Krüppel

Protein Interactions

Kruppel forms a bell-shape concentration gradient in a central position of the blastoderm. Genetic and molecular studies suggest that the Kr protein may act both as a positive and a negative regulator of transcription on several other genes of the zygotic segmentation hierarchy. The regulatory potential of Kr has been examined by a series of cotransfection experiments in the Drosophila Schneider cell line system. Different doses of Kr expression plasmid were tested for their ability to drive reporter gene expression mediated by a single 11-base pair Kr in vitro binding site common to several putative Kr target genes. Low amounts of Kr expression plasmid lead to transcriptional activation, whereas high amounts result in repression. Distinct portions of Kr, other than the DNA-binding domain, are required for gene activation and repression, suggesting that Kr itself can act as a concentration-dependent positive and negative regulator of transcription (Sauer, 1991).

Kruppel can both activate and repress gene expression through interaction with a single DNA-binding site. The opposite regulatory effects of Kf are concentration-dependent, and they require distinct portions of Kr such as the N-terminal region for activation and the C-terminal region for repression. Kr is able to form homodimers through sequences located within the C terminus. Dimerization requires Kruppel's 64 most C-terminal amino acids. When these sequences are fused to separated functional parts of the yeast transcription factor GAL4, they reconstitute a functional transcriptional activator upon dimerization in vivo. These results suggest that the Kr monomer is a transcriptional activator. At higher concentrations, Kr forms a homodimer and becomes a repressor that functions through the same target sequences as the activator (Sauer, 1993).

KR can associate with the transcription factors encoded by the gap genes knirps and hunchback which affect KR-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound HB protein or free KNI protein with distinct but different regions of KR results in the formation of DNA-bound transcriptional repressor complexes (Sauer, 1995a).

Krüppel can repress transcriptional activation by acting at a distance. More specifically, the N terminal repression domain of Krüppel can effectively silence activation by SP1, a glutamate rich activator, by directly interacting with SP1. KR does not work at a distance with the acidic activators (Licht, 1993).

Monomeric Kr can act as a transcriptional activator, while KR dimers formed at high concentration cause repression. KR-dependent control of transcription involves functional interactions with the basal transcription machinery. When ating from a site close to a basal promoter, monomeric KR interacts with TFIIB to activate transcription, while an interaction of the KR dimer with TFIIEß, a subunit of TFIIE, results in transcriptional repression. KR provides mainly, if not exclusively, repression through distant enhancers when acting within the segmentation gene cascade, possibly involving a quenching mechanism (Sauer, 1995b).

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

To assess the significance of the P-DLS-H motif contained in a repression domain in a C-terminal region of Krüppel, the activities were examined of a Gal4-Krüppel fusion protein that contains the C-terminal 101 amino acids residues from Krüppel. The chimeric coding sequence was expressed in ventral regions of transgenic embryos under the control of the twist promoter region. A lacZ reporter gene was introduced into embryos expressing this fusion protein. The reporter gene contains the eve stripe 2 and stripe 3 enhancers, and normally, it exhibits equally intense expression in both dorsal and ventral regions. The distal stripe 2 enhancer contains two tandem Gal4-binding sites (UAS), and when the lacZ reporter gene is crossed into embryos expressing the twist-gal4/Krüppel vector, it is repressed in the ventral mesoderm. The introduction of just three amino acid substitutions in the P-DLS-H motif (PEDLSMH to AAALSMH) eliminates the repression activity of the Krüppel fusion protein. The same substitutions also eliminate interactions between Krüppel and dCtBP in vitro. Similar results were obtained when the Gal4-Krüppel fusion protein was expressed in central regions of transgenic embryos using the Krüppel promoter region. The lacZ reporter gene used to assess the activity of this expression vector contains a modified rhomboid lateral stripe enhancer placed upstream of the proximal enhancer from the twist promoter. Normally, the reporter gene is expressed in lateral lines (mediated by the modified rhomboid enhancer) and the ventral mesoderm (twist enhancer). However, there is a gap in the lateral lines when the reporter gene is crossed into embryos expressing the Krüppel-gal4/Krüppel expression vector. This gap results from the binding of the Gal4-Krüppel fusion protein to UAS sites in the distal rhomboid enhancer. The gap is lost with a mutant fusion protein containing amino acid substitutions in the P-DLS-H motif, thereby indicating the importance of this motif in vivo (Nibu, 1998).

