Hairless

Targets of Activity

To examine the link between the Hairless misexpression phenotypes and Su(H)-dependent Notch activity, transgenic animals were generated carrying a lacZ reporter construct driven by the fusion between multimerized Su(H)-binding sites and an E(spl)mg promoter, a known Su(H) target. This construct consists almost exclusively of engineered Su(H)-binding sites. In a cell culture based reporter, the expression from the reporter construct is induced by the simultaneous expression of Su(H) and act.N, while the expression of any one construct alone fails to induce transcription. Strong lacZ expression is detected in the posterior part of the eye disc of late third instar transgenic larva. This expression is effectively suppressed by misexpression of H using the GAL4 line T113 and results in small eye discs, indicating that H overexpression can suppress Su(H)-dependent Notch signaling in vivo. The size of the eye is significantly affected and, in extreme cases, the eye is missing. In addition to small eyes, small wings and halteres are observed as well as more typical Notch loss-of-function phenotypes, such as extra thoracic bristles. The 'small eye' phenotype induced by H expression is not associated with severe eye roughness. This 'small eye' phenotype, together with the wing and haltere abnormalities, is reminiscent of Serrate loss-of-function mutations. To further explore the possibility that the observed eye phenotype reflects Ser-dependent Notch signaling, the genetic interactions were examined with Beaded of Goldschmidt (BdG), a dominant negative mutation of Ser known to affect wing margin development. In combination with BdG, strong synergistic effects are observed displaying phenotypes characteristic of Ser, such as small eyes, wings and halteres. Therefore, H misexpression can mimic Ser loss-of-function mutations, raising the possibility that Ser/Notch signaling may control eye morphogenesis (Go, 1998).

To further investigate the role Notch signaling plays in morphogenesis, the H and dominant negative N (d.n.N) transgenes were expressed at the dorsal/ventral (DV) compartment boundary of the wing disc using the vestigial-GAL4 driver. Misexpression of either H or d.n.N results in similar phenotypes, which range from wing margin notches to rudimentary wings. The effect of H misexpression can be suppressed by expressing activated Notch (act.N) and vice versa. For example, the lethality associated with misexpression of act.N is suppressed by simultaneous expression of H. Conversely, the phenotypes elicited by H misexpression are largely suppressed by act.N. This mutual suppression is observed with other GAL4 lines as well. Given that the actions of act.N and H seem to be manifested through Su(H), it is likely that the mutual suppression of act.N and H is also mediated by Su(H). It is noteworthy that, even though both H and d.n.N act as antagonists of Notch signaling and the phenotypes associated with their expression are similar, their interactions with act.N are different. While act.N is an effective suppressor of the phenotypes induced by H misexpression, it fails to suppress the effects of d.n.N (Go, 1998).

The relationship between Notch signaling and the expression of vg and wingless was examined, since the induction of both genes is considered to be essential for wing morphogenesis. When either d.n.N or H is misexpressed along the anterior/posterior (AP) boundary using the ptc-GAL4 line, expression from the vg DV boundary enhancer, as well as the wg enhancer, is effectively repressed near the intersection of the AP and DV boundaries. In contrast, the vg quadrant enhancer, which is normally silent at the intersection of the AP and DV boundaries, is induced by the identical constructs. Essentially the opposite effect is observed when act.N is misexpressed, demonstrating that Notch signaling has opposite effects on two distinct enhancers of vg (Go, 1998).

The effect of Notch activity on cell proliferation is not the simple consequence of vg induction Since vg is a direct target of Su(H)-dependent Notch signaling, it is possible that the mitogenic effect of Notch is mediated by the upregulation of vg. In this case, misexpression of Vg would be expected to result in phenotypes similar to those elicited by act.N. Misexpression of act.N in the dorsal side of the wing pouch, using the GAL4 line A9, induces expression from the vg DV boundary enhancer as well as the wg enhancer. The dorsal side of the wing pouch region appears enlarged. In contrast, when Vg is misexpressed in the same region, the dorsal side of the wing pouch becomes much smaller than the ventral side, while wg expression in the periphery of the dorsal side is suppressed. The loss of dorsal wing pouch induced by Vg misexpression is significantly rescued by expressing Wg simultaneously. This is consistent with the notion that the observed phenotype caused by misexpression of Vg is due to the repression of wg, whose expression in the wing pouch is more uniform at earlier stages. Misexpression of Wg alone on the dorsal side, unlike the misexpression of act.N, does not have a significant effect on cell proliferation in the wing pouch. These results indicate that the effect of act.N expression on mitosis is separable from vg induction. In addition, the results indicate that Vg is capable of repressing wg expression in the wing pouch, but not at the DV boundary (Go, 1998).

