genes that flank Ssdp both proximally and distally on the chromosome. One Ssdp EST (GM14473) was completely sequenced by the Drosophila Genome Project and corresponds to a transcription unit with a single intron of 1,581 bp. Alignments of the remaining ESTs show that Ssdp encodes at least two transcripts that derive by alternative splicing at the 3' end of the first exon (both transcripts splice to the same second exon). Twenty-two ESTs match the GM14473 sequence; in four ESTs (GH23938, RE28366, GH18277, and RE64068) the first exon is 505 bp shorter than the first exon of GM14473. The predicted ORFs for both transcripts are entirely within the common second exons, suggesting that both transcripts encode identical proteins (Chen, 2002).

Five alleles of Ssdp were available for this study. All of these are lethal when homozygous or hemizygous (heterozygous to a chromosomal deletion that includes Ssdp). Most of the homozygotes and hemizygotes die during the pupal stages. Likewise, most transheterozygotes of various combinations of Ssdp alleles die as pupae. A few Ssdp¹¹/Ssdp^BG01663 and Ssdp¹¹/Ssdp^KG03600 flies survive to eclose as adults with mild cuticular defects, including a slight distortion of the posterior scutellar bristles, often accompanied by duplication of the anterior scutellar bristles. The survival of many homozygous Ssdp mutants to late pupal stages could be due to the maternal expression of Ssdp. Mitotic recombination in the germ line was used to create oocytes that lack maternal contributions of either Ssdp³¹ or Ssdp^neo48. When fertilized by a sperm that lacks the Ssdp gene, the zygotes that lack both maternal and zygotic Ssdp die at the beginning of the second larval instar. Survival of these animals through embryogenesis may conceivably be sustained by residual activity of the Ssdp alleles that were used in this study. Paternal rescue of Ssdp exists; when oocytes that lack Ssdp are fertilized by a wild-type sperm, the Ssdp heterozygotes often survive to eclose as normal adults. These observations show that Ssdp is an essential gene (Chen, 2002).

Given the interactions between the vertebrate Ssdp and Ldb1 proteins, whether their Drosophila homologs might also interact in the context of the whole organism was tested. Previous work has shown that Chip (the Drosophila homolog of Ldb1) forms a dimer capable of binding two molecules of the LIM-homeodomain transcription factor Apterous. The Chip-Apterous tetramer activates transcription of a reporter gene in cultured cells and regulates the transcription of target genes involved in morphogenesis of the wing. Dlmo, a LIM-only protein, competes with Apterous for binding to Chip, and elevated levels of Dlmo lead to the displacement of Apterous from the complex, which renders the complex transcriptionally inactive, causing scalloped wings. Similar wing defects are displayed by homozygous apterous (ap) mutants and by double heterozygotes for mutations in ap and Chip (Chen, 2002).

Double heterozygotes for a Chip mutation and any of the five Ssdp alleles have scalloped wings; all of the single heterozygous mutants have normal wings. This genetic interaction is highly reminiscent of the genetic interaction displayed by double heterozygotes for Chip and ap, and suggests that Chip and Ssdp interact in vivo and that Ssdp is a positive cofactor required for normal function of the Chip-Apterous complex. Bx mutations, hypermorphic alleles of dlmo, also cause scalloped wings. Double heterozygotes for Bx mutations and any of the five Ssdp alleles display marked enhancement of the wing scalloping characteristic of Bx flies. Similar results were observed for several different Bx mutations. A similar enhancement of the wing scalloping of Bx/+ was reported in double heterozygotes for Bx and either Chip or ap mutations. No wing scalloping was observed, however, in double heterozygotes for any of the five Ssdp alleles and either ap⁴ or ap^56f. These observations support the model that Ssdp interacts in vivo with the Chip complex to regulate normal wing development (Chen, 2002).

It is not clear whether the in vivo functions of Ssdp are related to its ability to bind single-stranded DNA in vitro. The results imply that protein-protein interactions are essential for Ssdp function, but this implication does not preclude the possible importance of single-stranded nucleic acid interactions (Chen, 2002). Drosophila embryos defective in both maternal and zygotic Ssdp can develop into larvae. However, the alleles that were studied do not support subsequent development. Ssdp function in the fly may not be restricted to interactions with the Chip–Apterous complex, because the phenotype of the existing Ssdp mutants differs from that of known Chip and ap mutants. Furthermore, the facts that Ssdp genes are so well conserved throughout evolution, that they are expressed in a wide variety of cell types, and that they are indispensable for development seem to suggest that the encoded proteins function in many different transcriptional contexts (Chen, 2002).

Ssdp binds to Chip and regulates the activity of Apterous complexes in vivo

LIM-homeodomain transcription factors control a variety of developmental processes, and are assembled into functional complexes with the LIM-binding co-factor Ldb1 (in mouse) or Chip (in Drosophila). The identification and characterization is described of members of the Ssdp family of proteins, that are shown to interact with Ldb1 and Chip. The N terminus of Ssdp is highly conserved among species and binds a highly conserved domain within Ldb1/Chip that is distinct from the domains required for LIM binding and self-dimerization. In Drosophila, Ssdp is expressed in the developing nervous system and imaginal tissues, and it is capable of modifying the in vivo activity of complexes comprised of Chip and the LIM-homeodomain protein Apterous. Null mutations of the ssdp gene are cell-lethal in clones of cells within the developing wing disc. However, clones mutant for a hypomorphic allele give rise to ectopic margins, wing outgrowth and cell identity defects similar to those produced by mutant clones of Chip or apterous. Ssdp and Ldb/Chip each show structural similarity to two Arabidopsis proteins that cooperate with one another to regulate gene expression during flower development, suggesting that the molecular interactions between Ssdp and Ldb/Chip proteins are evolutionarily ancient and supply a fundamental function in the regulated control of transcription (van Meyel, 2003).

From a yeast two-hybrid screen to identify binding partners for mouse Ldb/NLI proteins, a murine homolog was isolated of avian sequence-specific single-stranded DNA-binding protein (SSDP). First identified in an experimental paradigm for induced differentiation of avian chondrocytes in culture, SSDP has been shown to selectively bind the promoter of the alpha2(I) collagen gene. Two mouse genes encoding highly similar proteins, Ssdp1 and Ssdp2 have been identified (van Meyel, 2003).

Both Ssdp2 and Ldb1 have orthologous counterparts in Drosophila, called Ssdp and Chip. Fly Ssdp residues 1-98 can bind strongly to Chip, and this interaction is dependent upon amino acids 387-426 of Chip. Chip residues 387-435 are 94% identical to Ldb1 amino acids 201-249, and this region has been named the Ldb1/Chip conserved domain (LCCD). Taken together, the results indicate that the N terminus of Ssdp proteins bind Ldb/Chip proteins in a region that is distinct from the two domains needed to form the tetrameric complex, namely the dimerization domain (DD) and the LIM interaction domain (LID) (van Meyel, 2003).

Searches of the NCBI databases indicate that Ssdp proteins comprise a family of highly related proteins in which there are four members in humans, three in mice and only one in Drosophila. Comparisons among primary sequences from Ssdp proteins from these and other species reveal a high degree of amino acid identity, particularly within the first 100 amino acids. Between flies and mice there is 90% identity over the N-terminal region. As is the case for all family members, the remainders of these proteins are characterized by an unusually high proportion of proline, glycine and methionine residues. For example, of the 352 amino acids of Drosophila Ssdp from amino acids 93-445, 21% are proline, 27% are glycine and 9% are methionine, for a total of 57% of all residues. Within this overall architecture, there are three small regions that are highly conserved across species (van Meyel, 2003).

There is also significant similarity in the N terminus of Ssdp family members to LEUNIG, a protein first identified in Arabidopsis and for which the N-terminal domain has been termed a LUFS domain, based on its similarity to other proteins in plants, Flo8 in yeast and to Ssdp. Although the LUFS domain remains functionally uncharacterized to date, it contains a Lissencephaly type 1-like homology motif (LisH) with a curious additional motif comprised at its core of the following sequence P-X-GFL-XX-WW-X-VFWD (van Meyel, 2003).

Like the LUFS domain of Ssdp proteins, the LCCD of Ldb1/Chip has been highly conserved through evolution, with 94% identity between mice and flies over a stretch of 49 amino acids (van Meyel, 2003).

The single ssdp gene in flies consists of two exons, the second of which contains the single open reading frame that encodes a 445 residue polypeptide. Epitope-tagged versions of Drosophila Ssdp were prepared in which five copies of the Myc epitope were fused to the C terminus of full-length Ssdp (SsdpFL) or a mutant lacking amino acids 2-92 (SsdpDelta2-92). These constructs were used to generate transgenic lines in which transgene expression is under the control of UAS sequences. Different GAL4 driver lines were used to express these recombinant proteins in a variety of cell types, including neurons and muscles. SsdpFL localizes to nuclei, with no staining in the cytoplasm. By contrast, SsdpDelta2-92 was found throughout the cytoplasm. Therefore nuclear localization of Ssdp is dependent upon the Chip-interacting LUFS domain, despite the fact that this region does not appear to encode a nuclear localization sequence (NLS). To address whether Chip, which does have an NLS, is required for translocation of Ssdp into the nucleus, tests were perfomed to see whether SsdpFL is properly localized to the nucleus in Chip^e5.5 null mutants. In contrast to wild-type, SsdpFL is distributed throughout the cytoplasm of cells lacking zygotic Chip. Occasionally, staining was detected in nuclei in addition to cytoplasmic staining. This may reflect residual activity in these embryos of maternally provided Chip. These results argue that nuclear targeting of Ssdp occurs through a Chip-dependent mechanism (van Meyel, 2003).

The pattern of ssdp expression was determined using in situ hybridization of digoxigenin-labeled antisense cRNA probes to embryos and third instar larvae. In embryos of syncytial blastoderm stage, ssdp transcript is ubiquitous, suggesting that there is maternal contribution. By the time of germband extension, although still widespread, expression appears to be enriched in the developing central nervous system (CNS). During germband retraction this enrichment of transcript in the embryonic CNS is more apparent, such that by stage 13-14 ssdp expression is largely restricted to the brain and ventral nerve cord. Closer examination of the pattern of expression in the ventral nerve cord suggests that expression occurs in all neurons of the CNS, with no major subclasses excluded. This pattern of expression is maintained through later stages of embryogenesis. In third instar larvae, ssdp is no longer detected in the ventral nerve cord, but moderate ssdp expression is observed in the optic lobes of the brain hemispheres. High levels of ssdp expression are observed in imaginal discs, including the anterior region of the antennal-eye disc, the wing and haltere discs and all leg discs, as well as in the salivary gland. With the exception of the eye-antennal disc, expression in imaginal discs is largely uniform (van Meyel, 2003).

To test the role of Ssdp in vivo, null mutations in the Drosophila ssdp gene were generated. P-element transposition was used to imprecisely excise the P-element EP(3)3097 and chromosomes were generated carrying deletions that were completely lethal in complementation tests with l(3)neo48. Several deletion lines were thus generated, including ssdp^L7 and ssdp^L5. DNA sequencing of the breakpoints of the ssdp^L7 deletion reveals that it is a complete null allele of ssdp and in all analyses where it has been examined, ssdp^L5 has had effects identical to those of ssdp^L7, arguing that it too is a null allele (van Meyel, 2003).

Each of the P-element and deletion alleles was tested in complementation analyses with the others and viability of the progeny was assessed at first and third instar larval stages using marked balancer chromosomes to distinguish homozygotes. The results indicate that the following allelic series exists with respect to increasing severity of the lethal phenotype: ssdp^l(3)neo48<ssdp^EP(3)3097 <ssdp^EP(3)3004<ssdp^L5 and ssdp^L7. In fact, the combination of EP(3)3097 and l(3)neo48 was not lethal in all cases, with 35% of EP(3)3097/1(3)neo48 individuals surviving through eclosion. Interestingly, most of these viable flies displayed mutant phenotypes, including wing blisters, a mild cleft in the notum along the AP axis, and thin, gnarled macrochaetae on the notum (van Meyel, 2003).

Ap is expressed in the dorsal compartment of the wing disc and is required to establish the DV affinity boundary, the wing margin, wing outgrowth and dorsal-specific wing structures such as sensory bristles. In the absence of Ap, the wing fails to develop. Ap functions through a tetrameric complex in which two molecules of Ap are bridged by a homodimer of Chip. Chip mutants interact genetically with ap to cause disruptions of the wing margin, and clones of Chip mutant cells in the wing disc behave like ap mutant clones, causing ectopic wing margins and outgrowths (van Meyel, 2003).

In contrast to a previous study, no phenotypes were detected in simple trans-heterozygous combinations of a null allele of Chip with any ssdp alleles used here, including ssdp^KG03600 and the two null alleles ssdp^L5 and ssdp^L7. Nor were any phenotypes detected in transheterozygous combinations of ap and ssdp. Thus, to address the role of Ssdp in the function of Chip/LIM-HD complexes in vivo, the GAL4-UAS system was used to reduce Ap/Chip complex activity to levels that would be sensitive to the effects of reducing ssdp gene dosage. ap^GAL4, a GAL4 P-element insertion in the ap gene, which faithfully expresses GAL4 in Ap-expressing cells, was used to drive expression of UAS transgenes in the dorsal compartment of the wing disc (van Meyel, 2003).

Over-expression of UAS-Chip disrupts wing patterning by titrating endogenous Ap into incomplete complexes in which LID domains of Chip molecules remain vacant. Relative to controls, such wings are small and lack regular structure, and the wing margin is poorly demarcated. These phenotypes resemble hypomorphic ap mutants, and can be completely suppressed by simultaneous overexpression of UAS-ap. This indicates that the stoichiometry between Ap and Chip is an important factor in the formation of functional complexes. The effect of removing one copy of the ssdp gene was examined; the resulting flies have little or no residual wing tissue, consistent with a further reduction of the activity of the complex (van Meyel, 2003).

Fusion of Chip and Ap into one chimeric molecule, called ChipDeltaLID:ApDeltaLIM, results in a hyperactive complex, since it is not susceptible to downregulation of activity imposed by LMO, a LIM-only factor that competes efficiently with Ap for binding with Chip. Flies that overexpress ChipDeltaLID:ApDeltaLIM have blistered wings in which the dorsal and ventral surfaces fail to fuse, and which are held upward and away from the thorax in a fashion resembling LMO loss-of-function mutants. Removal of one copy of ssdp suppresses the blistered wing phenotype and the surfaces fuse properly, although the wings remain held up. Thus, Ssdp can modify the activity of Chip/Ap tetrameric complexes of both reduced and hyperactive function (van Meyel, 2003).

Finally, the effects of Chip overexpression were compared with those produced by expression of a Chip variant lacking the LCCD (ChipDeltaLCCD). ChipDeltaLCCD is capable of self-dimerization and binding to Ap, but it cannot bind Ssdp. If Ssdp were required for function of the complex, ChipDeltaLCCD would be predicted to have a more potent dominant-negative effect on the function of the complex than would Chip itself, since the latter can still recruit Ssdp. Expression of ChipDeltaLCCD with ap^GAL4 consistently produced more extreme wing defects than Chip. ChipDeltaLCCD sequesters Ap into nonfunctional complexes, but it cannot bind Ssdp. Therefore, removal of one copy of ssdp would not be expected to suppress the phenotype caused by ChipDeltaLCCD, and indeed it does not. Collectively, these results argue that in addition to forming the dimeric bridge for two molecules of Ap, Chip also recruits Ssdp to the complex (van Meyel, 2003).

Clones of ap mutant cells in the dorsal compartment of the wing disc induce an ectopic wing margin and therefore ectopic wing outgrowth. These ap mutant cells differentiate ventral wing margin structures, despite the fact that they remain in the dorsal compartment. Chip mutant clones induced in the dorsal compartment give rise to strikingly similar phenotypes. The effects of Chip clones are influenced both by the timing of their induction as well as their position within the disc. For example, clones induced later (third instar) result in ectopic margin tissue, but do not lead to outgrowth (van Meyel, 2003).

If Ssdp were an additional member of the Ap/Chip complex, then mutations of ssdp would be predicted to give rise to mutant phenotypes similar to those of ap and Chip. To test this, the FRT/FLP recombinase system was used to induce clones of cells mutant for ssdp in an otherwise heterozygous animal. Clones were generated in larvae at second and third instar by heat-shock induction at 36 hours, 48 hours, 72 hours or 96 hours after egg laying (AEL). The effects of clone induction were observed in newly eclosed adults. Clones of mutant cells were identified by the presence of the cell-autonomous marker pawn (pwn). Each of the mutant alleles ssdp^L7, ssdp^L5 and ssdp^l(3)neo48 were tested, as was a control chromosome with no mutation, and the experiment was repeated on four separate occasions, each time observing many individuals of each genotype (van Meyel, 2003).

In controls, many clones of various sizes were induced, as evidenced by the presence of pwn mutant cells. These clones occurred on both the ventral and dorsal surfaces of the wing blade, but no mutant phenotypes were ever observed. By contrast, clones of cells mutant for either ssdp^L7 or ssdp^L5 (as marked by pwn) were never observed on either surface of the wing blade, indicating that both alleles have cell-lethal effects in the wing disc. In addition, there were fewer than the expected number of adults eclosing of the appropriate genotype for clone induction, suggesting that the cell-lethal effects, presumably in tissues other than the wing, led to decreased viability (van Meyel, 2003).

In contrast to the cell lethality associated with ssdp null alleles, there were striking phenotypes observed in clones of cells mutant for the hypomorphic ssdp^l(3)neo48 allele. Many pwn mutant clones located both ventrally and dorsally were observed. However, as for ap and Chip clones, associated phenotypes were found only when the clone arose on the dorsal surface of the wing. ssdp^l(3)neo48 clones induced earlier (at 36 hours and 48 hours AEL) give rise to ectopic margins and occasional wing outgrowth. The outgrowths were associated with ssdp mutant cells but were not entirely made up of them, indicating that, as for ap and Chip, outgrowth results from the induction of wild-type tissues in proximity to the clone. Clones induced at 72 hours and 96 hours AEL give rise to margin defects but not outgrowth, indicating that there is a temporal restriction on the extent to which ssdp mutation is capable of inducing outgrowth, similar to what has been shown for Chip (van Meyel, 2003).

Induction of ectopic margin bristles was the most commonly observed effect of dorsal ssdp mutant clones. They were primarily observed in proximity to a clone near the native anterior wing margin and comprised at least one row of extra sensory bristles. Most ectopic bristles were not marked by pwn, indicating they were induced by the neighboring mutant (pwn) cells. ssdp^l(3)neo48 mutant clones that occurred within the margin, rather than near it, resulted in the loss of dorsal-specific sensory bristles. Occasionally a large clone was observed to straddle the dorsoventral boundary, and in these instances, the entire margin, including some nearby non-margin tissue, was lost (van Meyel, 2003).

In general, there is a striking resemblance between the phenotypes resulting from ssdp^l(3)neo48 mutant clones and those reported for clones of Chip or ap. This provides strong evidence that Ssdp is an important additional component of Chip/Ap transcriptional complexes in vivo (van Meyel, 2003).

Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. This communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes, and this study shows that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. This study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication (Heitzler, 2003).

Chip^E is a viable allele of Chip that is associated with a point mutation in the LIM-interacting domain (LID), which specifically reduces interaction with the bHLH proteins Ac, Sc, and Da. As a consequence, the Chip^E mutation disrupts the functioning of the proneural complex encompassing Chip, Pnr, Ac/Sc, and Da. A homozygous Chip^E mutant shows thoracic cleft and loss of the DC bristles, similar to loss of function pnr alleles (Heitzler, 2003).