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

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

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

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

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

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

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

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

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

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

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

Krüppel and Snail can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. When located within an upstream enhancer, the repressor locally quenches nearby activators (by preventing them from binding or masking their activation surfaces) and permits other enhancers (acting from a considerable distance from a local repressor) to interact with the transcription complex, thus demonstrating enhancer autonomy. In contrast, when bound to promoter-proximal regions, the repressor functions in a dominant fashion and blocks multiple enhancers. Local quenching and dominant repression require close linkage (less than 100 base pairs) of the repressor within either upstream activators or in a proximal promoter adjacent to the the transcription complex. SNA acts on a 300bp rhomboid neuroectodermal enhancer, acting in a competition mechanism to prevent Dorsal activation, but SNA fails to prevent activation when SNA repressor sites are moved away from the closest activators. Likewise KR can repress DL activation of a rhomboid enhancer by a locally acting quenching mechanism (Gray, 1996).

It is conceivable that KNI, and other short-range repressors such as Krüppel and Snail recruit 'corepressors', which cause local changes in chromatin structure (e.g. positioning a nucleosome) or in some other way interfere with access to the DNA by activators or basal transcription factors. Alternatively, short-range repressors do not interact with neighboring activators, but instead might 'hitchhike' with neighboring activators, looping to contact the basal promoter, and then inhibit components of the transcription complex (Arnosti, 1996).

The general transcription factor TFIIE plays important roles at two distinct but sequential steps in transcription: the preinitiation complex formation and activation (open complex formation), and the transition from initiation to elongation. The large subunit of human TFIIE (TFIIEalpha) binds to and facilitates the enzymatic functions of TFIIH, but TFIIE also functions independent of TFIIH. To determine functional roles of the small subunit of human TFIIE (TFIIEbeta), deletion mutations were systematically introduced into putative structural motifs and characteristic sequences. All of these structures that lie within the central 227-amino acid region of TFIIEbeta are necessary and sufficient for both basal and activated transcription. Two C-terminal basic regions are essential for physical interaction with both TFIIEalpha and single-stranded DNA, as well as with other transcription factors, including the Drosophila transcriptional regulator Kruppel. In addition, the effects of the TFIIEbeta deletion mutations were analyzed on TFIIH-dependent phosphorylation of the C-terminal domain of RNA polymerase II and on wild type TFIIEbeta-driven basal transcription. Both responsible regions also map within the essential 227-amino acid region. These results suggest that TFIIE engages in communication with both transcription factors and promoter DNA via the TFIIEbeta subunit (Okamoto, 1998).

Drosophila mediator complex is used by Krüppel

To decipher the mechanistic roles of Mediator proteins in regulating developmental specific gene expression and compare them to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates promoter-specific activation signals to the basal transcription machinery (Park, 2001).

Previous studies in yeast and human cells have suggested that transcriptional activator proteins interact with Mediator complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes with the VP16 protein were examined. After incubation of nuclear extracts with an excess of GST fusion protein beads containing either wild-type or mutant (Delta456FP442) VP16 activation domain, the supernatants were analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating with GST-VP16 but not with GST-VP16^{Delta456FP442}. However, the amounts of dGCN5, dTAF_II40, dTAF_II250, and dTBP in the extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most strongly bound to and most readily recruited to the activation domain (Park, 2001).

In addition to the model VP16 activator derived from herpesvirus, dMediator interacts with Drosophila transcriptional activators Dorsal and heat shock factor (dHSF). When dMediator complex was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein G-agarose beads through anti-dSOH1 Ab and the binding of diverse ³⁵S-labeled Drosophila transcription factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).