Misexpression of Vg compared to act.N has opposite effects in the wing disc. Thus, Vg misexpression in the wing disc induces wg downregulation and small discs. In contrast, misexpression of Vg in the eye discs upregulates wg and results in a clear enlargement of the discs, demonstrating that Vg can either repress or induce wg expression in a context-dependent manner. The observed context-dependent effect of Vg on wg expression raises the possibility that Notch signaling may be capable of modulating the way Vg affects wg expression. This is of particular interest in view of the possibility that Vg does not suppress wg expression at the DV boundary because of the existing high level of Notch signaling activity. In fact, the simultaneous expression of act.N and Vg reveals a striking synergistic effect on cell proliferation. The most notable effects are in the eye discs, where tissue expressing the two proteins shows striking overgrowth associated with strong wg induction. The other discs are also clearly affected, displaying cellular overgrowth, but the effects are far less dramatic than in the eye discs. This overgrowth phenotype is also evident when act.N and Vg misexpression are driven by dpp-GAL4, even though the synergistic effect is less dramatic. In contrast, the effect of misexpression of Vg with dpp-GAL4 on wg induction and cell proliferation in the eye discs is, in some cases, significantly suppressed by simultaneous expression of H. These experiments demonstrate that the proliferative potential of certain tissues can be modulated by the synergistic action of Notch with other genes. Moreover, Notch signaling has now been identified as an important factor in the way Vg affects wg expression and cell proliferation at the DV boundary during wing morphogenesis (Go, 1998).

Two isoforms of Hairless are produced by differential translation initiation

Notch signals need to be tightly regulated, and in Drosophila they are antagonized by the Hairless (H) protein. H silences the activity of Notch target genes by transforming the Drosophila CSL protein, Suppressor of Hairless [Su(H)], from a transcriptional activator into a repressor while recruiting one of the corepressors CtBP or Groucho. The H protein has a calculated molecular mass of approximately 110 kDa and contains several functional domains apart from the two small corepressor-binding domains. However, although there is no indication for alternative splicing, two Hairless protein isoforms, H(p120) and H(p150), are observed throughout development. The smaller isoform derives from an internal ribosome entry site (IRES) within the ORF. The IRES is active in a heterologous assay and contains an essential, conserved structural element. The two Hairless isoforms have residual activity in vivo which is, however, reduced compared to a combination of both, which implies that both protein isoforms are necessary for WT function. In larval tissues, translation of the two isoforms is cell-cycle regulated: whereas the H(p150) isoform is translated during interphase, H(p120) is enriched during mitosis. Thus, the presence of either H isoform throughout the cell cycle allows efficient inhibition of Notch-regulated cell proliferation (Maier, 2002).

During all stages of the Drosophila life cycle except oogenesis, two H isoforms are produced, the long H^p150 isoform from the second M2, and less efficiently, from the first start codon M1 and the short H^p120 isoform from the third start codon M3. Strong evidence is provided that translation from M3 is directed by internal ribosome entry and not by cleavage of the longer protein isoform. In the latter case, no H^p120 isoform would be expected if M1 and M2 are mutated because translation should not occur at all; and secondly, a frame-shift construct should only provide the N-terminal peptides. The frame shift construct also excludes a leaky scanning mechanism, whereby the ribosome would pass by earlier start codons. Here, translation would terminate 45 codons behind M3 and 20 codons before the next in-frame start codon M4. Because backward scanning is less likely, the more closely located M4 should be preferred, which is not the case. Therefore, leaky scanning might lead to the production of H^p120 from a DeltaM1/M2 construct, but not from the frame shift construct (Maier, 2002).