To identify new factors that regulate this proneural complex, a screen was performed for second-site modifiers of the Chip^E phenotypes. One allele of osa (osa^E17) was found among the putative mutants. Osa^E17 corresponds to a loss-of-function allele, and homozygous embryos die with normal cuticle patterning. Both osa^E17 and null alleles of osa (osa⁶¹⁶ or osa¹⁴⁰⁶⁰) enhance the cleft but suppress the loss of DC bristle phenotypes of Chip^E flies. Indeed, Chip^E flies with only one copy of osa⁺ (Chip^E;osa⁶¹⁶/+) are weak and sterile but show wild-type DC bristle pattern (Heitzler, 2003).

These genetic interactions suggest that Osa can antagonize the function of Pnr. Moreover, overexpressed Osa (+/UAS-osa;Gal4-pnr^MD237/+) induces a thoracic cleft and the loss of DC bristles similar to the loss-of-function pnr alleles. In contrast, loss-of-function osa alleles display an excess of DC bristles similar to overexpressed Pnr. For example, (osa¹⁴⁰⁶⁰/+), (osa⁶¹⁶/+), and (osa^E17/+) flies exhibit respectively 2.35 ± 0.12, 2.38 ± 0.12, and 2.43 ± 0.17 DC bristles per heminotum (Oregon wild-type flies have 2.00 DC bristles/heminotum). Furthermore, transallelic combination of osa¹⁴⁰⁶⁰ with the hypomorphic osa^4H (osa^4H/osa¹⁴⁰⁶⁰) accentuates the excess of DC bristles compared with (osa¹⁴⁰⁶⁰/+). (osa^4H/osa¹⁴⁰⁶⁰) flies display 4.17 ± 0.19 DC bristles per heminotum. In contrast, (osa^4H/osa^4H) flies display 2.50 ± 0.11 DC bristles per hemithorax. The development of the extra DC bristles revealed by phenotypic analysis was compared with the positions of the DC bristle precursors detected with a LacZ insert, A101, in the neuralized gene that exhibits staining in all sensory organs. In (osa¹⁴⁰⁶⁰/osa^4H) discs, additional DC precursors are observed that lead to the excess of DC bristles. The pnr^D alleles encode Pnr proteins carrying a single amino acid substitution in the DNA binding domain that disrupts interaction with the U-shaped (Ush) antagonist. Consequently, Pnr^D constitutively activates ac/sc, leading to an excess of DC bristles. This excess is accentuated when osa function is simultaneously reduced (pnr^D1/osa⁶¹⁶) (Heitzler, 2003).

Since osa shows genetic interactions with trithorax group genes encoding components of the Brm complex like moira (mor) and brm, whether mutations in mor and brm suppress the Chip^E phenotype was investigated. Loss of one copy of brm⁺ in (Chip^E; brm²/+) flies suppresses the lack of DC bristles observed in Chip^E flies, similar to loss of one copy of osa⁺. This shows that brm and osa both act during Pnr-dependent patterning, in agreement with the fact that they have been shown to be associated in the Brm complex. In contrast, reducing the amount of Mor by half [(Chip^E;mor¹/+) flies] is not sufficient to modify the Chip^E phenotype. This does not definitely exclude the possibility that mor is directly or indirectly involved, via the Brm complex, in Pnr-dependent patterning (Heitzler, 2003).

The complete osa open reading frame of 2715 amino acids and the intronic splicing signals were PCR amplified from genomic DNA prepared from homozygous embryos (osa^E17 and osa¹⁴⁰⁶⁰) and homozygous first instar larvae (osa^4H). For osa¹⁴⁰⁶⁰ and osa^4H, the sequence analysis revealed a single mutation in the N terminus that causes a glutamine to stop codon substitution. The conceptual translation of osa¹⁴⁰⁶⁰ leads to a truncated Osa protein lacking both functional domains, whereas Osa^4H retains the ARID domain but lacks the C-terminal EHD. Wild-type osa function is necessary for patterning of the DC bristles. Although osa^E17 behaves as a stronger allele than osa¹⁴⁰⁶⁰ and osa^4H, molecular identity of the mutation is unknown. Hence, the osa^E17 phenotype may result from a mutation in regulatory sequences that affects osa expression (Heitzler, 2003).

It has been shown that a complex containing Pnr, Chip, and the (Ac/Sc)-Da heterodimer activates proneural expression in the DC proneural cluster and promotes development of the DC macrochaetae. Osa and Pnr/Chip have antagonistic activities during development because loss of osa function (osa^4H and osa¹⁴⁰⁶⁰) displays additional DC bristles. However, since the current study reveals that osa genetically interacts with pnr and Chip, it was asked whether Osa physically interacts with the Pnr and Chip proteins. Immunoprecipitations of protein extracts made from Cos cells cotransfected with expression vectors for tagged Osa and either Pnr or tagged Chip were immunoprecipitated. Because Osa is a large protein, several expression vectors encoding contiguous domains of Osa were used. Osa coimmunoprecipitates with Pnr and Chip and can be detected on Western blots with appropriate antibodies. The interactions appear to require the overlapping domains Osa E (His1733/Glu2550) and Osa F (Ala2339/Ala2715) corresponding to the EHD. Enhancer-promoter communication during proneural activation and development of the DC bristles requires regulatory sequences scattered over large distances and appears to be negatively regulated by interaction of Pnr and Chip with Osa through the EHD. Interestingly, the EHD is not conserved in yeast. In yeast, the UAS sequences are generally close to the promoter and there is no requirement for long-distance interactions. This observation could support the idea that the EHD is essential for long-distance enhancer-promoter communication. Alternatively, yeast may just lack proteins like Chip or Pnr (Heitzler, 2003).

The DNA-binding domain and the C-terminal region are essential for the function of Pnr during development of the DC sensory organs. The pnr^VX1 and pnr^VX4 alleles (collectively pnr^VX1/4) are characterized by frameshift deletions that remove two C-terminal alpha-helices and result in reduced proneural expression and loss of DC bristles (Heitzler, 2003).

The molecular interactions between Osa and Pnr^D1 and between Osa and Pnr^VX1 were investigated. Pnr^D1 protein interacts with the EHD as efficiently as wild-type Pnr. In contrast, the physical interaction is disrupted when the C terminus of Pnr encompassing the alpha-helices is removed. Because the C terminus of Pnr is required for the Pnr-Osa interaction in transfected cells extracts, the abilities of in vitro translated 35S-labeled Osa domains to bind to GST-C^TPnr attached to glutathione-bearing beads were investigated. Only Osa E and Osa F interact with the C terminus of Pnr. The interaction between Chip and Osa, and it was found that Osa associates with the N-terminal homodimerization domain of Chip, also required for the interaction between Chip and Pnr, was investigated. Furthermore, Osa E and Osa F also bind to immobilized GST-Chip. Deletion of the alpha helix H1 disrupts the interactions between Pnr and Osa. Interestingly, the same deletion also disrupts the interaction with Chip. Therefore, the functional antagonism between Chip and Osa during neural development may result from a competition between these proteins for association with Pnr. Alternatively, the deletion of H1 may affect the overall structure of the C terminus of Pnr and disrupt the physical interactions with Chip and Osa. To discriminate between these hypotheses, immunoprecipitations of protein extracts containing a constant amount of Pnr, a constant amount of the tagged Osa E domain, and increasing concentrations of Chip were performed. Pnr immunoprecipitates with immunoprecipitated tagged Osa E and the amount of Pnr immunoprecipitated increases in the presence of increasing concentrations of Chip. The presence of increasing amounts of Chip does not inhibit the Osa-Pnr interaction as would be expected if Osa and Chip were to compete for binding to Pnr. In contrast, it suggests that Chip and Pnr act together to recruit Osa and to target its activity and possibly the activity of the Brm complex to the ac/sc promoter sequences (Heitzler, 2003).

Using expression vectors encoding contiguous domains of Osa, it was shown that the EHD of Osa mediates interactions with Pnr and Chip. Because the EHD is lacking in the truncated Osa¹⁴⁰⁶⁰ and Osa^4H, it is hypothesized that the loss of interaction with Pnr and Chip are responsible for the excess of DC bristles observed in osa^4H and osa¹⁴⁰⁶⁰ (Heitzler, 2003).

To investigate whether these interactions between Osa, Pnr, and Chip function in vivo during DC bristle development, the effects of both loss of function and overexpression of osa were examined on the activity of a LacZ reporter whose expression is driven by a minimal promoter sequence of ac fused to the DC enhancer (transgenic line DC:ac-LacZ). It was found that expression of the LacZ transgene is increased in osa¹⁴⁰⁶⁰/osa^4H wing discs when compared with the wild-type control. For overexpression experiments, the UAS/GAL4 system was used, using as a driver the pnr^MD237 strain that carries a GAL4-containing transposon inserted in the pnr locus (driver: pnr-Gal4). This insert gives an expression pattern of Gal4 indistinguishable from that of pnr. It was found that overexpressed Osa leads to a strong reduction of LacZ staining in the DC area, consistent with the lack of DC bristles. Thus, overexpressed Osa represses activity of the ac promoter sequences required for DC ac/sc expression and development of the DC bristles. It has been previously reported that wingless expression is also required for patterning of the DC bristles. However, the repressing effect of Osa on development of the DC bristles is unlikely to be the result of an effect of Osa on wingless expression because overexpressed Osa driven by pnr^MD237 has no effect on the expression of a LacZ reporter inserted into the wingless locus. Thus, Osa acts through the DC enhancer of the ac/sc promoter sequences to repress ac/sc and neural development (Heitzler, 2003).

Chip^E disrupts the enhancer-promoter communication and strongly affects expression of the LacZ reporter driven by the ac promoter linked to the DC enhancer. Because null alleles of osa suppress the loss of DC bristles displayed by Chip^E, the consequences of reducing the dosage of osa was examined in Chip^E flies. The expression of the LacZ reporter is not affected in Chip^E flies when Osa concentration is simultaneously reduced (Heitzler, 2003).

In conclusion, Pnr function during proneural patterning is regulated by interaction with several transcription factors. Pnr function is negatively regulated by Ush, which interacts with its DNA-binding domain. Chip associates with the C terminus of Pnr, bridging Pnr at the DC enhancer with the AC/Sc-Da heterodimers bound at the proneural promoters, thus activating proneural gene expression. The current study reveals that Pnr function can also be regulated by interaction with Osa. Thus, Osa activity is specifically targeted to ac/sc promoter sequences and the binding of Osa therefore has a negative effect on Pnr function, leading to reduced expression of the proneural ac/sc genes. Osa belongs to Brm complexes, which are believed to play an essential role during chromatin remodeling necessary for gene expression. For example, in vitro transcription experiments with nucleosome assembled human beta-globin promoters have shown that the BRG1 and BAF155 subunits of the mammalian SWI/SNF homolog are essential to target chromatin remodeling and promote transcription initiation mediated by GATA-1. In contrast to what was observed in vitro, the current results suggest that in vivo the SWI/SNF complexes can also act to remodel chromatin in a way that represses transcription. Alternatively, the observed repression of proneural genes may simply define a novel function of Osa, independent of chromatin remodeling (Heitzler, 2003).

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO (Beadex). Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. A modular P-element based EP (enhancer/promoter) misexpression screen was conducted to look for genes involved in Apterous activity. Osa, a member of the Brahma chromatin-remodeling complex, was found to be a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. It is proposed that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression (Milan, 2004).

This study presents evidence that Osa, a member of a subset of Brahma chromatin remodeling complexes, behaves overall as a general activator of Apterous activity in the Drosophila wing. Overexpression of Osa rescues and loss of Osa enhances the Beadex¹ phenotype. It does so by modulating the expression levels of Apterous target genes, some of them being activated (e.g. Serrate and probably other unknown target genes) and some repressed (e.g. Delta, fringe). Chip has been shown to bind Osa. The fact that Osa has different effects on the transcription of Apterous target genes suggests that Chip recruits Osa to the promoters and in combination with other unknown factors mediates either transcriptional repression or activation. Osa mediates repression of both Apterous dependent and independent expression of fringe, suggesting a direct and probably Chip independent effect of Osa on fringe transcription (Milan, 2004).

Apterous activity is regulated during development by dLMO. Osa is required to mediate repression of dLMO expression. Since both early and late expression of dLMO depend on Chip, it is postulated that Chip forms a transcriptional complex with Apterous in the D compartment and an unknown transcription factor expressed in the wing pouch. Osa may interact with Chip thus recruiting the Brahma complex to the dLMO locus and remodeling chromatin in a way that limits dLMO transcriptional activation. High levels of dLMO protein reduce Apterous activity and the Notch dependent organizer is not properly induced along the DV boundary. Osa mediated repression of dLMO expression may ensure moderate levels of expression of dLMO in the wing, thus allowing proper wing development. Gain of function mutations that cause misexpression of vertebrate LMO proteins have been implicated in cancers of the lymphoid system. Truncating mutations in the human SWI-SNF complex, the human homologues of the Brahma complex, cause various types of human cancers. The SWI-SNF complex may be required to mediate repression of LMO expression in lymphoid tissues. Thus, it would be very interesting to analyze if truncating mutations in members of the human SWI-SNF complex cause higher levels of LMO expression and are associated with lymphoid malignancies (Milan, 2004).

It has been shown that the Brahma complex plays a general role in transcription by RNA Polymerase II. Then, is Osa having a general effect on the expression levels of every gene involved in wing patterning? Several observations indicate this is not the case. (1) Osa is a component of a subset of Brahma (Brm) chromatin complexes. (2) Brahma and Polycomb were shown to have non-overlapping binding patterns in polytenic chromosomes. Those genes involved in wing patterning and regulated by Polycomb (i.e. Hedgehog) may not be affected by overexpression of Osa. (3) Overexpression of Osa has different effects on the expression levels of Serrate, Delta and fringe. (4) Osa has been shown to specifically regulate the expression of Wingless target genes and the Achaete-scute complex genes, interestingly by restricting their expression levels (Milan, 2004).

Chip-mediated partnerships of the homeodomain proteins Bar and Aristaless with the LIM-HOM proteins Apterous and Lim1 regulate distal leg development

Proximodistal patterning in Drosophila requires division of the developing leg into increasingly smaller, discrete domains of gene function. The LIM-HOM transcription factors apterous (ap) and Lim1 (also known as dlim1), and the homeobox genes Bar and aristaless (al) are part of the gene battery required for the development of specific leg segments. Genetic results show that there are posttranslational interactions between Ap, Bar and the LIM-domain binding protein Chip in tarsus four, and between Al, Lim1 and Chip in the pretarsus, and that these interactions depend on the presence of balanced amounts of such proteins. In vitro protein binding is observed between Bar and Chip, Bar and Ap, Lim1 and Chip, and Al and Chip. Together with evidence for interactions between Ap and Chip, these results suggest that these transcription factors form protein complexes during leg development. It is proposed that the different developmental outcomes of LIM-HOM function are due to the precise identity and dosage of the interacting partners present in a given cell (Pueyo, 2004).

Biochemical studies in vitro have shown that LIM-HOM transcription factors confer little transcriptional activation of target genes on their own. LIM-HOM proteins interact with a variety of proteins, including members of the bHLH family, the POU family and also other LIM family members, to control specific developmental processes. It has been suggested that these protein interactions confer specificity and modulate LIM-HOM activity. For example, Dlmo proteins reduce LIM-HOM activity, and Lbd proteins such as Chip modulate LIM-HOM activity by acting as a bridge between LIM-HOM proteins and Chip-binding cofactors, thus leading to the formation of heteromeric complexes. LIM-HOM protein activity functions in different contexts is the development of Drosophila (Pueyo, 2004).

Bar and ap genes are expressed in the fourth tarsal segment and are required for its proper development, whereas the al and Lim1 genes are expressed and required in the pretarsus. All of these genes encode putative transcription factors and display canonical regulatory relationships. Thus, al activates lim1 expression and then both genes cooperate to repress Bar expression in the pretarsus. Reciprocally, Bar represses al and lim1 expression while activating the expression of ap in tarsus four. After the refinement of their gene expression domains by these regulatory interactions, Bar directs tarsus five development, whereas cooperation between al and lim1 directs pretarsus development, and cooperation between Bar and ap directs tarsus four. The results of this study offer more evidence for the existence of this regulatory network, but also suggest an interesting role for direct protein interactions in its mechanism (Pueyo, 2004).

The cooperation between Bar and Ap on the one hand, and Al and Lim1 on the other, is likely to be carried out by transcriptional complexes involving Chip. The Chip protein is required for development of the tarsus four, five and pretarsus, and Gst (Glutathione-S-transferase-Chip fusion construct) experiments reveal Chip's ability to bind Ap, Bar, Lim1 and Al. However, the results also show that modulation of LIM-HOM protein activity by Chip alone does not explain distal leg development. For example, Ap function is not modulated primarily by Chip and Dlmo. The relative amount of Chip and Ap has to be grossly unbalanced before a phenotype is obtained in the leg, and dlmo is not expressed or required in leg development. Furthermore, the interaction between Ap and Chip does not confer the developmental specificity that allows LIM-HOM proteins to produce different outcomes in different parts of the leg. (1) Ap and Chip also interact in the wing and the CNS. (2) A chimaeric Lim3-Ap protein containing the LIM domains of Lim3 and the HOM domain of Ap does not behave as a dominant negative when expressed in tarsus four, and is even able to fulfil Ap function and rescue ap mutants. In the distal leg, developmental specificity seems to be achieved at the level of DNA binding and the transcriptional control of targets genes, mediated by partnerships between LIM-HOM and HOM proteins (Pueyo, 2004).

The evidence for this is presented first by dosage interactions between LIM-HOM and HOM proteins. Whereas there seems to be a relative abundance of endogenous Ap in tarsus four, an excess of Bar or Chip leads to a mutant phenotype, which is rescued by restoring the normal balance between Ap, Bar and Chip proteins in co-expression experiments. The effects observed could be explained simply by independent competition and the binding of Bar and Ap to Chip, leading, for example, to an excess of Bar-Chip complexes and a reduction of the pool of Chip available for Ap-Chip complexes. However, this hypothesis alone does not explain the additional dominant-negative effects of ectopic LIM-HOM and HOM proteins in tarsus four (Lim3, Islet and Al), which are also not mediated by transcriptional regulation but are nonetheless rescued by co-expression of appropriate endogenous proteins. For example, ectopic expression of UAS-islet or UAS-Lim3 in the ap domain produces loss of tarsus four without affecting Ap or Bar expression, and simultaneous co-expression of UAS-Bar partially suppresses this phenotype. If the sole effect of both UAS-Bar and UAS-Lim3 or UAS-islet were to quench Chip away from Ap, then simultaneous co-expression of Bar and Lim3 or Islet should worsen the phenotype, not correct it as observed. Moreover, ectopic expression of Islet or Lim3 proteins is not corrected by simultaneous co-expression of either UAS-Chip or UAS-ap. Altogether these results show instead that UAS-islet and UAS-Lim3 must interfere posttranslationally with Bar. The most direct explanation is that Islet and Lim3 have the ability to quench Bar protein into a non-functional state. Interestingly, the hybrid UAS-Lim3:ap does not behave as dominant negative but as an endogenous Ap protein in these experiments, since it does not produce a mutant phenotype on its own and it rescues UAS-Bar overexpression. This suggests that the LIM domains are not very specific when it comes to interaction with Bar, and points to the involvement of a common LIM-binding intermediary such as Chip. These results suggest that a protein complex involving Ap, Chip and Bar is the correct functional state of these proteins in tarsus four, and deviations from this situation into separate Bar-Chip, Ap-Chip, or Bar-Chip-Lim3 or Bar-Chip-Islet complexes leads to a mutant phenotype (Pueyo, 2004).