To evaluate the requirement of dMediator for activated transcription in response to the Drosophila activator proteins that interact with dMediator, the ability of dMediator-deficient nuclear extracts to support transcriptional activation by the Dorsal and Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level of transcriptional activation is reduced significantly (five- and three-fold activations, respectively) in nuclear extract that has been depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by the sequence-specific transcription factor Even-skipped (Park, 2001).

dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer elements. However, the requirement of TAFs, or at least dTAF_II250, in activated transcription appears to be redundant in the in vitro transcription system used and affected by such factors as the core promoter organization or nucleosomal structure of transcriptional templates. Several TAF components in the TFIID complex indeed have biochemical activities and structural motifs adequate for the recognition of specialized settings of transcription templates. For example, certain TAFs recognize the Inr and DPE sequences located in many Drosophila core promoters and increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAF_II250, which has a HAT catalytic activity and also possesses a histone octamer-like module comprising the histone H2B-, H3-, and H4-like TAFs. Although not experimentally demonstrated, these TAFs may have some roles in the transcriptional regulation of nucleosomal templates (Park, 2001).

The sequence-specific transcription factors which interact physically with dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and Fushi-tarazu. These factors contain different types of activation domains (acidic and glutamine-rich domains). Most of these transcription factors have been shown to activate transcription either constitutively or inducibly. It is noteworthy that dHSF interacts with and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170), suggests that some function of Mediator is required for HSF-mediated transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is conserved between yeast and Drosophila, transcriptional activation by HSF might utilize the conserved Rgr1 components of the Mediator complexes (Park, 2001).

Although some human Mediator complexes appear to have a negative effect on activated transcription, dMediator does not exhibit such an activity in an in vitro transcription system reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is more plausible that the dMediator-Krüppel interaction observed is a part of the mechanism for transcriptional activation rather than transcriptional repression. Taken together with the fact that dMediator is dispensable for basal transcription, the lack of defect of the dMediator-depleted nuclear extracts on transcriptional repression by Even-skipped protein suggests that dMediator is required mainly for the mediation of transcriptional activation signals to the basal transcription machinery. Very recently, developmental roles of certain dMediator proteins found in the Drosophila genome database have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).

Like yeast Mediator, dMediator bind with the CTD repeats of Drosophila Pol II. This implies that though dMediator was purified separately from Pol II, these two complexes indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS. Such interactions may be involved in the regulation of Pol II preinitiation complex assembly. Related with this idea, it has been reported that in yeast, recruitment of general transcription factors such as TBP, TFIIB, and TFIIH to active promoters requires the function of Mediator. Also, TFIIE interacts with the Mediator protein Gal11. Further analyses will be required to clarify whether these interactions, observed both in yeast and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).

dMediator contains the protein kinase component Cdk8, which can phosphorylate serine residues in the CTD. This catalytic kinase subunit seems responsible, at least in part, for the Pol II phosphorylation by dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II repeats, suggesting the presence of a functional interaction between these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).

CtBP is essential when Krüppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation

Body pattern formation during early embryogenesis of Drosophila relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. This study identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation (Matyash, 2009).

This study provides evidence that Kr exerts transcriptional repression not only by association with the corepressor dCtBP but also by site-specific deacetylation of histones, a mechanism that involves an interaction between Kr and dSAP18. The dual mode of Kr-dependent repression might explain earlier studies showing that Kr represses eve stripe 2 expression, but not h stripe 7 expression, in a dCtBP-dependent manner. Consistent with these observations, a mutant Kr protein that lacks dCtBP-binding sites still associates with dSAP18, which in turn interacts with the Sin3A-HDAC1 repressor complex (Drosophila HDAC1 is Rpd3). dSAP18 was also shown to bind the homeodomain transcription factor Bicoid, causing repression of anterior gap genes such as hunchback in the late Drosophila blastoderm embryo. SAP18-dependent repression involves histone deacetylase both in flies and mammals, and SAP18 that links the HDAC1 complex with sequence-specific transcriptional repressors bound to chromatin is also found in plants. These results are consistent with such a SAP18-dependent mode of Kr-dependent repression that provides target gene-specific repression. Because both dCtBP and SAP18 are uniformly distributed in the embryo, it will be important to learn how the eve stripe 2 and the h stripe 7 enhancer distinguish between the dCtBP- or SAP18-dependent modes of repression. One possibility is that differential packing of the enhancer DNA into nucleosomes might account for the difference in susceptibility to the SAP18/HDAC1-mediated repression (Matyash, 2009).