Could the two isoforms possibly derive from different transcripts? The data provide good evidence to exclude both a hidden promoter for a shorter transcript as well as alternatively spliced mRNAs. Since both H isoforms appear at fairly constant ratios, the corresponding mRNAs should be as frequent, but neither a shortened mRNA nor alternative splice products have been isolated. Moreover, the M2-M3 interval contains no obvious candidate sequence elements. There are just five in frame AG dinucleotides, one of which lies between boxA and M3, but none could serve as splice acceptor lacking the relevant polypyrimidine stretch. Not surprisingly, a dicistronic construct without actin promoter shows rather low luciferase activity. Also, in vitro transcription of the full-length H cDNA by a T3 promoter still gives rise to both H isoforms. Leaky scanning in this case can again be excluded by the fact that mutation of boxA prevents H^p120 production. Finally, ectopic induction of the WT transcripts from a heat-inducible promoter gives rise to both isoforms in the expected ratio (Maier, 2002).

Other dipteran flies also show two H protein isoforms, and the largely diverged species Drosophila hydei produces two H proteins only slightly bigger than the Drosophila melanogaster proteins. In D. hydei, the third methionine is well conserved, and both isoforms are also generated in an in vitro transcription/translation assay, suggesting internal translation initiation as well. Quite strikingly, the H-IRES shares similarity with IRESs from the human ODC gene and from picorna viruses regarding the pyrimidine-rich region harboring the A-box in close proximity of the actual ribosome entry site. The A-box is essential for H-IRES recognition. In addition, there is a conspicuous Y-loop of 85 nt located 200 bases upstream of M3. Whether this structure, which is reminiscent of other cellular IRES sequences including that of the Drosophila Antp gene, is a relevant feature of the H-IRES remains to be determined (Maier, 2002).

Translation from the H-IRES results in a protein which is ~20% shortened at the N terminus. The N terminus itself seems not to contain a specific functional domain, as either isoform retains some WT activity. However, only constructs that provide both isoforms have normal activity, whereas those that provide only one isoform are significantly less active. This observation strongly implicates that both H isoforms are required together. Two, nonexclusive scenarios can be envisaged to accommodate this observation. First, H might act as a homo- or heterodimer (or multimer) involving the two isoforms, and heteromers might be functionally distinct from homomers. Second, H activity might also be required during times when normal translation is inhibited, like in cellular stress situations and/or during the mitotic phase of the cell cycle (Maier, 2002).

It is generally accepted that IRES sequences allow for cap-independent translation through G₂/M phase, when cap-dependent translation is inhibited. Several cases were described where an IRES directs cell-cycle-dependent translation of the respective mRNA, suggesting that this might represent a general paradigm. Lacking the possibility of synchronizing Drosophila cell cultures, whole larval tissue were used to address this question in situ. In agreement with the above idea, the H^p120 isoform, which is initiated from the IRES, is specifically enriched in mitotic cells that largely exclude the H^p150 translation product. This experiment was only possible by taking advantage of anti-A antisera, which allow the distinction between WT and overexpressed H protein. As cell-cycle-dependent differential stability of the two H isoforms seems not very likely, the idea is favored that H^p120 is translated during mitotic phases when H^p150 cannot be produced. In Drosophila, other functional IRES sequences have been identified before, but not with regard to cell-cycle-regulated translation. It will be interesting to see whether all of these mRNAs show a biphasic translation pattern also in Drosophila. After all, one might expect many more genes to contain IRES sequences if they need to be active throughout the cell cycle (Maier, 2002).

H acts as an antagonist of many Notch-dependent processes during Drosophila imaginal development including the regulation of cell proliferation. Overexpression of the activated Notch receptor results in profound overproliferation of a variety of tissues during imaginal development of Drosophila. This overgrowth is, in part, a result of the activation of Notch target genes, which themselves promote tissue growth, e.g., of vestigial or of the morphogen wingless. In mammals, Notch has been implicated more directly in the regulation of cell proliferation as a number of neoplasms involve ectopic Notch activation. Many genes involved in tumorigenesis are regulated at the translational level. Although not directly demonstrated for Notch, recent work suggests that the activated form of the Notch receptor might be generated independently of signaling by internal ribosome entry. It is tempting to speculate that this process is under cell-cycle control, and that H is regulated alike to antagonize the Notch pathway properly (Maier, 2002).