The notion of a protein complex involving Ap, Chip and Bar together is also supported by the Gst pull-down assays. The domain of Chip involved in Ap binding, the LIM interaction domain (LID), is not involved in Bar binding. However, the LID and the dimerisation domains of Chip are necessary to rescue the dominant-negative effect of UAS-Bar on tarsus four, suggesting a requirement for the formation of a complex with a LIM-HOM protein such as Ap. In agreement with this view, the Ap protein, and the LIM domains of Ap alone, are able to retain Bar protein in a Gst assay (Pueyo, 2004).

In the pretarsus, Al and Lim1 are possibly engaged in a partnership with Chip similar to that suggested for Ap, Chip and Bar. Synergistic cooperation between Al and Lim1 is required to direct pretarsus development and to repress Bar expression and function. Their cooperation entails a close functional relationship because a proper balance of Al, Lim1 and Chip is required, as is shown by the loss of pretarsal structures in UAS-Chip or UAS-Lim1 flies. Ectopic expression of LIM-HOM proteins in the pretarsus also disrupts pretarsal development without affecting Lim1 and Al expression. The possibility of direct protein interactions between Al, Lim1 and Chip is also suggested by the reciprocal ability of Al to interfere posttranscriptionally with Bar and Ap in tarsus four, and by the binding of Chip to Lim1 and to Al in in vitro experiments (Pueyo, 2004).

Comparison of tarsal development with other developmental processes illustrates how LIM-HOM proteins are versatile factors to regulate developmental processes. It had been observed that the outcome of LIM-HOM activity depends on their developmental context. This context can now be analysed as being composed of the presence, concentration and relative affinities of other LIM-HOM proteins, Ldb adaptors, and other cofactors such as LMO proteins and HOM proteins. It is proposed that the different developmental outcomes of LIM-HOM protein function could be due to the precise identity and dosage of cofactors available locally (Pueyo, 2004).

Ectopic expression experiments distort these contexts and lead to non-functional or misplaced LIM-HOM activities. In the wing, a finely balanced amount of functional Ap protein is modulated by Dlmo and Chip. Over-abundance of Chip stops the formation of functional tetramers in the wing but not in the CNS, where the relative amount of Ap, which is not modulated by Dlmo, is limiting for the formation of Ap-Chip functional complexes. In tarsus four, the Ap-Chip-Bar partnership is affected by experimentally induced over-abundance of Chip, presumably also because ectopic Ap-Chip tetramers typical of the CNS and the wing, and Bar-Chip complexes typical of tarsus five, are produced. Similarly, an excess of Bar might be interpreted by the cells as being a wrong developmental outcome, since high levels of Bar in the absence of Ap direct tarsus five development. Overexpression of Ap rescues this Bar dominant-negative effect, by restoring the relative amounts of Bar and Ap, which are determinant and limiting for tarsus four development. Finally, the dominant-negative effects produced by overexpression of either Chip or Lim1 in the pretarsus could either prevent the formation of Al-Chip-Lim1 complexes, or could favor the existence of Lim1-Chip complexes typical of the CNS (Pueyo, 2004).

The wing and the CNS models have postulated that Ap function is carried out by an Ap-Chip tetramer; however, the molecular scenario might be more complex. A new component of Ap-Chip complexes, named Ssdp, has been identified and is required for the nuclear localisation of the complex. Thus it is possible that an Ap-Chip tetramer also contains two molecules of Ssdp. In addition, different types of Chip-mediated transcriptional complexes and different regulators have been identified in other developmental contexts, such as in sensory organ development and thorax closure, in which the GATA factor Pannier forms a complex with Chip and with the bHLH protein Daughterless. Heterodimers of this complex are negatively regulated by a protein interaction with Osa. Thus, although the current results indicate that in different segments of the leg there exist specific interactions between LIM-HOM, Chip and HOM proteins, the involvement of further elements in these multiprotein complexes is not excluded (Pueyo, 2004).

The results support a partnership between HOM and LIM-HOM proteins in the specification of distinct segments of the leg, and the results are compatible with Ap-Chip-Bar, Bar-Chip and Lim1-Chip-Al forming transcriptional complexes. Although the characterisation of the target sequences, followed by further biochemical and molecular assays, is necessary to study the transcriptional mechanism of these interactions, it has been shown that LIM-HOM proteins can interact specifically and directly with other transcription factors to regulate particular genes. For instance, mouse Lim1 (Lhx1) interacts directly with the HOM protein Otx2. In addition, the bHLH E47 transcription factor interacts with Lmx1, and both synergistically activate the insulin gene. This interaction is specific to Lmx1, since E47 is unable to interact with other LIM-HOM proteins such as Islet. Moreover, Chip is able to bind to other Prd-HOM proteins, such as Otd, Bcd and Fz, to activate downstream genes. Chip also complexes with Lhx3 and the HOM protein P-Otx, increasing their transcriptional activity. The current results reinforce the notion of Chip as a multifunctional transcriptional adaptor that has specific domains involved in each interaction (Pueyo, 2004).

Experiments in Drosophila have demonstrated a conservation of LIM-HOM activity at the functional and developmental level in the CNS between Drosophila and vertebrates. In addition, xenorescue experiments have shown that the mechanism of action of Ap and its vertebrate homolog Lhx2 is very conserved in Drosophila wings, whereas ectopic expression of dominant-negative forms of chick Lim1, Chip, Ap and Lhx2 mimic both Ap and Lhx2 loss-of-function phenotypes. The developmental role of Ap, Bar and Al in the fly leg, and their putative molecular interactions may also have been conserved because their vertebrate homologs Lhx2, Barx and Al4 are also co-expressed in the limb bud. It is expected that the interactions between the LIM-HOM and Prd-HOM proteins shown here represent a conserved mechanism to specify different cellular fates during animal development (Pueyo, 2004).

The Drosophila LIM-homeodomain protein Islet antagonizes proneural cell specification in the peripheral nervous system: Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction

The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix-loop-helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in clusters of cells (proneural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorsocentral enhancer (DC) of ac/sc. The LIM-homeodomain (LIM-HD) transcription factor Islet (Isl) was identified by genetic screening and its role in the thoracic prepatterning was investigated. isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) proneural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases proneural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC proneural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction. It is proposed that sensory organ prepatterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying prepatterns within the different proneural clusters (Biryukova, 2005).

During Drosophila development, the expression of transcription factors divides the dorsal thorax into three domains -- one median and two lateral domains. The lateral domains are specified by the homeobox-containing proteins of the iroquois-complex (iro), whereas the GATA factor Pnr is required to establish the median domain. Within the mesothorax, Pnr together with U-shaped (Ush) and Chip plays a key role in dorsal closure. This report presents evidence that Isl is an essential regulator of the dorso-median patterning of the thorax. isl⁻ clones generated adjacent to the thoracic midline, induce a strong cleft, suggesting that Isl is required for proper dorsal closure during metamorphosis. Ectopic expression of Pnr leads to wing-to-thorax transformations, consistent with its role as medio-dorsal patterning factor. Ectopic Isl expression does not exhibit this phenotype, excluding the LIM-HD factor from a direct function as a prothoracic selector. Pnr is also known to activate wingless (wg) in dorsal thorax. isl loss-of-function has no significant effect on wg expression. However, overexpressed Isl strongly reduces the size of the wg thoracic stripe. This result is consistent with a repressive activity of Isl on Pnr (Biryukova, 2005).

Iro proteins and Pnr are direct activators of the proneural genes in their respective domains. Pnr binds directly to the DC enhancer of ac/sc, providing therefore region-specific control of the proneural prepatterning. Flies with reduced or lack of Pnr function fail to activate ac/sc and to develop DC and SC sensory organs. The proneural activity of Pnr is antagonized by Ush, the vertebrate homologue of the FOG (friend of GATA). Ush is expressed only in the dorsal-most cells of the medial region. As a consequence, the segregation of the sensory organ precursors occurs along two stripes at the border of the medial domain of Pnr expression, where Ush is absent or insufficient to repress Pnr (Biryukova, 2005).

Several lines of evidence indicate that Isl interferes with the proneural activity of Pnr as a repressor. (1) isl loss-of-function mutants show an opposite phenotype with regard to Pnr or Chip loss-of-function mutants: an excess of DC and SC sensory organs. (2) A genetic synergism exists between Pnr^D and isl⁻ alleles. This genetic interaction is less sensitive than that between Pnr^D and ush⁻, implying an alternative route for Isl to modulate the Pnr proneural activity. (3) Isl is coexpressed with Pnr within the posterior mesothorax. (4) Isl modulates the activity of a DC:ac-lacZ reporter. Loss-of-function isl mutants expand the DC:ac-lacZ expression as in ush⁻ or Pnr^D constitutive mutants, whereas overexpressed Isl reduces the DC:ac-lacZ expression (Biryukova, 2005).

In the DC region, the regulation of Pnr concentration is critical for the proper position and shape of the DC proneural cluster. Isl expression overlaps with the dorsal-most domain of Pnr and DC proneural activity coincides with the posterior border of Isl expression. Therefore, it proposed that both Isl and Ush restrict Pnr activity in the mesothorax. Interestingly, the regulation of the concentration of the mammalian Pnr ortholog, GATA-1, is similarly critical for proper erythroid, megakaryocytic, eosinophilic and mast cell lineages (Biryukova, 2005).

Ush behaves as either an activator or a repressor of Pnr, depending on developmental context. No evidence was found for a direct Isl-Ush interaction by GST pull down assay: Ush, Pnr and Isl could be co-immunoprecipitated from transient transfected S2 cells. Both Ush and Isl may behave as positive cofactors of Pnr for nonneural activities, such as cardiac development, embryonic dorsal closure and metamorphosis. Several reports emphasize the role of the Pnr homolog, GATA-1 and Isl1 in human blood disorders. It seems likely that GATA:Islet interactions represent a conserved mechanism to specify different cell fates in humans and other organisms (Biryukova, 2005).

Isl proteins are known as positive regulators of transcription in vertebrates. In flies, Isl mediates repression of Pnr-driven proneural activity via binding to the DNA-binding domain of Pnr. Interestingly, these interactions are less specific than for the Pnr-Ush interaction, where the amino-terminal zinc finger of Pnr is specifically involved (Biryukova, 2005).

Genetic analyses of mutants reveal that the DC and the SC proneural clusters show differential sensitivities during neurogenesis. Ush mutants display ectopic DC bristles and a few additional SC bristles. This phenotype is similar to Pnr^D constitutive mutants, in which Pnr-Ush interactions are greatly reduced. In contrast, isl mutants show the opposite phenotype, with a large excess of SC bristles and a few additional DC bristles. The Chip^E mutant exhibits antagonistic phenotypes: lack of DC bristles, reflecting Pnr loss-of-function and an excess of SC bristles, reflecting Isl loss-of-function. The differential topography of DC and SC enhancer binding sites presumably underlies differential transcription-complex binding affinities (Biryukova, 2005).

Chip is the ortholog of Ldb factors that are ubiquitous multiadaptor proteins in vertebrates. Each Ldb-dependent developmental event is specified by modification of the transcriptional complex and is dependent on the stoichiometry of the region-specific Ldb partners. During normal development of the thorax, different partners of Chip (i.e., Isl, Ap and Pnr) are expressed in the same region. The Chip^E mutant is highly sensitive to the dosage of these factors. In Chip^E flies, removing one copy of either Pnr or Isl causes pupal lethality associated with extreme morphogenetic phenotypes. Removing one copy of Ap, however, rescues the Pnr-dependent phenotypes of Chip^E flies. Taken together, these results indicate selective competition between the different partners of Chip, suggesting that hierarchical protein interactions depending on differential affinities and the strict stoichiometry of Chip and its partners, are critical to establish proper transcriptional codes within different proneural fields (Biryukova, 2005).

isl mutants were isolated in genetic screens for dominant enhancers of the Chip^E phenotype. This study demonstrates that the LIM-HD transcription factor Isl can bind to the LID of Chip. The binding of the LID domain of Chip with LIM domains has been conserved throughout evolution as has Chip binding with bHLHs proteins. LID contains two subdomains: a small N-terminal hydrophobic β patch (VMVV) followed by a large α helix. Chip^E mutation has a single substitution that changes an Arg to Trp (R504W) in the middle of the α helix. This residue is highly conserved among species and mediates high-affinity contact with the LIM domains. Interestingly, the R504W substitution in Chip abolishes, or strongly reduces, both interactions with the bHLHs and also interactions with Isl. This result implies that bHLHs and Isl recognize the same site within the LID domain of Chip. The data argue that competition between bHLHs and Isl for the LID domain of Chip may be critical for modulating the activity of transcription complexes during development. In vertebrates, the NLI homolog of Chip mediates direct coupling of the proneural bHLH factors Ngn2, NeuroM and the LIM-HD transcription factors (Isl1 and Lhx3). This interaction leads to transcriptional synergism and the synchronization of motor neuron subtype specification with neurogenesis in the embryonic spinal cord of chicken. This work demonstrates that Isl is able to interfere with proneural activity of Chip-Pnr-bHLH transcription complex and therefore, Isl is thought to be able to antagonize proneural specification (Biryukova, 2005).

Interestingly, the Chip^E mutation has little or no effect on interactions with other LIM-containing factors, such as Ap and dLMO, suggesting that different factors have different affinities with the Chip LID domain. Therefore, the Chip^E mutation changes the hierarchy of the affinities among the different partners of Chip in the mesothorax (Biryukova, 2005).

A transcription-complex 'cassette' model is proposed for the specification of region-specific patterns of specialized cell types. In this model, the presence of one of a number of alternative binding factors modifies the specificity of a core transcription complex. This model makes the prediction that, while the core components of the transcription complex will be strongly conserved in evolution, the specificity cassette components will vary significantly between species showing divergent morphogenetic patterns. Comparison of these variable components in related species should provide insights into the fundamental mechanisms of encoding the pattern of differentiated cell types within morphogenetic fields (Biryukova, 2005).

Toutatis, a TIP5-related protein, positively regulates Pannier function during Drosophila neural development

The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc) complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, Toutatis (Tou; see Teutates the supposed deified spirit of male tribal unity in ancient Celtic polytheism, best known under the name Toutatis, through the Gaulish catchphrase "By Toutatis!", invented for the Asterix comics by Goscinni and Uderzo), which physically interacts with both Pnr and Chip, was identified. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

Transcriptional activation of many developmentally regulated genes is mediated by proteins binding to enhancers scattered over the genome, raising the question on how long-range activation is restricted to the relevant target promoter. Numerous studies have highlighted the essential role of boundaries, which maintain domains independent of their surrounding (Vanolst, 2005).

The patterning of the large sensory bristles (macrochaetae) on the thorax of Drosophila melanogaster is a powerful model to study how enhancers communicate with promoters during regulation of gene expression. Each macrochaeta derives from a precursor cell selected from a group of equivalent ac-sc-expressing cells, the proneural cluster. ac and sc encode basic helix-loop-helix proteins (bHLH) that heterodimerize with Daughterless (Da) to activate expression of downstream genes required for neural fate. Transcription of ac and sc in the different sites of the imaginal disc is initiated by enhancers of the ac-sc complex and the expression is maintained throughout development by autoregulation mediated by the (Ac-Sc)-Da heterodimers binding to E boxes within the ac-sc promoters. Each enhancer interacts with specific transcription factors that are expressed in broader domains than the proneural clusters and define the bristle prepattern. Thus, the GATA factor Pannier (Pnr) binds to the dorsocentral (DC) enhancer and activates proneural expression to promote development of DC sensory organs. The Drosophila LIM-domain-binding protein 1 (Ldb1), Chip physically interacts both with Pnr and the (Ac-Sc)-Da heterodimer to give a multiprotein proneural complex which facilitates the enhancer-promoter communication (Vanolst, 2005 and references therein).

Chromatin plays a crucial role in control of eukaryotic gene expression and is a highly dynamic structure at promoters. In Drosophila, the polycomb (Pc) group and the trithorax (Trx) group proteins are chromatin components that maintain stable states of gene expression and are involved in various complexes. The Pc group proteins are required to maintain repression of homeotic genes such as Ultrabithorax, presumably by inducing a repressive chromatin structure. Members of the Trx group were identified by their ability to suppress dominant Polycomb phenotypes. Evidence has been provided that enhancer-promoter communication during Pnr-driven proneural development is negatively regulated by the Brahma (Brm) chromatin remodelling complex, homologous to the yeast SWI/SNF complex (Vanolst, 2005).

Evidence is presented that Toutatis (Tou), a protein that associates both with Pnr and Chip and that positively regulates activity of the proneural complex encompassing Pnr and Chip during enhancer-promoter communication. Tou has been previously identified in a genetic screen for dominant modifiers of the extra-sex-combs phenotype displayed by mutant of polyhomeotic (ph), a member of the Pc group in Drosophila. Tou shares functional domains with Acf1, a subunit of both the human and Drosophila ACF (ATP-utilizing chromatin assembly and remodelling factor) and CHRAC (chromatin accessibility complex), and with TIP5 of NoRC (nucleolar remodelling complex). Hence, Tou regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling. Moreover, Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is also necessary for activation of ac-sc and neural development. Since Iswi is shown to physically interact with Tou, Pnr and Chip, it is suggested that a complex encompassing Tou and Iswi directly regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

In Drosophila, Chip has been postulated to be a facilitator required both for activity of the DC enhancer of the ac-sc complex. Enhancer-promoter communication at the ac-sc complex is negatively regulated by the Brm complex whose activity is targeted to the ac-sc promoter sequences through dimerization of the Osa subunit with both Pnr and Chip. The Brm complex is thought to remodel chromatin in a way that represses transcription (Vanolst, 2005).

Tou and Iswi appear to act together as subunits of a multiprotein complex to positively regulate activity of Pnr and Chip during enhancer-promoter communication. Tou and Iswi therefore display opposite activity to that of the Brm complex, raising questions about their molecular function during neural development. Tou shares essential functional domains with members of the WAL family of chromatin remodelling proteins, including Acf1 of ACF and CHRAC. Importantly, Acf1 and TIP5 associate in vivo with Iswi, showing that Iswi can mediate both activation and repression of gene expression. Tou positively regulates Pnr/Chip function during the period of ac-sc expression in neural development, and it associates with Iswi. Since Iswi also positively regulates Pnr/Chip function, it is hypothesized that a complex encompassing Tou and Iswi acts during long-range activation of proneural expression, possibly through chromatin remodelling. Further studies will help to resolve this issue (Vanolst, 2005).

Interestingly, Chip and Pnr seem to play similar roles both during recruitment of the Brm complex and recruitment of Tou and Iswi, since they dimerize with Osa, Tou and Iswi. In addition, Pnr and Chip apparently cooperate to strengthen the physical association with Osa and Tou. However, Osa, on the one hand, and Tou and Iswi, on the other, display antagonistic activities during neural development. Since they are ubiquitously expressed, accurate regulation of ac-sc expression would require a strict control of the stoichiometry between Osa, Tou and Iswi. It remains to be investigated whether the functional antagonism between Osa and Tou/Iswi relies on a molecular competition for association with Pnr and Chip. Determination of this would require a complete molecular definition of the putative complex encompassing Tou and Iswi, together with a full understanding of how this complex and the Brm complex molecularly interact with the proneural complex to regulate enhancer-promoter communication during development (Vanolst, 2005).

