Interactive Fly, Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

Protein Interactions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

CtBP and Hairy

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

CtBP and Brinker

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

CtBP and Teashirt

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

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

CtBP and Hairless

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

CtBP and Snail

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

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

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

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

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

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

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

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

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

YY1 DNA binding and PcG recruitment requires CtBP

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Hairless induces cell death by downregulation of EGFR signalling activity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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