Protein Interactions

Su(H) binds directly to the ankyrin repeats of the intracellular domain of Notch. When Delta expressing cells interact with Notch expressing cells, cytoplasmic Notch-Su(H) interaction is relieved by direct interaction of Notch with Deltex, and Su(H) is subsequently detected mostly in the nucleus (Fortini, 1994). Hairless gene product can inhibit the DNA binding of Su(H) through direct protein-protein interaction. Thus Hairless acts as a negative regulator of Su(H) activity (Brou, 1994).

Hairless, a major antagonist of the Notch signaling-pathway in Drosophila, associates with Suppressor of Hairless [Su(H)], thereby inhibiting trans-activation of Notch target genes. These molecular interactions could occur either at the step of signal transduction in the cytoplasm or during implementation of the signal within the nucleus. The subcellular distribution of Hairless was examined. Hairless is a low abundant, ubiquitous protein that is cytosolic as well as nuclear. High levels of Hairless cause nuclear retention of Su(H); loss of Hairless reduces the amount of Su(H) in the nucleus (Maier, 1999).

In order to examine the Hairless expression pattern, antisera were raised that were directed against, respectively, the central portion of Hairless, termed anti-A, and the C-terminal part, termed anti-B. Upon forced expression, both antisera detect ectopic Hairless with high specificity almost exclusively within nuclei. Induction of Hairless from a heat shock controlled transgene results in a strong nuclear accumulation of Hairless in somatic tissues, like the follicle cells of ovaries. Nuclear accumulation of Hairless is also observed, when ectopic expression is forced from a UAS-H transgene in a pair rule pattern in the embryo or along the antero-posterior boundary in wing imaginal discs. Thus, although anti-A does not recognize nuclear Hairless in wild type, it does detect ectopically expressed Hairless in a sub-cellular distribution identical to that of anti-B. This implies that nuclear Hairless at wild type levels is modified or bound by another nuclear protein, which is present in limiting amounts and able to mask the anti-A epitopes upon binding to Hairless. Su(H) has been shown previously to bind to Hairless in the vicinity of the A-domain. It was then asked whether the anti-A epitope of Hairless is demasked in cells mutant for Su(H). However, in embryos devoid of Su(H) protein, anti-A exclusively detects cytoplasmic Hairless. The same is observed in Su(H) clones induced in imaginal discs. Thus, Su(H) binding plays no major role in the recognition of Hairless by anti-A, and it remains undecided, whether masking of Hairless by protein(s) other than Su(H) or its modification accounts for the differential recognition by the two antisera (Maier, 1999).

The reverse experiment was performed, asking whether Su(H) protein distribution depends on Hairless. Mutant cell clones devoid of Hairless show a markedly reduced amount of nuclear Su(H) protein. Conversely, ectopic expression of Hairless causes Su(H) to accumulate strongly in the nuclei of Hairless overexpressing cells. Calculations on Su(H) distribution show that this accumulation is not simply due to an overall increase of Su(H) in the Hairless expressing cells but mainly caused by an increased nuclear vs. cytoplasmic ratio, compared to the surrounding wild type cells. In situ hybridization of such discs with a Su(H) probe reveals no ectopic Su(H) transcription. Thus, the increase of nuclear Su(H) in response to Hairless must occur primarily at the level of protein. As a result of the ectopic Hairless expression along the antero-posterior border, adult wings are notched at the tip, resulting from interference of Hairless with the Notch-dependent process of wing margin formation (Maier, 1999).

Hairless subcellular distribution is consistent with antagonizing Su(H) activity in either compartment. The finding that levels of nuclear Su(H) accumulation are linked to Hairless levels places this interaction within the nucleus. It remains for future studies to determine whether nuclear retention prevents Su(H) from shuttling back to the membrane, recharging another Notch receptor or whether nuclear accumulation reflects a dual function of Su(H) in Drosophila, as suggested for the mammalian homolog, which acts in repression and activation of Notch target genes (Maier, 1999).