Biochemical analysis of Iswi and Iswi-containing complexes, together with genetic studies of Iswi and associated proteins in flies and in budding yeast, has revealed roles for Iswi in a wide variety of nuclear processes, including transcriptional regulation, chromosome organization and DNA replication. Accordingly, Iswi was found to be a subunit of various complexes, including NURF (nucleosome remodelling factor), ACF and CHRAC. Iswi-containing complexes were primarily recognized as factors that facilitate in vitro transcription from chromatin templates. However, genetic analysis in Drosophila and in Saccharomyces cerevisiae have provided evidence that Iswi-containing complexes are involved in both transcriptional activation and repression in vivo. For example, immunostaining of Drosophila polytene chromosomes of salivary glands showed that Iswi is associated with hundreds of euchromatic sites in a pattern that is non-overlapping with RNA polymerase II. It suggests that Iswi may play a general role in transcriptional repression. In contrast, it was also demonstrated that expression of engrailed and Ultrabithorax are severely compromised in Iswi-mutant Drosophila larvae. Recent studies have also shown that a mouse Iswi-containing complex, NoRC, plays an essential role during repression of transcription of the rDNA locus by RNA polymerase I. Tou, a protein that is structurally related to the TIP5 subunit of NoRC. Tou positively regulates enhancer-promoter communication during Pnr-driven proneural development and its activity is targeted to the ac-sc promoter sequences through dimerization with Pnr and Chip. Evidence is provided that Iswi is required during neural development. Overexpression of Iswi^K159R in the precursor cells of the sensory organs using the sca^Gal4 driver leads to flies lacking multiple bristles, suggesting that Iswi functions late during neural development, essential for either cell viability or division of the precursor cell. Using the Iswi¹/Iswi² transheterozygous combination and individuals overexpressing Iswi^K159R in earlier stages of development and in less restricted patterns, it has been shown that Iswi also regulates ac-sc expression. Interestingly, the regulation is probably direct since Iswi associates with the transcription factors Pnr and Chip, known to promote ac-sc expression at the DC site. Since Iswi interacts with Tou, it is proposed that Tou and Iswi may positively regulate activity of Pnr and Chip during enhancer-promoter communication, possibly as subunits of a multiprotein complex involved in chromatin remodelling (Vanolst, 2005).

Drosophila LIM-only is a positive regulator of transcription during thoracic bristle development

The Drosophila LIM-only (Lmo) protein DLMO functions as a negative regulator of transcription during development of the fly wing. This study reports a novel role of Dlmo as a positive regulator of transcription during the development of thoracic sensory bristles. New dlmo mutants, which lack some thoracic dorsocentral (DC) bristles, were isolated. This phenotype is typical of malfunction of a thoracic multiprotein transcription complex, composed of Chip, Pannier (Pnr), Achaete (Ac), and Daughterless (Da). Genetic interactions reveal that dlmo synergizes with pnr and ac to promote the development of thoracic DC bristles. Moreover, loss-of-function of dlmo reduces the expression of a reporter target gene of this complex in vivo. Using the GAL4-UAS system it was also shown that dlmo is spatially expressed where this complex is known to be active. Glutathione-S-transferase (GST)-pulldown assays showed that Dlmo can physically bind Chip and Pnr through either of the two LIM domains of Dlmo, suggesting that Dlmo might function as part of this transcription complex in vivo. It is proposed that Dlmo exerts its positive effect on DC bristle development by serving as a bridging molecule between components of the thoracic transcription complex (Zenvirt, 2008).

The results presented in this study uncover a novel role of Dlmo in regulation of the development of the thoracic DC bristles. Homozygous, or hemizygous, loss-of-function (dlmo^hdp) mutants lack the anterior pair of the DC bristles. Moreover, these dlmo mutants displayed genetic interactions with mutants in genes known to regulate DC bristle development, such as pnr and ac, to reduce the number of DC bristles. Consistently, overexpression alleles of dlmo (dlmo^Bx) also exhibited genetic interactions with these pnr and ac mutants, resulting in an increased number of bristles. In addition, the finding that overexpression of pnr under the regulation of dlmo-GAL4 affects DC bristle development suggests that dlmo is expressed in the region of the wing disc that gives rise to these bristles (Zenvirt, 2008).

These results suggest a role of Dlmo in positive regulation of transcription. The negative role of Dlmo in modulation of transcription during Drosophila wing development has been well documented. The findings indicate that in another context, namely in regulation of DC bristle development by the Chip, Pnr, Ac and Da (CPAD) complex, Dlmo has another role, as a positive regulator of transcription. Lowering the level of Dlmo (in dlmo^hdp mutants) results in a reduction in the expression of a reporter driven by regulatory sequences of a bona fide target gene of the CPAD transcription complex, suggesting that Dlmo is a positive regulator of CPAD-dependent transcription. While the mechanism by which Dlmo positively regulates transcription in the context of the CPAD complex remains to be elucidated, a first clue to this mechanism may lie in the finding that Dlmo can bind constituent proteins of this complex, including Pnr and Chip, in vitro. Should these interactions also take place in vivo, Dlmo may exert its positive role in transcriptional regulation as a component of the CPAD complex (Zenvirt, 2008).

Insights into the mechanism of positive transcriptional regulation by Dlmo can be gleaned from LMO2, one of the mammalian homologs of Dlmo. LMO2 was demonstrated to participate in a multiprotein transcription complex that contains Ldb1, a GATA factor (GATA-1 or GATA-2), and the bHLH transcription factors TAL1 and E2A, which are homologous, respectively, to the fly components of the CPAD complex, Chip, Pannier, Achaete, and Daughterless. Various lines of evidence indicate that in mammals LMO2 serves as a bridge between components of the complex, and silencing of LMO2 causes disruption of the complex and decreases in the activation of transcription of its target genes, just as does silencing of Ldb1 or Tal1. Similarly to LMO2, Dlmo might serve as a bridge between components of the CPAD complex. LIM domains are protein-interaction modules and could serve Dlmo to bind components of the CPAD complex. This suggestion is supported by the finding that each single LIM domain of Dlmo is capable of binding components of the CPAD complex in vitro, and it agrees with similar reports on other LIM-containing proteins. Notably, a single LIM domain from LMO2 and LMO4 is sufficient to interact with Ldb1 or the related protein CLIM-1a. However, both LIM domains are required for the highest-affinity interactions (Zenvirt, 2008).

This proposed mode of action of Dlmo, as a bridging molecule, which binds a different protein through each one of its LIM domains, predicts that a Dlmo molecule with one defective LIM domain and one intact LIM domain would bind only one protein at a time and not be able to bridge between molecules. Indeed, in these new dlmo mutants it was found that deletions that span the second zinc finger of the second LIM domain of Dlmo, namely dlmo^hdp48-1 and dlmo^hdp185-1, resulted in dlmo loss-of-function mutations. These mutants display partial loss of thoracic DC bristles along with reduced expression of a target gene of the thoracic transcription complex. Interestingly, the wing size of these mutants is normal, unlike the small wings of mutants with lesions in the 5'-UTR of Dlmo, such as dlmo^hdp58-1, dlmo^hdp67-2, and dlmo^hdpR590. This may suggest that the defective Dlmo protein, which has only a single intact LIM domain, is sufficient for its function in the context of the wing, where Dlmo acts as a negative regulator that binds only one protein (CHIP), but is not sufficient when Dlmo acts as a bridging molecule in the thoracic CPAD transcription complex. Finally, the finding that Dlmo can bind other Dlmo molecules to generate homodimers or multimers might provide Dlmo with a greater flexibility of bridging between distant components of the complex. This possibility remains to be examined (Zenvirt, 2008).

In conclusion, Dlmo appears to have a dual role in regulation of transcription, depending on the context. Such a phenomenon has been documented for other transcription cofactors, whose dual function in transcription regulation varies according to their binding partners, the specific tissue, or the developmental stage. Likewise, these results indicate Dlmo has such a dual role, being a negative regulator with respect to the Ap-Chip complex and a positive regulator in the context of the CPAD complex (Zenvirt, 2008).

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. This study used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses (van Tienen, 2017).

The Wnt/β-catenin signaling cascade is an ancient cell communication pathway that operates context-dependent transcriptional switches to control animal development and tissue homeostasis. Deregulation of the pathway in adult tissues can lead to many different cancers, most notably colorectal cancer. Wnt-induced transcription is mediated by T cell factors (TCF1/3/4, LEF1) bound to Wnt-responsive enhancers, but their activity depends on the co-activator β-catenin (Armadillo in Drosophila), which is rapidly degraded in unstimulated cells. Absence of β-catenin thus defines the OFF state of these enhancers, which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain. This domain tetramerizes to promote transcriptional repression (Chodaparambil, 2014), which leads to chromatin compaction apparently assisted by the interaction between Groucho/TLE and histone deacetylases (HDACs) (van Tienen, 2017).

Wnt signaling relieves this repression by blocking the degradation of β-catenin, which thus accumulates and binds to TCF, converting the Wnt-responsive enhancers into the ON state. This involves the binding of β-catenin to various transcriptional co-activators via its C-terminus, most notably to the CREB-binding protein (CBP) histone acetyltransferase or its p300 paralog, resulting in the transcription of the linked Wnt target genes. Subsequent reversion to the OFF state (for example, by negative feedback from high Wnt signaling levels near Wnt-producing cells, or upon cessation of signaling) involves Groucho/TLE-dependent silencing, but also requires the Osa/ARID1 subunit of the BAF (also known as SWI/SNF) chromatin remodeling complex which binds to β-catenin through its BRG/BRM subunit. Cancer genome sequencing has uncovered a widespread tumor suppressor role of the BAF complex, which guards against numerous cancers including colorectal cancer, with >20% of all cancers exhibiting at least one inactivating mutation in one of its subunits, most notably in ARID1A. Thus, it appears that failure of Wnt-inducible enhancers to respond to negative feedback imposed by the BAF complex strongly predisposes to cancer (van Tienen, 2017).

How β-catenin overcomes Groucho/TLE-dependent repression remains unclear, especially since β-catenin and TLE bind to TCF simultaneously (Chodaparambil, 2014). Therefore, the simplest model envisaging competition between β-catenin and TLE cannot explain this switch, which implies that co-factors are required. One of these is Pygo, a chromatin reader binding to histone H3 tail methylated at lysine 4 (H3K4m) via its C-terminal PHD finger (Fiedler, 2008). In Drosophila where Pygo was discovered as an essential co-factor for activated Armadillo, its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression. It has been discovered recently that Pygo associates with TCF enhancers via its highly conserved N-terminal NPF motif that binds directly to the ChiLS complex, composed of a dimer of Chip/LDB (LIM domain-binding protein) and a tetramer of SSDP (single-stranded DNA-binding protein, also known as SSBP). Notably, ChiLS also binds to other enhancer-bound NPF factors such as Osa/ARID1 and RUNX, and to the C-terminal WD40 domain of Groucho/TLE, and thus forms the core module of a multiprotein complex termed 'Wnt enhanceosome' (Fiedler, 2015). This study proposed that Pygo renders this complex Wnt-responsive by capturing Armadillo/β-catenin through the Legless adaptor (whose orthologs in humans are BCL9 and B9L, also known as BCL9-2). The salient feature of this model is that the Wnt enhanceosome keeps TCF target genes repressed prior to Wnt signaling while at the same time priming them for subsequent Wnt induction, and for timely shut-down via negative feedback depending on Osa/ARID1 (Fiedler, 2015; van Tienen, 2017 and references therein).

This study assessed the function of Legless and BCL9/B9L within the Wnt enhanceosome. Using a proximity-labeling proteomics approach (called BioID) in human embryonic kidney (HEK293) cells, a compelling association was uncovered between BCL9/B9L and the core Wnt enhanceosome components, regardless of Wnt signaling. Co-immunoprecipitation (coIP) and in vitro binding assays based on Nuclear Magnetic Resonance (NMR) revealed that BCL9 and B9L associate with TLE3 through their C-termini, and that they bind directly to Chip/LDB-SSDP via their evolutionary conserved homology domain 3 (HD3). These elements are outside the sequences mediating the adaptor function between Pygo and Armadillo/β-catenin, but they are similarly important for Wnt responses during Drosophila development and in human cells, as is shown by CRISPR/Cas9-based genome editing. The results consolidate and refine the Wnt enhanceosome model, indicating a constitutive scaffolding function of BCL9/B9L within this complex. The evidence further suggests that BCL9/B9L but not Pygo undergoes a β-catenin-dependent rearrangement within the enhanceosome upon Wnt signaling (see Model of the Wnt enhanceosome), gaining proximity to TCF, which might trigger enhanceosome switching (van Tienen, 2017).

This study has uncovered genetic and physical interactions between two constitutive core components of the Wnt enhanceosome and the C-terminus of Legless/BCL9. The first of these is ChiLS, the core module of the Wnt enhanceosome (Fiedler, 2015): ChiLS is a direct and specific ligand of the α-helical HD3 element of B9L and, likely, of other Legless/BCL9 orthologs, given the strong sequence conservation of this α-helix. The physiological relevance of this interaction with ChiLS is underscored by genetic analysis in flies. The evidence thus implicates HD3 as an evolutionary conserved contact point between Legless/BCL9 and ChiLS, although the primary link between these two proteins appears to be provided by Pygo (van Tienen, 2017).

A second link between the Legless/BCL9 C-terminus and the Wnt enhanceosome is mediated by the WD40 domain of TLE/Groucho. Given evidence from RIME, this link is also likely to be direct although, for technical reasons, it has not been possible to prove this. The function of the C-terminus of Legless/BCL9 for transducing Wnt signals was revealed by the wg-like phenotypes in Drosophila larvae and flies and by their defective transcriptional Wg responses, and by the loss of transcriptional Wnt responses in BCL9/B9L-deleted human cells. The evidence indicates that Legless/BCL9 undergoes three separate functionally relevant interactions with distinct components of the Wnt enhanceosomewith Pygo, ChiLS and Groucho/TLE. Importantly, BioID revealed that these interactions are constitutive, preceding Wnt signaling, and that they hardly change upon Wnt stimulation. Taken together with its multivalent interactions with the Wnt enhanceosome, this is consistent with Legless/BCL9 being a core component of this complex, providing a scaffolding function that facilitates its assembly and/or maintains its cohesion (van Tienen, 2017).

Following Wnt stimulation, Legless/BCL9 undergoes an additional physiologically relevant interaction, by binding to (stabilized) Armadillo/β-catenin via HD2. Legless/BCL9 thus confers Wnt-responsiveness on the Wnt enhanceosome through its ability to capture Armadillo/β-catenin. In other words, in addition to scaffolding the enhanceosome, Legless/BCL9 also earmarks this complex for Wnt responses. Intriguingly, the BioID data indicated that the capture of β-catenin by Legless/BCL9 triggers its rearrangement within the complex, apposing its C-terminus to TCF. This apparent β-catenin-dependent apposition is consistent with structural data showing that BCL9/B9L HD2 is closely apposed to TCF when in a ternary complex with β-catenin. The evidence supports the notion of Legless/BCL9 acting as an Armadillo loading factor, facilitating access of Armadillo/β-catenin to TCF, but argues against the original co-activator hypothesis which posited that Legless/BCL9 is recruited to TCF by Armadillo/β-catenin exclusively in Wnt-stimulated cells. Whatever the case, the β-catenin-dependent apposition of the Legless/BCL9 C-terminus to TCF is likely to trigger Wnt enhanceosome switching from OFF to ON, resulting in the relief of Groucho/TLE-dependent repression and culminating in the Wnt-dependent transcriptional activation of linked target genes (van Tienen, 2017).

This transition of the Wnt enhanceosome from OFF to ON is accompanied by a proximity gain between Legless/BCL9 and CBP/p300, likely to reflect at least in part its de novo binding to Armadillo/β-catenin. However, the evidence indicates that CBP/p300 is associated with the Wnt enhanceosome prior to Wnt signaling, possibly via direct binding to B9L as suggested by RIME, and that the docking of Armadillo/β-catenin to the Wnt enhanceosome strengthens its association with CBP/p300, and/or directs the histone acetyltransferase activity of CBP/p300 towards its substrates, primarily the histone tails. By acetylating these tails, CBP/p300 appears to promote Wnt-dependent transcription in flies and human cells. Indeed, CBP-dependent histone acetylation has been observed at Wg target enhancers in Drosophila although, interestingly, this preceded transcriptional activation. This is consistent with BioID data, indicating constitutive association of CBP/p300 with the Wnt enhanceosome (van Tienen, 2017).

It seems plausible that histone acetylation at Wnt target enhancers is instrumental in antagonizing the compaction of their chromatin imposed by Groucho/TLE, which depends on its tetramerization via its Q domain as well as its binding to HDACs. Indeed, HDACs were found near the bottom of the BioID lists, and one of the top hits identified by B9L was GSE1, a subunit of the BRAF-HDAC complex. However, CBP/p300 also has non-histone substrates within the Wnt enhanceosome, including dTCF in Drosophila whose Armadillo-binding site can be acetylated by dCBP, which thus blocks the binding between the two proteins and antagonizes Wg responses. It thus regulates Wnt-dependent transcription positively as well as negatively, similarly to Groucho/TLE which not only silences Wnt target genes but also earmarks them for Wnt inducibility, as a core component of the Wnt enhanceosome. It is intriguing that both bimodal regulators are associated constitutively with this complex. A corollary is that the docking of Armadillo/β-catenin to the Wnt enhanceosome changes their substrate specificities and/or activities (van Tienen, 2017).

An important refinement of the initial enhanceosome model is with regard to the BAF complex, which appears to be a constitutive component of the Wnt enhanceosome, as indicated by BioID data. This complex is highly conserved from yeast to humans, and it contains 15 subunits in human cells (Kadoch, 2015), including the DNA-binding Osa/ARID1 subunit. A wealth of evidence from studies in flies and mammals indicates that this complex primarily antagonizes Polycomb-mediated silencing of genes, most notably of the INK4A locus which encodes an anti-proliferative factor, which could explain why the BAF complex functions as a tumor suppressor in many tissues. However, recall that this complex also specifically antagonizes Armadillo/β-catenin-mediated transcription, likely via its BRG/BRM subunit which directly binds to β-catenin. Evidence from studies in Drosophila of Wg-responsive enhancers suggests that this complex mediates a negative feedback from high Wg signaling levels near Wg-producing cells which results in re-repression, imposed by the Brinker homeodomain repressor and its Armadillo-binding Teashirt co-repressor. The same factors may also install silencing on Wnt-responsive enhancers upon cessation of Wnt signaling. Notably, mammals do not have a Brinker ortholog, which could explain some of the apparent functional differences between flies and mammals with regard to the BAF complex (Kadoch, 2015). However, the closest mammalian relatives of Teashirt are the Homothorax/MEIS proteins, a family of homeodomain proteins whose expression can be Wnt-inducible. They are thus candidates for Wnt-induced repressors that confer BAF-dependent feedback inhibition (van Tienen, 2017).

Notably, none of BioID lists contained RUNX proteins. Based on functional evidence from Drosophila midgut enhancers, it is proposed that these proteins (which bind to both enhancers and Groucho/TLE) are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development, or in uncommitted progenitor cells of specific cell lineages (Fiedler, 2015). However, HEK293 cells are epithelial cells and may thus not express any RUNX factors. In any case, the negative BioID results suggest that RUNX factors function in a hit-and-run fashion. Evidently, the Wnt enhanceosome complex, once assembled at Wnt-responsive enhancers, can switch between ON and OFF states without RUNX (van Tienen, 2017).