dSAP18 binds to three distinct regions of Kr, including the 42-amino acid-long repressor region, which is conserved in Kr homologs of all Drosophila species. However, as observed for dCtBP, dSAP18 alone cannot account for Kr-dependent repression of h7-lacZ, because prolonged expression of Kr is able to overcome the lack of dSAP18 activity as observed for the h7 element in dSAP18 mutants. Therefore, it is likely that the full spectrum of Kr-dependent repression is mediated redundantly, employing at least two different corepressors that involve different modes of repression (Matyash, 2009). In vitro, dSAP18 binds to the sequence motif ³⁴⁴RRRHHL³⁴⁹ of Kr and to a similar motif (¹⁴³RRRRHKI¹⁴⁹) of Bicoid; the latter is consistent with the results reported by Zhu (2001). In both proteins, the dSAP18-binding sites are localized in the C-terminal portion of their DNA-binding domains. Thus, when acting from weak binding sites in vivo, transcription factors might be able to form strong complexes with dSAP18. In fact, Bicoid-dependent repression of hunchback, which depends on both SAP18 and HDAC1 (Singh, 2005), occurs only at the very anterior tip of blastoderm embryos where the Bicoid concentration is highest and the target gene enhancers contain multiple weak Bicoid-binding sites (Matyash, 2009).

dSAP18 also interacts with the histone-specific H3K27 methyl-transferase E(z) (Enhancer of zeste) (Wang, 2002), a component of the polycomb group protein complex, and with the GAGA factor, a transcription factor of the trxG (trithorax group) protein complex. Thus, dSAP18 is capable of interacting with two regulatory protein complexes that have antagonistic functions in gene regulation. Whereas the polycomb group complex acts as a repressor of homeotic genes in ectopic locations, the trxG complex is required for activation and maintenance of their transcription. However, this clear-cut distinction between polycomb group and trxG functions has been questioned, because polycomb group and trxG group members were shown to act both as context-dependent repressors and activators of transcription, and factors with such dual functions include both the E(z) and GAGA factor proteins. In fact, interactions between dSAP18 and GAGA factor at the iab-6 element of the bithorax complex, for example, were shown to cause transcriptional activation and not repression (Matyash, 2009).

This study suggests that Kr mediates repression through at least two pathways involving either dCtBP or SAP18. dCtBP-dependent and -independent repression of the transcription factors Knirps and Hairless exert quantitative effects, whereas Kr distinguishes dCtBP and dSAP18 recruitment at different enhancers. It was observed, however, that the loss of SAP18 activity does not affect the pattern of eve stripe expression and that prolonged Kr can suppress h7-lacZ expression in the absence of dSAP18. Thus, although both dSAP18 and dCtBP act independently from each other, the two corepressors, or other yet unknown corepressors, can functionally substitute for each other under forced conditions. However, their mode of repression appears to involve different mechanisms. One mechanism is exemplified by the dCtBP-dependent repression of eve-stripe 2 and not yet established at the molecular level. dCtBP-dependent repression does not act via unleashing local heterochromatization, does not require dHDAC1 activity, and is insensitive to the HDAC inhibitor TSA. Consistently, coimmunoprecipitation studies failed to detect HDAC activity in the dCtBP immunoprecipitates, histone H3 remained acetylated in dCtBP-deficient embryos, and transcription was not repressed. Other studies, however, implied an association of dCtBP with HDACs. Thus, the mechanism of the dCtBP mode of repression is not yet fully understood (Matyash, 2009).

The results of this study showing a lack of H3 deacetylation at the eve stripe 2 enhancer in response to Kr repression are consistent with the argument that eve stripe 2-mediated repression involves the corepressor CtBP. The second, dCtBP-independent mode of Kr-dependent repression, as exemplified by the h stripe 7 element (and possibly also eve stripes 1, 3, and 4) does require both dSAP18 and HDAC1 activities. In support of this mode of repression, the following phenomena were observed in Kr-overexpressing embryos (1) a dSAP18-dependent loss of K^9,14H3 acetylation on the h stripe 7 element, (2) an increased resistance of the h7 enhancer DNA to sonication, and (3) SAP18-dependent repression of the h7 reporter gene in response to Kr activity. These Kr-dependent effects were dependent on HDAC1 enzymatic activity as revealed by experiments using the HDAC1 inhibitor, TSA. These results therefore suggest that dSAP18-dependent repression by Kr involves structural changes of chromatin, such as compaction or condensation, likely to be caused by site-specific heterochromatization in response to enhancer-specific HDAC1 activity (Matyash, 2009).

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