In summary, this study has uncovered a fundamental role to Legless/BCL9 as a scaffold of the Wnt enhanceosome, far beyond its role in linking Armadillo/β-catenin to Pygo. Indeed, the function of Legless/BCL9 may extend beyond transcriptional Wnt responses, as indicated by the unexpected discovery of its strong association with nuclear co-receptor complexes. Potentially, these associations underlie the observed cross-talk between Wnt/β-catenin and nuclear hormone receptor signaling, documented extensively in the literature, including evidence for direct activation of the androgen receptor by β-catenin. Furthermore, a strong association between TLE1 and the estrogen receptor has been discovered in breast cancer cells, where TLE1 assists the estrogen receptor in its interaction with chromatin and its proliferation-promoting function. This is reminiscent of the role of Groucho/TLE as a cornerstone of the Wnt enhanceosome, proposed to earmark TCF enhancers for subsequent β-catenin docking and transcriptional Wnt responses (Fiedler, 2015). It will be interesting to test experimentally the putative roles of BCL9/B9L and Pygo in enabling cross-talk between β-catenin and nuclear hormone receptor signaling, both during normal development and in cancer (van Tienen, 2017).

Drosophila eye developmental defect caused by elevated Lmx1a activity is reliant on chip expression

The LIM-homeodomain (LIM-HD) family member Lmx1a has been successfully used to induce dopaminergic neurons from other cell types, thus showing significant implications in replacement therapies of Parkinson's disease, but the underlying mechanism remains elusive. This study used Drosophila eye as a model system to investigate how forced expression of dLmx1a (CG4328) and Lmx1alpha (CG32105), the fly homologs of human Lmx1a, alters cell identify. Ectopic expression of dLmx1a suppresses the formation of Drosophila eye tissue; the LIM and HD were found to be two essential domains. dLmx1a requires and physically binds to Chip, a well-known cofactor of LIM-HD proteins. Chip connects two dLmx1a proteins to form a functional tetrameric complex. In addition, evidence is provided showing that dLmx1a expression results in the suppression of two retina determination gene eyes absent (eya) and string (stg). Taken together, these findings identified Chip as a novel partner of dLmx1a to alter cell differentiation in Drosophila eye through repressing eya and stg expression, and provide an animal model for further understanding the molecular mechanism whereby Lmx1a determines cell fate (Wang, 2015).

DEVELOPMENTAL BIOLOGY

Embryonic

Chip protein is maternally contributed during oogenesis. From the early cellular blastoderm stage through gastrulation to the end of embryogenesis, Chip protein is present in most, if not all nuclei, including the pole cell nuclei. Staining is undetectable or very weak in syncytial blastoderm nuclei until just before cellularization, although Chip must be present at low levels, because lack of Chip activity affects expression of segmentation genes at this stage (Morcillo, 1997).

Larval

Chip is present in the nuclei of larval tissue, including imaginal discs, fat body, and salivary gland. Chip associates with a very large number of specific sites on larval polytene chromsomes (Morcillo, 1997).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).

acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).

In addition to the eight genes described below, five other TFs were examined that were not pursued because of the lack of expression in GH146-PNs at 18 hr APF (aristaless and pdm-1) or the lack of targeting defects in homozygous mutant PNs (abrupt [ab^k02807], kruppel [Kr¹], and Dichaete [Dichaete⁸⁷]) (Komiyama, 2007).

LIM-HD factors and PN targeting: LIM-homeodomain (LIM-HD) TFs are involved in multiple events during neuronal development. Most functions of LIM-HD factors require the LIM domain-binding cofactor, which is represented in Drosophila by ubiquitously expressed Chip. Chip antibody revealed ubiquitous expression of Chip in cells around the antennal lobe (AL) including all GH146-PNs at 18 hr APF (Komiyama, 2007).

The requirement of Chip in PN dendritic targeting was tested. Wild-type adPNs, lPNs, and vPNs target stereotyped sets of glomeruli. PNs homozygous for a Chip null allele (Chip^e5.5) failed to target most of the correct glomeruli and occupied inappropriate glomeruli. Most adPN and lPN clones (12/13) also mistargeted a fraction of dendrites to the structure ventral to the AL, the suboesophaegeal ganglion (SOG). Thus, Chip is required for targeting specificity of most, if not all, PN classes studied here, and Chip-interacting proteins including LIM-HD factors likely play important roles in PN dendritic targeting (Komiyama, 2007).

Five LIM-HD factors have been characterized in Drosophila: apterous, arrowhead, islet, lim1, and lim3. apterous, arrowhead, or lim3 were not pursued because they are not expressed in GH146-PNs at 18 hr APF (apterous) or they do not have targeting defects in PNs homozygous for null alleles (lim3^37Bd6 and awh¹⁶) (Komiyama, 2007).

Islet antibody detected Islet expression in ~50% adPNs and most lPNs but not in vPNs at 18 hr APF and adult. isl^−/− adPNs failed to target many (but not all) of the normal target glomeruli, including VA1lm, VA3, and VM7. In addition, DA1, a lPN target, was often specifically mistargeted. Defects of isl^−/− lPNs were very similar to Chip^−/− lPN defects. A fraction of dendrites often mistargeted to the SOG. Within the AL, dendrites were diffusely spread, although DA1 and DL3 were always correctly innervated. Targeting of isl^−/− vPNs was normal, consistent with their lack of Islet expression (Komiyama, 2007).

Lim1 antibody revealed Lim1 expression in most or all vPNs, but not in adPNs or lPNs in adults. The expression pattern appears similar at 18 hr APF, although vPNs are difficult to identify unambiguously at early stages. lim1^−/− adPNs showed no defects, consistent with the lack of Lim1 expression. lim1^−/− lPNs rarely showed a cell number decrease, but in clones in which the cell number was normal, lim1^−/− lPNs targeted correct glomeruli. In contrast, lim1^−/− vPNs showed a specific targeting defect. Wild-type vPNs innervate DA1 and VA1lm densely because of the single vPNs that specifically innervate these glomeruli, in addition to the diffuse innervation all over the AL contributed by the pan-AL vPN. In lim1^−/− vPNs, DA1 innervation was greatly reduced and sometimes undetectable. Therefore, lim1 is required for dendritic targeting by a single vPN class, vDA1, despite its general expression in vPNs. lim1 might be redundant with other factors in non-DA1 vPNs. It was note that phenotypes of islet and lim1 combined are only a subset of the Chip phenotype. Additional Chip phenotype may be explained by non-Lim-HD molecules interacting with Chip (Komiyama, 2007).

EFFECTS OF MUTATION

Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein in dorsal cells. dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the Chip co-factor, and suggest that the Ap and Chip proteins form a multimeric functional complex. In support of this model, it has been shown that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression (Fernández-Fúnez, 1998).

Chip mutations behave as strong enhancers of wing phenotypes produced by hypomorphic ap mutations. This synergistic interaction suggests that Chip and ap have related functions. To investigate further the function of Chip during wing development, genetic mosaics were generated by induced mitotic recombination using the Minute technique. Clones of Chip mutant cells in the wing ventral compartment show a wild-type phenotype and appear with normal frequencies, indicating that Chip is not required in this compartment. In contrast, Chip clones in the dorsal compartment are associated with wing outgrowths and ectopic wing margins. Cells within these clones have a ventral identity revealed by the morphology of the wing margin bristles they differentiate. Normal cells abutting the mutant clones are induced to form the dorsal structures characteristic of the wing margin. The ectopic margins can be visualized in undifferentiated imaginal discs with the use of molecular markers that label the wing margin. The largest wing outgrowths correspond to clones far from the normal wing margins. These clones cause the outgrowth of wild-type tissue, with the mutant clones located at the tip of the outgrowth . Thus, although Chip is expressed in all wing cells, Chip mutations produce specific phenotypes that are indistinguishable from ap phenotypes in clones. One possibility is that normal Chip function is required for ap expression. To test this possibility, ap expression was monitored in Chip mutant clones induced in wing imaginal discs. Ap protein is shown to accumulate normally in Chip mutant cells. Thus Chip does not regulate ap expression but it does show genetic interactions with ap, and Chip also produces the same mutant phenotypes in genetic mosaics. Taken together, these results are consistent with the hypothesis that Chip encodes a co-factor required for ap function as a dorsal selector gene (Fernández-Fúnez, 1998).

If Ap and Chip physically interact forming a functional complex, their stoichiometry may be important for the formation of the complex and for dorso-ventral patterning. To test whether the levels of Chip expression are important for dorso-ventral patterning, Chip was overexpressed in various patterns. Overexpression of Chip using a decapentaplegic GAL4 driver results in wing outgrowths, the creation of an ectopic wing margin on the dorsal compartment and a cut in the wing. These phenotypes are also evident in imaginal discs using wingless-lacZ (wglacZ) expression as a marker of the wing margin. Because lack of ap function in clones also causes wing outgrowths and ectopic wing margins, the distribution of Ap protein was examined in these wing imaginal discs. Chip overexpression does not alter the distribution of Ap protein; these results indicate that the phenotypes produced by Chip overexpression are not caused by Chip repressing ap. Interestingly, overexpression of ap using the drivers dppGAL4 or apGAL4 described above does not result in wing abnormalities in the dorsal compartment. Thus overexpression of Chip results in the same phenotype as its lack of function, i.e. transformation of dorsal into ventral cells, and, as a consequence, wing margin formation and outgrowth. These observations suggest that the relative amounts of Ap and Chip are important for dorso-ventral patterning (Fernández-Fúnez, 1998).

If the relative amounts of Ap and Chip are critical for dorso-ventral patterning, then it should be possible to rescue the excess of Chip phenotype by overexpressing ap. The proposed domain structure of the LDB/NLI family of proteins provides a conceptual framework to understand the phenotypes produced by altering the dosage of Chip and ap. The presence of homodimerization and LIM-interacting domains in LDB/NLI proteins suggests that LDB and LIM domain proteins may form tetrameric complexes. These complexes would be formed by two LDB molecules interacting through the N-terminal homodimerization domain; in addition, each LDB molecule would interact with a LIM domain through the C-terminal LIM-interacting domain. The occurrence of these complexes has been demonstrated between murine LDB and the hamster LIM/homeodomain protein LMX1. In the case of Chip-Ap, this tetrameric complex may be the functional complex carrying out the dorso-ventral patterning functions. This model predicts that Chip overexpression would lead to the formation of non-functional complexes, and it also predicts that functional Chip-Ap complexes would be reconstituted by overexpressing ap in addition to Chip. To test this model, a GAL4 insertion in ap was used. Expression from this GAL4 driver faithfully reproduces ap expression in the wing imaginal disc. When Chip is expressed from the ap promoter, the wing is reduced or eliminated depending on the UAS:Chip transgenic line used. Most lines reduce the wings, whereas the strongest line completely eliminates them. The reduced wing phenotype can be completely rescued by ap overexpression. These results provide further evidence for the idea that the stoichiometry of Ap and Chip is critical for dorso-ventral patterning (Fernández-Fúnez, 1998).

Cuticle of embryos lacking maternal Chip exhibit normal dorsal-ventral polarity, but have severe segmentation defects. Most cuticles have a single fused, irregularly shaped patch of ventral denticles. A few rare embryos display a pair-rule-like phenotype, with approximately half the normal number of segments (Morcillo, 1997).

The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the Drosophila Suppressor of Hairy-wing protein (Su[Hw]). Su[Hw] binds a DNA sequence in the gypsy retrotransposon and prevents activation of promoter-enhancers that are distal to a gypsy insertion in a gene without affecting promoter-enhancers that are proximal to gypsy (next to the structural gene). Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in either scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).

Chip may encode an enhancer-facilitator, acting to facilitate the activity of distal enhancers. The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila Suppressor of Hairy-wing protein [su(Hw)] that binds to gypsy retrovirus insertions between enhancers and promoters. su(Hw) bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. Chip is needed for the wing margin enhancer of cut. The Chip mutation dominantly enhances the mutant phenotypes displayed by partially suppressing gypsy insertions in both cut and Ultrabithorax and is a homozygous larval lethal, indicating that Chip regulates multiple genes. Chip is normally required for the wing margin enhancer function of cut because Chip mutations enhance the cut wing phenotype of a cut mutation. Heterozygotes for Chip display cut wing phenotypes when either scalloped or mastermind (mam) is also a heterozygous mutant. Both Sc and Mam are known to regulate the cut distal enhancer, but in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. Thus, in a heterozygous Chip mutant, a heterozygous gypsy insertion in cut displays a cut wing phenotype, whereas a heterozygous enhancer deletion does not. Dependence on the nature of the heterozygous lesion in the regulatory region strongly suggests that Chip directly regulates cut. More strikingly, it indicates that in a Chip heterozygote, a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) bound to a gypsy insertion in one cut allele acts in a transvection-like manner (interchromosomally) to block the wing enhancer in the wild-type cut allele on a second chromosome. This implicates Chip in enhancer-promoter communication (Morcillo, 1997 and references).

LIM-homeodomain transcription factors are expressed in subsets of neurons and are required for correct axon guidance and neurotransmitter identity. The LIM-homeodomain family member Apterous requires the LIM-binding protein Chip to execute patterned outgrowth of the Drosophila wing. To determine whether Chip is a general cofactor for diverse LIM-homeodomain functions in vivo, its role in the embryonic nervous system was studied. Loss-of-function Chip mutations cause defects in neurotransmitter production that mimic apterous and islet mutants. Chip is also required cell-autonomously by Apterous-expressing neurons for proper axon guidance, and requires both a homodimerization domain and a LIM interaction domain to function appropriately. Using a Chip/Apterous chimeric molecule lacking domains normally required for their interaction, the complex was reconstituted and the axon guidance defects of apterous mutants, of Chip mutants and of embryos doubly mutant for both apterous and Chip were rescued. These results indicate that Chip participates in a range of developmental programs controlled by LIM-homeodomain proteins and that a tetrameric complex comprising two Apterous molecules bridged by a Chip homodimer is the functional unit through which Apterous acts during neuronal differentiation (van Meyel, 2000).

Chip is expressed in most, if not all, embryonic and larval tissues. In wild-type embryos, strong, nuclear Chip expression is found throughout the developing VNC with no apparent subclasses of neurons excluded. A substantial fraction of embryonic Chip is contributed maternally during oogenesis, and this maternally derived expression is required for early embryonic segmentation. To estimate the relative contribution of zygotic and maternally derived Chip to the embryonic VNC, homozygous embryos mutant for a Chip null allele were examined. Derived from an intercross of heterozygous parents, mutants are expected to retain half the maternal and not any zygotic Chip expression. Little reduction of staining in mutant embryos is observed, relative to Chip/+ heterozygotes. Thus it appears a substantial fraction of Chip in the VNC is provided maternally. Co-labelling embryos with anti-Ap and anti-Chip antibodies reveals that Chip expression overlaps with all the Ap neurons of the developing VNC (van Meyel, 2000).

If Chip were required for Ap function, elimination of Chip might be expected to result in an ap-like phenotype. The requirement of maternally supplied Chip in segmentation precluded an examination of the effects of eliminating both maternal and zygotic Chip on neuronal development. Thus, neurotransmitter expression and axon guidance were examined in Chip mutants in which half of the maternal and all of the zygotic Chip expression were absent. In each thoracic hemisegment of the VNC, ap is expressed in a lateral cluster of four neurons, one of which is the Tv neuroendocrine cell that expresses the neurotransmitter dFMRFa. In wild-type embryos, there are a total of six Tv cells, one in each thoracic hemisegment. In ap mutants, the Tv neurons are present, but half of all Tv neurons stochastically fail to express dFMRFa. This regulation of dFMRFa by ap is transcriptional, since expression of a fusion transgene comprising a 446 bp Tv neuron-specific enhancer of the dFMRFa gene driving beta-galactosidase (Tv-lacZ) is similarly reduced in ap mutants. Ap binds in vitro to each of three sequences within the enhancer, and mutagenesis of these sites has confirmed that these sequences are important for Tv-lacZ expression in vivo (van Meyel, 2000).

To determine whether reduction of Chip results in an ap-like reduction in transcriptional activation of dFMRFa, expression of the Tv-lacZ reporter transgene was assayed in wild-type, ap and Chip mutant embryos. Both ap and Chip mutant embryos show decreased Tv-lacZ activity in Tv neurons relative to wild-type controls, implicating Chip in the establishment of this Ap-regulated aspect of neuronal differentiation. The reduction of Tv-lacZ activity is less severe in Chip null mutants than ap null mutants, probably because of the maternally supplied Chip remaining in Chip mutants. In embryos homozygous for an antimorphic Chip mutation, Tv-lacZ expression is reduced further than Chip null mutants but not to the level of ap mutants (van Meyel, 2000).

Like Ap, the LIM HD protein Isl also regulates neurotransmitter identity of embryonic neurons. There are three dopaminergic cells per segment of the VNC, one unpaired midline cell and a pair of dorsal lateral cells, all of which express Isl protein and thus represent a subset of the isl interneurons. isl mutants show loss of expression of tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of dopamine. To test the role of Chip in the expression of TH, late-stage wild-type and Chip mutant embryos were stained with anti-TH antibodies. Homozygous Chip mutant embryos retain TH expression in the ventral unpaired midline cells, but few of the dorsal lateral cells express TH, and in those that do, TH levels are significantly reduced relative to wild-type. In embryos homozygous for a Chip antimorph, TH expression is greatly diminished in both the ventral midline and dorsal lateral dopaminergic neurons. While it is clear that the paired dorsal TH cells are more sensitive to the reduction in Chip dosage than the unpaired ventral cells, the effects of the antimorphic Chip allele suggest that TH production in the latter cells is also dependent on Chip. From these results, together with the above results on the expression of FMRFamide, it is concluded that Chip is required for both Ap- and Islet-regulated neurotransmitter production in the CNS (van Meyel, 2000).

tailup, a LIM-HD gene, and Iro-C cooperate in Drosophila dorsal mesothorax specification

The LIM-HD gene tailup has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Sequence-specific single-stranded DNA-binding protein (Ssdp) are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a `pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. These data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate (de Navascues, 2007).

Tup has been categorised as a prepattern factor that controls the expression of the proneural achaete-scute genes in the third instar wing disc. This study shows that tup functions earlier in the development of the dorsal mesothorax. Loss of tup causes a range of phenotypes, which taken together indicate interference with the assignment of cells to form notum. Thus, depending on the time of induction of the clones and their location multiple effects are observed; the formation of notum-like cuticle with altered cell-cell adhesion properties, the generation of ectopic wing-hinge structures including tegulae, sclerites or sensilla typical of the proximal wing, or even the loss of the entire heminotum. Consistent with these adult phenotypes, in third instar wing discs tup mutant cells can upregulate genes typically expressed at high levels in the wing-hinge territory of the disc, such as zfh2, msh, sal and the lacZ insertion line l(2)09261. Concomitantly, notum-expressed genes such as eyg, ush and pnr are generally repressed, although in some cases tup cells may abnormally express notum and hinge genes together. These data indicate that notum tup cells undergo transformation towards either an altered notum fate or a hinge fate. Moreover, the activation of hinge markers in wild-type cells surrounding some tup clones might reflect the presence of ectopic notum/hinge borders, which are known to promote non-autonomous effects (de Navascues, 2007).

Unequivocal notum-to-hinge transformations are consistently observed in clones induced during the first larval instar. In later-induced clones, this phenotype becomes less manifest and the modified notum cuticle phenotype becomes prevalent. Accordingly, the upregulation of hinge marker genes and the converse downregulation of notum genes in the notum territory are most consistently observed in first instar-induced clones. This suggests that the requirement for the 'pronotum' function of tup progressively decreases as development advances. Lesions associated with tup clones can appear anywhere within the notum, although each particular phenotype shows a degree of topographic specificity. Interestingly, the activation of hinge genes and the repression of notum genes are best shown in early-induced clones located in the presumptive medial notum. Probably, these clones, which are normally large, do not yield adult structures, since the expected large regions of mutant cuticle have not been recovered. The clones might give rise to flies lacking part or most of a heminotum. The dynamic expression pattern of tup fits well with the spatial distribution of these phenotypes and the early requirement for tup function for the development of the notum. Indeed, tup is expressed very early in the wing disc, when it has less than 100 cells, and the expression occurs within the region that will form the notum. It is concluded that, similar to other LIM-HD factors such as Ap and the vertebrate Tup homologue Isl1, Tup is required for the proper specification of not only cell types, but also developing territories (de Navascues, 2007).

Tup is known to bind the co-factor Chip. Since, in dorsal compartment specification, Chip functions in a 2Ap-2Chip-2Sspd hexamer, it was asked whether a similar 2Tup-2Chip-2Sspd complex might mediate Tup function in notum specification. The results support this interpretation. The loss of either Chip or Ssdp upregulates hinge genes (zfh2, msh), represses a notum marker (eyg), and induces cuticular defects similar to those associated with tup clones. Moreover, an excess of Chip would be expected to titrate Tup and/or Ssdp in incomplete complexes and mimic the loss-of-function phenotype of notum-to-hinge transformation, as was experimentally observed (de Navascues, 2007).

By contrast, during the later process of sensory organ formation, Tup appears to act by sequestering both Chip and Pnr, thus preventing activation of the proneural genes achaete-scute. This negative function of Tup does not seem relevant for notum specification, where both Tup and Chip work as positive effectors. Moreover, the Tup homeodomain is dispensable for titrating Chip and Pnr, but this is not the case for its 'pronotum' function. Interestingly, a missense mutation within the LIM-interacting interacting domain of Chip (Chip^E) severely reduces its ability to interact with Tup and suppresses the negative regulation by Tup of bristle formation. However, homozygous Chip^E flies have no defects in notum specification. This suggests that a residual interaction between Chip^E and Tup might persist, as additionally suggested by the suppression of the extra bristles present in Chip^E individuals by UAS-tup overexpression. A weak interaction between Tup and Chip, which might only permit the formation of low levels of hexameric complex, might still allow proper notum specification. This suggestion agrees with the fact that tup^d03613, a strong hypomorphic allele (as substantiated by its embryonic lethality over the null tup^ex4, allows proper notum formation in homozygosis (de Navascues, 2007).

Similarly to tup, Iro-C also has a 'pronotum' function. However, their roles are not entirely equivalent. Anywhere within the notum territory, loss of Iro-C during first or second instar induces a clear switch to hinge fate. By contrast, loss of tup causes an assortment of different combinations of derepressed hinge genes and repressed notum genes. Moreover, many tup clones induced during the second larval instar, and even some induced in the first, can develop recognisable notum cuticle. Thus, it is proposed that tup reinforces/stabilises the commitment of cells to develop as notum, a commitment imposed mainly by Iro-C. This reinforcement or stabilisation might be most necessary in the proximal part of the disc, where expression of ara/caup ceases after the second instar, but that of tup persists. This might account for the derepression of hinge genes being most manifest in this region. Depending on the location and time of Tup deprival, its loss may be inconsequential or lead to a partial or even a complete loss of notum commitment. Such diversity of consequences led to an exploration of whether tup might act on target genes by affecting chromatin remodelling. However, no genetic interactions have been found with Polycomb (Pc, Scr+Pcl+esc) or trithorax (trx, osa, brm, Trl, lawc) group genes (de Navascues, 2007).

In contrast to the absolute requirement for Iro-C for notum specification, overexpression of UAS-ara can impose a notum fate only on the wing anlage, and only when provided early in the development of the disc. An extra notum with mirror-image disposition versus the extant notum is generated at the expense of the wing, a phenotype identical to that resulting from early deprivation of Wg function. Since UAS-ara overexpression can interfere with wg expression, Wg deprival probably explains the formation of the extra notum. Thus, by itself, overexpression of UASara probably lacks a genuine potential for imposing the notum fate. Similar notum duplications arise upon early and strong overexpression of UAS-tup (MD638, dpp-Gal4 and ptc-Gal4 drivers) and, again, they probably result from inhibition of Wg activity. Consistent with this interpretation, weaker and later expression of either UAS-tup or UAS-ara (C765 driver) has little or no capacity to promote notum fate. However, when coexpressed, these transgenes are effective in imposing the notum fate and this should not be attributed to Wg depletion. Indeed, the transformation consists of an expansion of the notum tissue, rather than a notum duplication. Moreover, as detected by the onset of the ectopic expression of notum markers (eyg, DC-lacZ), the transformation occurs in late third instar discs (J.deN., unpublished) that have a nearly wild-type morphology and a distinguishable wing pouch. This indicates that these markers are activated in territories previously specified as wing, hinge or pleura, and subsequently forced to acquire notum identity. Moreover, overexpression of the Wg pathway antagonists UAS-Axin or UAS-dTCF^DN (dTCF or pan with the same driver failed to transform wing towards notum. Finally, the activation of eyg and the formation of notum tissue in the sternopleurite, a derivative of the leg disc, also attest to the capacity of tup plus ara to commit cells to develop as notum (de Navascues, 2007).

It is well established that signalling by the EGFR pathway is essential for notum development. Its inhibition prevents activation of Iro-C and the growth of the notum territory. By contrast, Dpp negatively regulates Iro-C and restricts its domain of expression at both its distal and proximal borders. The data indicate a novel function of Dpp in notum development; namely, the activation or maintenance of tup expression in second and third instar discs. In the notum region of the early disc, Dpp signalling occurs at low levels, but the results suggest that these are sufficient for activating tup. Expression of tup is largely independent on EGFR signalling. Thus, EGFR and Dpp signalling seem to cooperate in specifying notum identity to the cells of the proximal part of the disc by activating their respective 'pronotum' downstream genes, Iro-C and tup (de Navascues, 2007).

Chip is required for posteclosion behavior in Drosophila

Neurons acquire their molecular, neurochemical, and connectional features during development as a result of complex regulatory mechanisms. This study shows that a ubiquitous, multifunctional protein cofactor, Chip, plays a critical role in a set of neurons in Drosophila that control the well described posteclosion behavior. Newly eclosed flies normally expand their wings and display tanning and hardening of their cuticle. Using multiple approaches to interfere with Chip function, it was found that these processes do not occur without normal activity of this protein. Furthermore, the nature of the deficit was identified to be an absence of Bursicon in the hemolymph of newly eclosed flies, whereas the responsivity to Bursicon in these flies remains normal. Chip interacts with transcription factors of the LIM-HD (LIM-homeodomain) family, and one member, dIslet, was identified as a potential partner of Chip in this process. These findings provide the first evidence of transcriptional mechanisms involved in the development of the neuronal circuit that regulates posteclosion behavior in Drosophila (Hari, 2008).

This study used a selective overexpression strategy to identify a novel function of Chip in a set of neurons that control a stereotyped behavioral program. Chip is a widely expressed multidomain cofactor molecule that interacts with many transcription factors. It can function both as a transcriptional coactivator and a bridging factor between proteins that bind to distal enhancers and the core transcription machinery. Identifying specific functions of such a protein is confounded by the superposition of a multitude of effects. The mutations in Chip cause early lethality precluding the examination of later functions. Because the molecule exists as a part of multiple complexes, even simultaneously within the same cell, altering the level of one class of interactors can potentially disrupt several functions. This study has elucidated a highly specific role of Chip in a particular class of neurons in Drosophila and implicated a known LIM-HD partner of Chip, Islet, in this function (Hari, 2008).

It is proposed that the defect is attributable to a failure in the release rather than in the production or the responsiveness to the neurohormone Bursicon. How might Bursicon release be controlled as a result of Chip function in development? The hemolymph transfer experiments provide a unique insight into this puzzle. The literature describes a model wherein posteclosion wing expansion requires a combination of a neural signal from the brain as well as Bursicon release. The results extend the understanding of how this interplay of activity and secreted factors is set up in development. It appears that, several days before eclosion, Chip is able to regulate an as-yet-unidentified event in the CCAP neurons, such that the hemolymph contains adequate levels of Bursicon after eclosion. The CCAP-expressing neurons are divided into at least two interacting subpopulations, only one of which secretes Bursicon. The other subpopulation does not secrete Bursicon but is implicated in regulating its release. Chip may therefore mediate the formation of proper connectivity among CCAP neurons, which eventually ensures timely Bursicon release several days later. Supporting this scenario, Chip has been reported to regulate axon pathfinding and proper innervation of targets in other systems. The data are suggestive of Chip requirement in the early period of puparium formation, which fits well with a report that CCAP neurons undergo extensive remodeling in this period of metamorphosis. The importance of this connectivity is underscored by the identification of several other genes in a gain-of-function screen, which displayed a simultaneous disruption of both posteclosion wing expansion and the pattern of CCAP neuron innervation. Therefore, thes findings motivate an examination of Chip function in regulating the connectivity of CCAP neurons, a role that directly links this key aspect of neuronal development with the control of posteclosion behavior in Drosophila (Hari, 2008).

Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. This study shows that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam is able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. Although it has been shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. These data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner (Dutta, 2008).

Wts was identified as a protein that is expressed during autophagic cell death of Drosophila larval salivary glands with a high-throughput proteomics approach. This was surprising, given that wts RNA was not detected with DNA microarrays. Therefore, this study investigated whether Wts is present in salivary glands, and it was determined to be constitutively expressed at stages before and after the rise in ecdysone that triggers autophagic cell death. Animals that are homozygous for the hypomorphic wts^P2 allele, which is caused by a P element insertion, are defective in salivary gland cell death (Martin, 2007). Significantly two forms of Hpo are expressed during stages preceding salivary gland cell death, suggesting that phosphorylated Hpo is present in these cells and that this signaling pathway is activated (Dutta, 2008).

These studies indicate that Wts and other core components of this tumor-suppressor pathway are required for autophagic cell death of Drosophila salivary glands. wts is required for cell growth arrest and for proper regulation of caspases and autophagy, which contribute to the destruction of salivary glands. Although it is well known that cell division, cell growth, and cell death are important regulators of tissue and tumor size, it has been unclear whether a mechanistic relationship exists between cell growth and control of cell death (Dutta, 2008).

It is possible that wts and associated downstream growth-regulatory mechanisms could suppress cell death in other animals and cell types. Autophagic cell-death morphology has been reported in diverse taxa, but little is known about the mechanisms that control this form of cell death, and this lack of understanding is probably related to the limited investigation of physiologically relevant models of this process (Dutta, 2008).

This study used steroid-activated autophagic cell death of salivary glands as a system to study the relationship between cell growth and cell death. It is logical that cell growth influences cell death in salivary glands, given that autophagy is known to be regulated by class I PI3K signaling, which contributes to the death of these cells (Berry, 2007). It is unclear whether growth arrest is a determinant of autophagic cell death in other cell types and animals, and this question is important to resolve because of the importance of growth and autophagy in multiple disorders, including cancer. wts mutant salivary gland cells fail to arrest growth at the onset of puparium formation, and this suppresses the induction of autophagy. The inhibitor of apoptosis DIAP1 influences salivary gland cell death and is one of the best-characterized target genes of the Wts signaling pathway, but DIAP1 levels are not altered in wts mutant salivary glands. Significantly, the data provide the first evidence that Wts regulates autophagy and support previous studies indicating that caspases and autophagy function in an additive manner during autophagic cell death. Given the importance of both the Wts pathway and autophagy in human health, it is critical to determine whether this relationship exists in other cells (Dutta, 2008).

Cell growth and division are often considered to be synonymous, even though they are controlled by independent mechanisms. The Wts signaling pathway must influence cell growth, but most studies have emphasized the influence of this pathway on cell division and death. bantam is the only previously studied gene that is regulated by the Wts pathway and that is known to regulate cell growth. However, the mechanism of bantam action remains obscure. The current studies suggest the possibility that Wts may regulate growth via different mechanisms and that the nature of this regulation may depend on cell context. It is premature to conclude that bantam regulates a completely novel cell growth program, but the fact that misexpression of bantam stimulates cell growth in the absence of changes in a phosphoinositide marker and that chico and TOR fail to suppress the bantam-induced salivary gland-persistence phenotype minimally suggests that this microRNA regulates genes downstream of TOR. Significant progress has been made in the identification of microRNA targets, and future studies should resolve the mechanism underlying bantam regulation of cell growth (Dutta, 2008).

Recent studies of Wts signaling in Drosophila have identified a linear pathway that terminates with Yki and Sd regulation of effector genes that influence cell growth, cell division, and cell death. These studies indicate that the Wts pathway may not always regulate downstream effector genes via Yki and Sd, given that Yki expression was not able to phenocopy the wts mutant salivary gland destruction and expression of Sd induced premature degradation of salivary glands. Although bantam expression is sufficient to induce growth and inhibit cell death in salivary glands, bantam function is not required for the wts mutant phenotype. wts mutant salivary glands possess altered markers of PI3K signaling, and their defect in cell death is suppressed by chico and TOR. Combined, these results indicate that Wts regulates cell growth and cell death via a PI3K-dependent, and Yki- and Sd-independent, mechanism. Future studies will determine whether Wts regulates cell growth in a PI3K-dependent manner in other cells and animals (Dutta, 2008).

The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. The transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. Two LIM-homeodomain proteins, Arrowhead and Lim1, are shown to be expressed in the region of the eye-antennal disc affected in Chip mutants, and both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue (Roignant, 2009).

Regionalization of the eye-antennal disc is a progressive process in which selector genes and signaling pathways specify the fates of different head structures. Clones of eye-antennal disc cells induced during the second larval instar can contribute to multiple organs, indicating that these cells retain developmental plasticity at this stage. The anteroposterior boundary of the wing disc is established much earlier; expression of the selector gene engrailed (en) specifically in the posterior cells during embryogenesis generates an affinity border that keeps the two compartments clonally separated. By contrast, the eye selector gene ey is uniformly expressed throughout the early eye-antennal disc, and only retracts to the eye field in the second instar. It was initially proposed that localized Notch signaling controls this retraction, as expression of dominant-negative forms of Notch in the eye disc abolishes ey expression and leads to antennal duplications. However, a later study demonstrated that loss of Notch function does not affect ey expression directly, but reduces cell proliferation in the retinal field, preventing the initiation of eya expression. This study shows that Chip and Lim1 are both necessary to repress ey expression in the anterior of the antennal disc. Additional factors probably help to restrict ey expression to the eye disc, because ey expression does not extend throughout the normal Lim1 expression domain in Lim1 or Chip mutant clones in the antennal disc (Roignant, 2009).

Since Lim1 mutant clones always misexpress Ey, but rarely misexpress Eya and never differentiate ectopic photoreceptors, additional proteins must interact with Chip to repress retinal differentiation. Awh is a good candidate because it is expressed at the ventral margin of the eye-antennal disc, its misexpression in the retina represses photoreceptor differentiation in a Chip-dependent manner, and loss of both Lim1 and Awh leads to ectopic photoreceptor differentiation in the ventral eye-antennal disc. Since ectopic photoreceptors differentiate only in the absence of both Lim1 and Awh, whereas Ey expansion is observed in Lim1 single mutants, Awh must control the expression of target genes other than ey. It may negatively regulate other genes involved in retinal determination, such as eya, or positively regulate genes important for head capsule development, such as Deformed and odd-paired (Roignant, 2009).

Like Chip, Hth is required to prevent retinal differentiation at the ventral eye-antennal disc boundary. Investigation of the relationship between Chip and Hth indicates that Chip is not required for Hth expression or activity. The ability of Hth to repress photoreceptor differentiation in Chip mutant clones rules out the possibility that Chip acts as a co-factor for Hth or an essential downstream mediator of its effects. The normal expression of Hth and its target gene wg in Chip mutant clones also make it unlikely that Chip controls the expression of Hth or its co-factor Exd. However, the possibility that Hth and Chip act in parallel poses the paradox that misexpressed Hth is sufficient to repress photoreceptor development in the eye field in the absence of Chip, but endogenous Hth is insufficient to do so in the head field. It is possible that Hth expression levels in the head field early in development are too low to repress the eye fate in the absence of Chip. Consistent with this hypothesis, it was found that overexpression of Hth in Chip mutant cells prevents ectopic photoreceptor differentiation. Similarly, overexpression of Awh or Lim1 prevents ectopic photoreceptor differentiation in hth mutant cells, suggesting that endogenous levels of these LIM-HD proteins are not sufficient to compensate for the absence of Hth. The two classes of transcription factors may normally act on different sets of target genes, but show some cross-regulatory ability when overexpressed (Roignant, 2009).

The boundary between the eye and the dorsal head appears to be established differently from the boundary in the ventral region. The LIM-HD gene tup is expressed at the dorsal eye-antennal disc boundary, in a pattern resembling the mirror image of the Awh pattern, and is capable of repressing photoreceptor development in a Chip-dependent manner. However, loss of Chip in this region does not lead to ectopic eye formation, although it can cause overgrowth and mispatterning of the head. In the absence of Chip, the GATA transcription factor Pannier (Pnr) and its target gene wg may be sufficient to maintain dorsal head fate. The ventral margin of the eye-antennal disc may be particularly susceptible to ectopic photoreceptor differentiation because of the high level of Dpp signaling there. A 5' enhancer element has been shown to direct dpp expression specifically in the ventral marginal peripodial epithelium of the eye-antennal disc. The ability of Dpp and Ey to synergize to drive retinal differentiation therefore makes it critical to repress Ey in this region, which is fated to form head capsule (Roignant, 2009).

In addition, this domain of Dpp overlaps with Wg present at the anterior lateral margin of the eye disc; the combination of these two growth factors induces proximodistal growth of the leg. One function of Chip and its partner proteins might thus be to repress the outgrowth that would otherwise be triggered by the combination of Dpp and Wg. Unlike growth of the wild-type eye disc, growth of Chip mutant regions appears to be Notch-independent, as they do not contain a fng expression boundary and do not show activation of the Notch target genes E(spl)mβ or eyg. Notch has been thought to trigger growth by inducing the expression of the JAK/STAT ligand Unpaired (Upd); however, a recent report describes an earlier function for Upd upstream of Notch, raising the possibility that upd expression is activated independently of Notch in Chip mutant clones. As hth mutant clones, or clones lacking the Odd skipped family member Bowl, frequently show ectopic ventral photoreceptor differentiation but rarely induce outgrowths like those seen in Chip mutants, the functions of Chip in growth and differentiation are likely to be separable (Roignant, 2009).

LIM-HD proteins also set developmental boundaries in other imaginal discs, acting in concert with other classes of transcription factors. In the wing disc, Tup specifies the notum in collaboration with homeodomain transcription factors of the Iroquois complex, and Ap specifies the dorsal compartment. Ap interacts with the homeodomain protein Bar and Lim1 with Aristaless to establish specific tarsal segments within the leg disc. LIM-HD proteins have also been implicated in vertebrate eye development, although those that have been studied appear to play positive roles. The Ap homologue Lhx2 is expressed within the mouse retinal field at the neural plate stage, and contributes to the expression of Pax6, Six3 and Rx. Lmx1b, the homologue of CG32105, is required for the development of anterior eye structures such as the cornea and iris, and is mutated in human patients with nail-patella syndrome, often characterized by glaucoma. Within the retina, loss of Lim1 results in mispositioning of horizontal cells within the amacrine cell laye. Drosophila Lim3 shows photoreceptor-specific expression, and might therefore have a positive function in eye development (Roignant, 2009).

In the central nervous system, LIM-HD proteins act combinatorially to specify different neuronal cell fates. In both Drosophila and vertebrates, combinations of Islet and Lhx3/4/Lim3 proteins regulate motoneuron specification and pathfinding. The ability of Chip to interact with LIM-HD proteins and other transcription factors as well as to dimerize enables it to form heteromeric transcription factor complexes. In the wing disc, the active complex is a tetramer containing two subunits each of Chip and Ap, whereas in motoneuron development the Chip homologue NLI can form either a tetramer with Lhx3 or a hexamer containing both Isl1 and Lhx3. The finding that Lim1 and Awh act redundantly to prevent eye development in the ventral head primordium, whereas Chip is absolutely required, seems most consistent with regulation of distinct subsets of target genes by independent Chip-Awh and Chip-Lim1 complexes; however, a contribution from a complex containing all three proteins, or even additional transcription factors, cannot be ruled out. The role of the Chip co-factor may be to coordinate multiple transcriptional regulatory complexes to restrict developmental fates within the eye-antennal imaginal disc, allowing it to give rise to the head cuticle as well as distinct external sensory structures (Roignant, 2009).

EVOLUTIONARY HOMOLOGS

An evolutionarily conserved Lhx2-Ldb1 interaction regulates the acquisition of hippocampal cell fate and regional identity

Protein cofactor Ldb1 regulates cell fate specification by interacting with LIM-homeodomain (LIM-HD) proteins in a tetrameric complex consisting of an LDB:LDB dimer that bridges two LIM-HD molecules, a mechanism first demonstrated in the Drosophila wing disc. This study demonstrates conservation of this interaction in the regulation of mammalian hippocampal development, which is profoundly defective upon loss of either Lhx2 (see Drosophila Apterous) or Ldb1 (see Drosophila Chip). Electroporation of a chimeric construct that encodes the Lhx2-HD and Ldb1-DD (dimerization domain) in a single transcript cell-autonomously rescues a comprehensive range of hippocampal deficits in the mouse Ldb1 mutant, including the acquisition of field-specific molecular identity and the regulation of the neuron-glia cell fate switch. This demonstrates that the LHX:LDB complex is an evolutionarily conserved molecular regulatory device that controls complex aspects of regional cell identity in the developing brain (Kinare, 2020).

LIM homeodomain (LIM-HD) and nuclear LIM-only proteins play important roles in a variety of developmental processes in animals. In some cases their activities are modulated by a nuclear LIM binding protein family called Ldb/NLI/Clim, exemplified by Drosophila Chip. The Ldb/NLI/Clim ortholog ldb-1 of the nematode C. elegans has been characterized. Two alternatively spliced variants exist, which differ in their amino-termini. The ldb-1 ortholog of Caenorhabditis briggsae has the same structure as that of C. elegans and is highly conserved throughout the open reading frame, while conservation to fly and vertebrate proteins is restricted to specific domains: the dimerization domain, the nuclear localization sequence, and the LIM interaction domain. C. elegans ldb-1 is expressed in neurogenic tissues in embryos, in all neurons in larval and adult stages, and in vulval cells, gonadal sheath cells, and some body muscle cells. C. elegans LDB-1 is able to specifically bind LIM domains in yeast two-hybrid assays. RNA inactivation studies suggest that C. elegans ldb-1 is not required for the differentiation of neurons that express the respective LIM-HD genes or for LIM-HD gene autoregulation. In contrast, ldb-1 is necessary for several neuronal functions mediated by LIM-HD proteins, including the transcriptional activation of mec-2, the mechanosensory neuron-specific stomatin (Cassata, 2000).

LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lim-11 (most closely related to Drosophila Lim1) have been shown to alter the cell division pattern of a subset of the 2º lineage vulval cells. Multiple functions of lin-11 during vulval development have been demonstrated. The fate of vulval cells was examined in lin-11 mutant animals using five cellular markers: lin-11 is necessary for the patterning of both 1º and 2º lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, lin-11 is shown to be initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2º lineage cells. Using a conditional RNAi approach, it has been shown that lin-11 regulates vulval morphogenesis. LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning (Gupta, 2003).

LIM homeodomain proteins are developmental regulators whose functions depend on synergism with LIM domain binding proteins (Ldb proteins); they are homologs of Drosophila Chip. Three members of the ldb gene family (Ldb1, Ldb2 and Ldb3) from the zebrafish, Danio rerio, share 95%, 73% and 62% amino acid identity with mouse Ldb1, respectively. In overlay assays, Ldb proteins bind LIM homeodomain proteins and LMO1, but not zyxin or MLP. Whole mount in situ hybridization showed that zebrafish ldb1 is expressed ubiquitously from gastrulation onward. Ldb2 is ubiquitous at gastrulation, and later is found in many tissues, especially the anterior central nervous system (CNS) and vasculature, but not all tissues; Ldb3 mRNA is expressed primarily in the anterior CNS. The expression of individual lbd and LIM proteins correlates in various regions and stages of embryogenesis. For example, lbd1 and ldh2 are expressed in the shield as are lim1 and lim6. Both lbd2 and lbd3 expression in the telencephalon overlap with that of lim5 and lim6, and three genes, lbd2, lim3 and lim5, are coexpressed in the epiphysis (Toyama 1998).

The crucial involvement of CLIM/NLI/Ldb cofactors for the exertion of the biological activity of LIM homeodomain transcription factors (LIM-HD) has been demonstrated. CLIM cofactors are widely expressed during zebrafish development with high protein levels in specific neuronal cell types where LIM-HD proteins of the Isl class are synthesized. The overexpression of a dominant-negative CLIM molecule (DN-CLIM) that contains the LIM interaction domain (LID) during early developmental stages of zebrafish embryos results in an impairment of eye and midbrain-hindbrain boundary (MHB) development and disturbances in the formation of the anterior midline. On a cellular level it has been shown that the outgrowth of peripheral but not central axons from Rohon Beard (RB) and trigeminal sensory neurons is inhibited by DN-CLIM overexpression. A further critical role of CLIM cofactors has been demonstrated for axonal outgrowth of motor neurons. Additionally, DN-CLIM overexpression causes an increase of Isl-protein expression levels in specific neuronal cell types, likely due to a protection of the DN-CLIM/LIM-HD complex from proteasomal degradation. These results demonstrate multiple roles of the CLIM cofactor family for the development of entire organs, axonal outgrowth of specific neurons and protein expression levels (Becker, 2002).

A novel protein and a new LIM-domain-binding factor, Ldb1, has been isolated on the basis of its ability to interact with the LIM-HD protein Lhx1 (Lim1). High-affinity binding by Ldb1 requires paired LIM domains and is restricted to the related subgroup of LIM domains found in LIM-HD and LMO proteins (see Drosophila Muscle LIM protein at 60A). The highly conserved Xenopus Ldb protein XLdb1, interacts with Xlim-1, the Xenopus orthologue of Lhx1. When injected into Xenopus embryos, XLdb1 (or Ldb1) can synergize with Xlim-1 in the formation of partial secondary axes and in activation of the genes encoding goosecoid, chordin, NCAM and XCG7, demonstrating a functional as well as a physical interaction between the two proteins (Agulnick, 1996).

The homeobox genes Xlim-1 and goosecoid are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Since gsc is a possible target gene for Xlim-1, the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages was studied by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins show that the Xlim-1 homeodomain and the Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element does not bind Xlim-1 or PV.1 but does bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. Using animal cap assays, it has been shown that the endogenous gsc gene is synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene is activated by Xlim-1/3m, and this activation is suppressed by the posterior factor Xbra. Based on these data, a model is proposed for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation (Mochizuki, 2000).

Thus Gsc protein is capable of inhibiting the activity of its own promoter in assays using reporters activated by Xlim-1, Ldb1, and Otx2. Otx2 and Gsc belong to the same homeodomain group in that both have a lysine residue at position 50 of the homeodomain and share binding specificity for TAATCT and TAATCC. Since these two proteins recognize similar target sequences, there may be competition between Otx2 and Gsc for binding to the C site of distal element, with Otx2 having activating and Gsc inhibiting effects. Inhibition of the mouse and human gsc promoter by Gsc requires the proximal element, suggesting that Gsc inhibition, just like Xlim-1 activation, involves multiple sites in the complex gsc promoter. Repression of the gsc promoter by Gsc and PV.1 proteins is similarly effective under the experimental conditions employed, but the biological roles of the two proteins are different. In the case of Gsc autoinhibition, the rationale may be to provide a feedback loop to limit gsc expression. In contrast, PV.1, closely related to Xvent-1, is expressed ventrally as a consequence of BMP signaling in a region of the embryo where gsc is not expressed. It appears that PV.1 is a repressor protein whose function is to maintain the character of ventral mesoderm by inhibiting gsc expression in the non-organizer regions of the marginal zone. Similarly, Xbra may inhibit Gsc function in the notochord where gsc expression diminishes during gastrulation. The ability of Xbra to repress otx2 expression and of Gsc to repress Xbra expression may play a role in restricting gsc expression to the prechordal plate and Xbra expression to the notochord at mid- to late gastrulation. However, because Xbra is a transcriptional activator, it is assumed that otx2 repression is indirect (Mochizuki, 2000).

These transcription factor interactions have been incorporated into a model of dorsoventral and anteroposterior patterning in the gastrula embryo. In the prechordal plate, Xlim-1 and Ldb1, in addition to contributing to chordin induction, maintain the expression of otx2 and of gsc; the autoinhibitory action of the latter is counteracted by the activating function of Otx2, while Xbra expression is suppressed by Gsc. In the notochord, the high initial level of Xbra prevents otx2 gene activation by Xlim-1 plus Ldb1, and in the absence of Otx2, the gsc gene turns itself off by autorepression. Note that in the early gastrula, gsc is active in the entire organizer, but its expression fades in posterior axial mesoderm as gastrulation proceeds. In ventral mesoderm, the strong repression of gsc and otx2 by PV.1/Xvent-1 and Xbra maintains the ventral character of this tissue. Clearly, this scheme is incomplete in that additional factors are undoubtedly involved, yet it provides a cogent model for the interactions of the factors considered in this paper during axial patterning in the gastrula (Mochizuki, 2000).

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions; interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. A novel protein, nuclear LIM interactor (NLI) has been isolated that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions (Jurata, 1996).

Two highly homologous proteins specifically interact with the LIM domains of P-Lim/Lhx3 and several other LIM homeodomain factors. Transcripts encoding these factors can be detected as early as mouse E8.5, with maximal expression observed in regions of the embryo in which the LIM homeodomain factors P-Lim/Lhx3, Isl-1, and LH-2 are selectively expressed. These proteins can potentiate transactivation by P-Lim/Lhx-3 and are required for a synergistic activation of the glycoprotein hormone alpha-subunit promoter by P-Lim/Lhx3 and a pituitary Otx class homeodomain transcription factor (P-OTX/Ptx1), with which they also specifically associate. The two new genes are referred to as CLIM-1 and CLIM-2 (cofactor of LIM homeodomain proteins). The CLIM proteins are required for a transcriptional synergy between P-Lim/Lhx3 and P-OTX/Ptx1. The fact that CLIM-encoded mRNAs show a widely overlapping expression pattern with Otx1 and Otx2 in the developing mouse brain suggests that the CLIM protein family may play critical roles in the functional relationships of LIM homeoproteins and additional Otx factors (Bach, 1997).

The mesenchymal cells that contribute to oral and facial hard tissues are derived from cranial neural crest cells, whereas limb mesenchyme cells are derived from axial mesoderm. The outgrowth of facial processes has been compared with limb bud outgrowth; the tooth bud enamel knot has been identified as a signaling center with similarities to both the limb ZPA and AER signaling centers. In addition, several homeobox-containing genes have been implicated in both branchial arch and limb development, such as members of the Msx, Dlx and Lim-homeobox families. The expression of the Lim-domain gene Lhx-6, and its closely associated family member Lhx-7 are largely restricted to anterior mesenchymal cells of the mandibular and maxillary arches. Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud mesenchyme in association with Lim genes Lhx-6 and Lmx-1, respectively. In common with both these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face and the limb buds Fgf-8 is identified as the likely candidate signaling molecule that regulates Clim-2 expression. In the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface binding protein that has been considered to be a hyaluronan receptor. Blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. CD44 has been shown to be required for presentation of Fgf-8 to its receptor, rather than as a hyaluronan receptor. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and mandibular arch development (Tucker, 1999).

LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified and widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. A 38-amino-acid fragment of NLI is sufficient for the association of NLI with nuclear LIM domains. NLI has been shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI is not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis reveals higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. NLI forms complexes with Lmx1 on the rat insulin I promoter and inhibits the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses that require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions (Jurata, 1997).

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. A widely expressed protein, Ldb1, has been identified whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibits cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibits erythroid differentiation. These studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state (Visvader, 1997).

The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukemias, is normally expressed in hematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the interaction is unexplained. In erythroid cells Lmo2 forms a novel DNA-binding complex with GATA-1, TAL1, E2A, and the recently identified LIM-binding protein, Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box (CAGGTG), followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex, which may play a role in hematopoiesis (Wadman, 1997).

LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Evidence is presented that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex (see Drosophila Sin3A). A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. It is concluded that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain (Bach, 1999).

Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of either Islet-2 or the LIM-interacting domain of Ldb proteins prevents binding of Islet-2 to Ldb proteins in vitro and causes similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons. Inhibition of Islet-2 translation with antisense morpholino oligonucleotide against Islet-2 mRNA reproduces the defects caused by overexpression of LIM domains of Isl-2. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains, derived from two different Islet-1 family members, demonstrate that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors (Segawa, 2001).

Repression of Islet-2 function either by overexpressing the LIM domains of Isl-2 or injecting antisense against Islet-2 mRNA impairs outgrowth of the peripheral axons from the primary sensory neurons, while keeping their central axons intact. In vitro study using the explants of the chick dorsal root ganglion (DRG) has demonstrated that the DRG neurons extend their axons into the peripheral tissue only in the presence of the nerve growth factor (NGF), while they extend the axons into the CNS tissue irrespective of the presence of NGF. This suggests that NGF may selectively promote the peripheral outgrowth of the DRG neurons. Recently, double mutant mice for the genes encoding the proapoptotic BCL-2 homolog BAX and NGF/TrkA were generated. All DRG neurons (which would normally die in the absence of the NGF/TrkA signaling) survive if BAX is also eliminated. In BAX^-/-;NGF^-/- or BAX^-/-;TrkA^-/- mice, only the peripheral axons of the DRG neurons are lost, while their central axons remain intact. Therefore, the NGF/TrkA signaling regulates outgrowth of the peripheral axons from the DRG neurons. In view of this, it would be intriguing to examine whether Islet-2 is involved in the NGF/TrkA signaling (Segawa, 2001).

The interactions of distinct cofactor complexes with transcription factors are decisive determinants for the regulation of gene expression. Depending on the bound cofactor, transcription factors can have either repressing or transactivating activities. To allow a switch between these different states, regulated cofactor exchange has been proposed; however, little is known about the molecular mechanisms that are involved in this process. LIM homeodomain (LIM-HD) transcription factors associate with RLIM (RING finger LIM domain-binding protein) and with CLIM (cofactor of LIM-HD proteins; also known as NLI, Ldb and Chip) cofactors. The co-repressor RLIM inhibits the function of LIM-HD transcription factors, whereas interaction with CLIM proteins is important for the exertion of the biological activity conferred by LIM-HD transcription factors. RLIM has been identified as a ubiquitin protein ligase that is able to target CLIM cofactors for degradation through the 26S proteasome pathway. Furthermore, this study demonstrates a ubiquitination-dependent association of RLIM with LIM-HD proteins in the presence of CLIM cofactors. These data provide a mechanistic basis for cofactor exchange on DNA-bound transcription factors, and probably represent a general mechanism of transcriptional regulation (Ostendorff, 2002).

The LIM domain-binding protein 1 (Ldb1) is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. Targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype. There is no heart anlage and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. There are also severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells and the amnion. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in Ldb1 mutant embryos. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning regulated by Ldb1 (Mukhopadhyay, 2003).

The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remain largely unknown. The Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM has been isolated and its functional interactions with Xlim-1 and Ldb1 have been explored. Although XRnf12 functions as an E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. A high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1, suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12 throughout early embryogenesis, Rnf12/RLIM is proposed as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues (Hiratani, 2003).

Structural basis for the recognition of ldb1 by the N-terminal LIM domains of LMO2 and LMO4

LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1-LID). This study reports the solution structures of two LMO:ldb1 complexes (PDB: 1M3V and 1J2O) and shows that ldb1-LID binds to the N-terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a beta-hairpin in LIM1 domains. These findings constitute the first molecular definition of LIM-mediated protein-protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology (Deane, 2003).

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). A high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, has been determined in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains (Deane, 2004).

Identification of the key LMO2-binding determinants on Ldb1

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. This study shows using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1. However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1, whereas the second LIM domain increases binding by an order of magnitude. Mutagenesis was used in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding 'hot spots' within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia (Ryan, 2006).

Role of LDB1 in the transition from chromatin looping to transcription activation

Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1) protein is recruited to the beta-globin locus via LMO2 (see Drosophila Beadex) and is required for looping of the beta-globin locus control region (LCR) to the active beta-globin promoter. This study shows that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2, sufficient to completely restore LCR-promoter looping and transcription in LDB1-depleted cells. The looping function of the DD is unique and irreplaceable by heterologous DDs. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the coregulators FOG1 (U-shaped in Drosophila) and the nucleosome remodeling and deacetylating (NuRD) complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior, as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1 in these processes (Krivega, 2014).

SCL assembles a multifactorial complex that determines glycophorin A expression

SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. This study identified the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and shows that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, these observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression (Lahlil, 2004).

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) have been shown to interact with Ldb1 and are also important in developmental programs. Two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins (Xu, 2007).

Ssdp1 regulates head morphogenesis of mouse embryos by activating the Lim1-Ldb1 complex

The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is regulated by their interactions with various factors that bind to the LIM domain. Reduced expression of single-stranded DNA-binding protein 1 (Ssdp1), which encodes a co-factor of LIM domain interacting protein 1 (Ldb1), in the mouse mutant headshrinker (hsk) disrupts anterior head development by partially mimicking Lim1 mutants. Although the anterior visceral endoderm and the anterior definitive endoderm, which together comprise the head organizer, are able to form normally in Ssdp1^hsk/hsk mutants, development of the prechordal plate was compromised. Head development is partially initiated in Ssdp1^hsk/hsk mutants, but neuroectoderm tissue anterior to the midbrain-hindbrain boundary is lost, without a concomitant increase in apoptosis. Cell proliferation is globally reduced in Ssdp1^hsk/hsk mutants, and approximately half also exhibit smaller body size, similar to the phenotype observed in Lim1 and Ldb1 mutants. Ssdp1 contains an activation domain and is able to enhance transcriptional activation through a Lim1-Ldb1 complex in transfected cells, and Ssdp1 interacts genetically with Lim1 and Ldb1 in both head development and body growth. These results suggest that Ssdp1 regulates the development of late head organizer tissues and body growth by functioning as an essential activator component of a Lim1 complex through interaction with Ldb1 (Nishioka, 2005).

Ssdp1 mutants exhibit a global reduction in cell proliferation after E8.5 and an increase in apoptosis in somites at E9.0. These changes may be at the root of the abnormalities such as growth retardation and kinked neural tube that were observed in Ssdp1 mutants. Although the mechanism by which Ssdp1 regulates cell proliferation is unknown at present, growth retardation of Ssdp1^+/hsk;Lim1^+/- and Ssdp1^+/hsk;Ldb1^+/- compound mutants suggests involvement of a Ssdp1-Lim1-Ldb1 complex in this process. A shortened body axis was also observed in embryos lacking either Ldb1 or Lim1, supporting this hypothesis. However, if the Lim1 complex plays a major role in the regulation of cell proliferation and cell death, it must be through an indirect mechanism, since Lim1 is not expressed in all of the affected cells. It is conceivable that defective gastrulation movements or the inability of cells with reduced Lim1 complex activity to induce lateral plate mesoderm genes secondarily affects the proliferation and survival of surrounding cells. Furthermore, it is possible that Ssdp1 may also function independently of Lim1, in which case the Ldb1-Ssdp1 complex may regulate cell proliferation in a cell-autonomous manner by controlling the activities of transcription factors involved in cell cycle regulation and cell survival. Alternatively, Ssdp1 might play a direct role in the DNA replication process as a single stranded DNA-binding protein (Nishioka, 2005).

Analysis of hsk mutants shows that disruption of the Ssdp1 gene and the resulting reduction in Ssdp1 expression causes defects in the prechordal plate development and anterior truncations, with some mutants also exhibiting smaller body size. In vitro data have demonstrated that Ssdp1 acts as a coactivator that enhances transcriptional activation by the Lim1-Ldb1 complex. Moreover, genetic interactions between Ssdp1 and Lim1 or Ldb1 suggest that the phenotypes observed in Ssdp1 mutants very probably reflect reduced activity of a Lim1 complex. Together, these data demonstrate that Ssdp1 acts as an essential activator component of a Ssdp1-Lim1-Ldb1 complex in the development of the prechordal plate and body growth (Nishioka, 2005).

Novel binding partners of Ldb1 are required for haematopoietic development

Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. This study has identified a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although the complex was originally identified in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 it is postulated that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells (Meier, 2006).

The Lim-only protein LMO2 acts as a positive regulator of erythroid differentiation

LMO2, a member of the LIM-only protein family, is essential for the regulation of hematopoietic stem cells and formation of erythroid cells. It is found in a transcriptional complex comprising LMO2, TAL1, E47, GATA-1, and LDB1 which regulates erythroid genes. While TAL1 has been shown to induce erythroid differentiation, LMO2 appears to suppress fetal erythropoiesis. In addition to LMO2, the closely related LMO4 gene is expressed in hematopoietic cells, but has unknown functions. This study demonstrates that LMO2 and LMO4 are expressed at the same level in erythroid colonies from mouse bone marrow, implying a function in erythroid differentiation. However, while LMO2 induced erythroid differentiation, LMO4 had no such effect. Interestingly, both LMO2 and TAL1 were able to partially suppress myeloid differentiation, implying that they activate erythroid differentiation in uncommitted bone marrow progenitors. Both LMO2 and LMO4 interacted strongly to LDB1, which was required for their localization to the nucleus (Hansson, 2007).

A LIM-complex directed regulatory network to segregate the identity of neuronal subtypes

Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. This study shows how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, this study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops (Lee, 2008).

A SELEX study revealed that the Lhx3-binding sites deviate between HxRE and TeRE in sequence. As HxRE is recognized by MN-hexamer but not by V2-tetramer, the conformation of Lhx3 in MN-hexamer and V2-tetramer is likely different. This may involve an allosteric structural change in the DNA binding domain of Lhx3 in MN-hexamer, induced by the Isl1:Lhx3 interaction. As MN-hexamer is assembled only in MNs, HxRE-containing genes would be stimulated specifically in MNs but not in V2-INs. In contrast, TeRE is activated by both V2-tetramer and MN-hexamer in vitro, suggesting that TeRE-containing V2 genes could be inappropriately induced in MNs. However, TeRE is a V2-specific response element in the developing embryos due to a collaborative action of Hb9 and LMO4 to silence TeRE in MNs. Thus, these studies demonstrate that DNA-REs for specific transcription complexes are sufficient to confer gene expressions to proper cell types in developing embryos (Lee, 2008).

Although Lhx4 and their cofactor NLI (Ldb, CLIM, Chip) dimerization is dispensable for the DNA-binding activity of V2-tetramer and MN-hexamer, it is essential for their robust transactivation. Thus, reiterated TeRE^1/2s and HxRE^1/2s are necessary for functional TeREs and HxREs. Indeed, the MN-specific enhancer of Hb9 has two functional HxRE^1/2s spaced ~150 nt apart. Similarly, three evolutionarily conserved TeRE^1/2 sequences were found in the Chx10-TeRE region. Three possible advantages can be proposed for the NLI dimerization in V2-tetramer and MN-hexamer. First, the requirement for multiple repeats of TeRE^1/2 and HxRE^1/2 may impose higher stringency for functional target gene selection. Second, NLI dimerization bridges two NLI-interacting transcription factors bound to their DNA-binding sites separated by a relatively long spacer region in Drosophila (Heitzler, 2003; Morcillo, 1997). This raises the possibility that V2-tetramer and MN-hexamer may integrate the transcriptional activity of multiple TeRE^1/2s or HxRE^1/2s located within a single target gene or across multiple target genes. For instance, both Chx10 and Lin-52 have additional TeRE^1/2s within their gene. Thus, it will be interesting to test whether the Chx10-TeRE region is required to regulate both Chx10 and Lin-52 by V2-tetramer and whether the multiple TeRE^1/2s throughout these two genes enable V2-tetramer to temporally coordinate expression of these genes during development. Third, NLI dimerization may potentiate the transcriptional activity of LIM-complexes by stabilizing the LIM-complexes and/or facilitating recruitment of transcriptional coactivators and chromatin remodeling complexes. Indeed, single-stranded DNA-binding proteins have been found to interact with NLI and augment the transactivation of NLI-containing complexes (Lee, 2008).

DNA-REs affect the protein-protein interaction properties of their cognate transcription factors. Sox2 and Pou factor family members Oct1 or Oct4 dimerize onto different DNA-REs in distinct conformational arrangements, offering one molecular explanation for the wide spectrum of developmental functions for Sox/Pou factors. Thus, it will be interesting to interrogate the role of TeRE and HxRE on the spatial alignment of DNA-protein complex of Lhx3/TeRE^1/2 and of Isl1:Lhx3/HxRE^1/2 (Lee, 2008).

As p2 and pMN cells are exposed to relatively similar concentration of Shh, deregulation of the transcriptional events downstream of Shh often results in V2-MN fate conversion or hybrid phenotypes. The initial segregation of V2 and MN pathways appears to involve transcriptional crossrepression of progenitor factors Irx3 and Olig2 in neuroepithelial cells. However, additional mechanisms are likely to be needed, as both differentiating V2 and MN cells express Lhx3/4, which are necessary for their cell fates. The results reveal an efficient feedforward gene regulatory circuitry in which a cell-type specific LIM-complex triggers expression of a transcriptional repressor, which in turn binds and represses the DNA-RE of another related LIM-complex, thereby blocking the unwanted choice of alternative fates. This likely contributes to establishing a precise cell identity once a specific LIM-complex is assembled and activates the downstream target genes in neural precursors (Lee, 2008).

First, MN-hexamer upregulates Hb9, which in turn refines MN-gene expression by silencing V2-genes. Hb9 selectively binds TeREs, which prevents undesirable recruitment of MN-hexamer (and any V2-tetramer) to TeREs and represses their inappropriate activation in embryonic MNs. Indeed, V2 genes are upregulated in MNs lacking Hb9. Hb9, a transcriptional repressor, may suppress TeRE-containing V2 genes by recruiting corepressors to their TeREs. Mnr2, an Hb9 paralog, is also known to recruit Ctbp-like corepressor. Thus, TeRE-containing V2 genes are likely to be activated by V2-tetramer in V2-INs, while they are simultaneously silenced by Hb9 in MNs (Lee, 2008).

Second, V2-tetramer directly binds Chx10-TeRE and upregulates Chx10 in chick spinal cord, suggesting that Chx10 is a direct target gene of V2-tetramer. Although Chx10 regulates retinal development, neither its function in the developing spinal cord nor its in vivo target genes are known. Interestingly, Chx10 binds Hb9-HxRE through its homeodomain and represses both the basal and MN-hexamer-induced levels of transcriptional activity mediated by HxRE, consistent with a previous report that Chx10 primarily functions as a transcriptional repressor. In V2 cells, Chx10 could be necessary to completely shut off any leaky expression of HxRE-containing MN genes or to actively block erroneous activation of MN genes by other transcription factors shared between V2-INs and MNs via recruiting corepressors to HxREs. Overall, repression of HxRE by Chx10 is likely to contribute to further refining V2 identity by suppressing unwanted MN-gene expression in V2 cells. Analysis of V2 specification in Chx10 mutant embryos should help examine this possibility genetically (Lee, 2008).

Overall, these studies reveal a sequential regulatory cascade of gene expression that operates to ensure the high fidelity in gene regulation required to specify two closely related, but distinct neural subtypes during vertebrate CNS development. This cascade resembles feedforward loops described in other organisms such as E. Coli, yeast, C. elegans and Drosophila. In particular, this study highlights the key role for DNA-REs in feedfoward gene regulatory loop and combinatorial transcription code, which have been underappreciated previously. Both HxRE and TeRE function as binary switches in the developing spinal cord; i.e., TeRE is off-switch in MNs and on-switch in V2-INs, while HxRE is on-switch in MNs and off-switch in V2-INs. Importantly, these strategies should reinforce the distinct gene expression outcomes in MNs and V2-INs, as expression of HxRE- and TeRE-containing genes would be precisely coregulated to opposite directions depending on the cell context. Thus, cell-type-specific DNA-RE alone is capable of decoding all the cell-fate-specifying genetic programs installed in each cell type, sensing both transcriptional activation and repression machineries. This model also predicts that a set of genes with TeRE or HxRE would be synonymously regulated during cell fate specification. Thus, the defined consensus TeRE and HxRE sequences could be useful in bioinformatics approaches to find a group of genes, which are specifically expressed in V2-INs and MNs and direct V2 and MN differentiations and maturations. Together, these findings provide a prototypic gene regulatory network for cell-type specification in development, which involves feedforward gene regulatory loops (Lee, 2008).

Hb9-MNe of Hb9 gene consists of functional HxRE and E-box elements that recruit proneural basic helix-loop-helix (bHLH) factors. MN-hexamer transcriptionally synergizes with proneural bHLH factors Ngn2 and NeuroM to fully activate Hb9 gene and subsequently specify MNs in the developing spinal cord and P19 cells. This synergistic interaction of MN-hexamer and Ngn2/NeuroM requires DNA bindings of these transcription factors in proximity. Thus, full activation of Chx10-TeRE in the neural tube may also need other transcription factors bound elsewhere in Chx10. It will be interesting to test whether V2-tetramer indeed cooperates with other transcription factors involved in V2 specification, such as Mash1, GATA2, FoxN4, or SCL, to promote V2-IN fate (Lee, 2008).

LMO4 disrupts the assembly of V2-tetramer in newborn MNs by displacing Lhx3 from NLI and suppresses V2-IN development in chick embryos. Among LMOs, LMO4 is most highly expressed in differentiating MNs in chick and mouse embryos and it binds NLI with a 2-fold higher affinity than LMO2. Thus, LMO4 is a good candidate to regulate the formation of LIM-complexes in MNs. Under the condition of 1:1 interactions, the affinities of Lhx3 and Isl1 for NLI binding are comparable, suggesting that LMO4 inhibits similarly the formation of V2-tetramer and MN-hexamer through competition for NLI binding. However, the data indicate that LMO4 functions as a selective competitor to disrupt V2-tetramer over MN-hexamer assembly. Interestingly, the binding of NLI and Isl1 without Lhx3 is also sensitive to LMO4, suggesting that the resistance of NLI:Isl1-Lhx3 binding to LMO4 is not simply due to the differences in the binding affinities between NLI:Lhx3 interaction and NLI:Isl1 interaction. Rather, the differences in the complex architecture of MN-hexamer and V2-tetramer may contribute to the distinct sensitivity of the two complexes to LMO4. Relative to V2-tetramer, MN-hexamer is a higher-order multiprotein complex. Thus, it could be more stable through multiple protein-protein interactions and less sensitive to a competitor such as LMO4. In MNs, LMO4 should increase the population of MN-hexamer, as MN-hexamer is formed at the expense of V2-tetramer. Consistently, deletion of LMO4 results in progressive increase of V2-INs. Although V2-MN hybrid cells are consistently found in LMO4 mutants, the phenotype is relatively subtle in LMO4 single mutant and greatly enhanced in LMO4:Hb9 compound mutants. Thus, LMO4 may provide a fine-tuning mechanism to control the stoichiometry of LIM-complexes in the developing spinal cord by increasing MN-hexamer concentration in MNs (Lee, 2008).

The results demonstrate that Hb9 and LMO4 cooperate to silence V2 genes. Why is the cooperative action of Hb9 and LMO4 necessary to inhibit V2 genes in MNs? LMO4 seems to function as a modulator, rather than an active MN fate selector, to promote MN-hexamer formation over other possible LIM-complexes in MNs. Likewise, although Hb9 is dominant over MN-hexamer for TeRE binding and thus blocks the access of MN-hexamer to TeRE-containing V2 genes, Hb9 may have intrinsically modest affinity to TeRE (weaker than V2-tetramer). Thus, Hb9 alone could be inefficient in blocking binding of V2-tetramer to TeRE, and may not completely shut down V2-gene expression in MNs, unless LMO4 helps Hb9 to bind TeRE more readily by destabilizing V2-tetramer, Hb9's competitor to bind TeRE. The loss of LMO4 and Hb9 in LMO4:Hb9-DKO likely permits both V2-tetramer and MN-hexamer to bind TeREs and upregulate V2 genes. As a consequence, both TeRE-containing V2 genes and HxRE-containing MN genes are activated in MNs, thereby resulting in MN-V2 hybrid cells. Thus, the functional cooperation between Hb9 and LMO4 is expected to be a critical component in the overall strategy to suppress expression of V2 genes in MNs. Together, these findings underscore the importance of actively suppressing alternative fate choice to generate correct neuronal subtype (Lee, 2008).

The data demonstrate that transcriptionally active endogenous MN-hexamer is assembled in MMCm-MNs. Interestingly, LMO4 is maintained mainly in MMCm-MNs among motor columns. Thus, the HxRE may mediate not only MN specification but also postmitotic MN diversification to MMCm cells and LMO4 may also antagonize the unwanted formation of V2-tetramer in MMCm cells. V2-INs also undergo further diversification to excitatory Chx10⁺ V2a-INs and inhibitory GATA2/3⁺ V2b-INs. Analogous to MN development, Lhx3 is maintained in V2a-INs, but extinguished in V2b-INs. Thus, TeRE activity could be maintained only in V2a subtype in which V2-tetramer is assembled. In comparison, V2b-INs express GATA2/3, SCL, and LMO4, which could assemble a transactivating complex similar to a hematopoietic complex containing NLI, GATA1, SCL, and LMO2. This raises the possibility that LMO4 might control the V2 subtype segregation by acting as an activator in V2b and a repressor in V2a (Lee, 2008).

In summary, these studies have established that subtle differences in DNA-REs can direct segregation of lineage-specific transcription pathways in the developing nervous system by concertedly mobilizing the action of transcriptional activators and repressors. This regulatory network likely represents a prototypic genetic mechanism for segregating related but distinct cell fates during the nervous system development. Importantly, this knowledge should provide a rational strategy to direct stem/progenitor cells into MNs in vitro (Lee, 2008).

The stage-dependent roles of Ldb1 and functional redundancy with Ldb2 in mammalian retinogenesis

The Lim domain binding proteins (Ldbs; see Drosophila Chip) are key cofactor proteins that assemble with LIM domains of the LMO/LIM-HD family to form functional complexes that regulate cell proliferation and differentiation throughout the CNS. Ldb1 interacts with Lhx2 (see Drosophila Apterous) in the embryonic mouse retina, and both Ldb1 and Ldb2, probably functioning with Lhx2 in a complex, play a key role in maintaining the pool of retinal progenitor cells. This is accomplished by controlling the expression of the homeodomain factors Vsx2 (see Drosophila Vsx1) and Rax, as well as components of the Notch and Hedgehog signaling pathways. Furthermore, the Ldb1/Ldb2 mediated complex, is essential for generation of early-born photoreceptors through the regulation of Rax and Crx (an Orthodenticle homolog). Finally, functional redundancy between Ldb1 and Ldb2 was demonstrated. Ldb1 can fully compensate the loss of Ldb2 during all phases of retinal development, whereas Ldb2 alone is sufficient to sustain activity of Lhx2 in both early and late-stage RPCs and in Muller glia. In contrast, loss of Ldb1 disrupts activity of the LIM domain factors in neuronal precursors. These findings uncover an intricate regulatory network mediated by Ldb1 and Ldb2 that promotes RPC proliferation and multipotency, and also controls specification of the major cell types of the mammalian retina (Gueta, 2016).

Ldb1 is essential for development of Nkx2.1 lineage derived GABAergic and cholinergic neurons in the telencephalon

The progenitor zones of the embryonic mouse ventral telencephalon give rise to GABAergic and cholinergic neurons. Two LIM-homeodomain (LIM-HD) transcription factors, Lhx6 and Lhx8, that are downstream of Nkx2.1, are critical for the development of telencephalic GABAergic and cholinergic neurons. This study investigated the role of Ldb1, a nuclear protein that binds directly to all LIM-HD factors, in the development of these ventral telencephalon derived neurons. Ldb1 is expressed in the Nkx2.1 cell lineage during embryonic development and in mature neurons. Conditional deletion of Ldb1 causes defects in the expression of a series of genes in the ventral telencephalon and severe impairment in the tangential migration of cortical interneurons from the ventral telencephalon. Similar to the phenotypes observed in Lhx6 or Lhx8 mutant mice, the Ldb1 conditional mutants show a reduction in the number of both GABAergic and cholinergic neurons in the telencephalon. Furthermore, defects were shown in the development of the parvalbumin-positive neurons in the globus pallidus and striatum of the Ldb1 mutants. These results provide evidence that Ldb1 plays an essential role as a transcription co-regulator of Lhx6 and Lhx8 in the control of mammalian telencephalon development (Zhao, 2014).

Crystal structure of human LDB1 in complex with SSBP2

The Lim domain binding proteins (LDB1 and LDB2 in human and Chip in Drosophila) play critical roles in cell fate decisions through partnership with multiple Lim-homeobox and Lim-only proteins in diverse developmental systems including cardiogenesis, neurogenesis, and hematopoiesis. In mammalian erythroid cells, LDB1 dimerization supports long-range connections between enhancers and genes involved in erythropoiesis, including the beta-globin genes. Single-stranded DNA binding proteins (SSBPs) interact specifically with the LDB/Chip conserved domain (LCCD) of LDB proteins and stabilize LDBs by preventing their proteasomal degradation, thus promoting their functions in gene regulation. The structural basis for LDB1 self-interaction and interface with SSBPs is unclear. This study reports a crystal structure of the human LDB1/SSBP2 complex at 2.8-Å resolution. The LDB1 dimerization domain (DD) contains an N-terminal nuclear transport factor 2 (NTF2)-like subdomain and a small helix 4-helix 5 subdomain, which together form the LDB1 dimerization interface. The 2 LCCDs in the symmetric LDB1 dimer flank the core DDs, with each LCCD forming extensive interactions with an SSBP2 dimer. The conserved linker between LDB1 DD and LCCD covers a potential ligand-binding pocket of the LDB1 NTF2-like subdomain and may serve as a regulatory site for LDB1 structure and function. This structural and biochemical data provide a much-anticipated structural basis for understanding how LDB1 and the LDB1/SSBP interactions form the structural core of diverse complexes mediating cell choice decisions and long-range enhancer-promoter interactions (Wang, 2019).

REFERENCES

Search PubMed for articles about Drosophila Chip

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