Hairless is a member of the Notch signaling pathway, where it acts as antagonist by binding to Suppressor of Hairless [Su(H)], thereby inhibiting Notch target gene activation. The pathway and its members are highly conserved in metazoans from worms to humans. However, a Hairless ortholog from another species has not yet been identified. The identification of Hairless in largely diverged species by cross-hybridization has failed so far, probably due to a low degree of conservation. Therefore, D. hydei Hairless, where a Hairless mutation has been described, was examined. The D. hydei Hairless ortholog is reasonably well conserved with regard to gene structure and expression (69% identity). The prospective Hairless protein orthologs share several highly conserved regions that are separated by quite diverged stretches. As is to be expected, the largest region of high conservation corresponds to the Su(H) binding domain. This region is also functionally conserved, since this D. hydei protein domain binds very strongly to the D. melanogaster Su(H) protein. The other conserved regions support structure-function analysis since these regions nicely correspond to previously defined, functionally important protein domains. Most notably, the very most C-terminal domain, which is very sensitive to structural alterations, is nearly identical between the two species. In summary, this evolutionary study improves the knowledge on functionally significant domains of the Hairless protein, and may be helpful for the future identification of homologs in other animals, especially in vertebrates (Marquart, 1999).

Two different activities of Suppressor of Hairless during wing development in Drosophila

The Notch pathway plays a crucial and universal role in the assignation of cell fates during development. In Drosophila, Notch is a transmembrane protein that acts as a receptor of two ligands, Serrate and Delta. The current model of Notch signal transduction proposes that Notch is activated upon binding its ligands and that this leads to the cleavage and release of its intracellular domain (also called Nintra). Nintra translocates to the nucleus where it forms a dimeric transcription activator with the Su(H) protein. In contrast with this activation model, experiments with the vertebrate homolog of Su(H), CBF1, suggest that, in vertebrates, Nintra converts CBF1 from a repressor into an activator. The role of Su(H) in Notch signaling during the development of the wing of Drosophila has been assessed. The results show that, during this process, Su(H) can activate the expression of some Notch target genes and that it can do so without the activation of the Notch pathway or the presence of Nintra. In contrast, the activation of other Notch target genes requires both Su(H) and Nintra, and, in the absence of Nintra, Su(H) acts as a repressor. The Hairless protein interacts with Notch signaling during wing development and inhibits the activity of Su(H). These results suggest that, in Drosophila, the activation of Su(H) by Notch involves the release of Su(H) from an inhibitory complex, which contains the Hairless protein. After its release Su(H) can activate gene expression in the absence of Nintra (Klein, 2000).

Since Su(H) is expressed ubiquitously and continuously, the fact that Su(H) can promote transcription without the presence of Nintra suggests that the activity of Su(H) must be suppressed in the absence of Notch activation during normal development. One possible mechanism for this is the binding of an inhibitory factor. A candidate for this function is the Hairless (H) protein, which has been shown to interact with Su(H) and antagonize its DNA-binding activity. Furthermore, several reports show that H antagonizes Notch signaling during adult PNS development. To test whether H is an antagonist of Su(H) also during wing development, H mutant clones were induced in the wing pouch and it was asked whether genes dependent only on Su(H) activity are expressed in these clones. If H regulates the activity of Su(H), the removal of H might lead to the activation of Su(H) and result in the expression of its targets, e.g. the vestigial boundary enhancer (vgBE) and Serrate. Both are ectopically activated in H mutant clones. The ectopic expression of the vgBE in the clones varies and is strongest near the DV boundary. This graded expression is possibly due to the requirement of a diffusible factor coming from the DV boundary. One candidate for this is Wg, which seems to be required for the proper expression of the vgBE. The cells in the H mutant clones do not express cut or wg, which are dependent on the presence of Nintra, suggesting that Notch is not activated in these clones (Klein, 2000).

The loss of H function seems to elicit Su(H)-dependent target gene expression in the wing pouch, a region probably devoid of Notch activity. This suggests that the inactivation of H is sufficient to activate Su(H). To test further this conclusion, an examination was performed to see whether the activity of the vgBE is maintained in H mutant wing pouches if Notch is concomitantly removed. For this, Notch mutant clones were induced in H mutant wing discs. In H mutant wing pouches, weak ubiquitous expression of the vgBE is observed throughout the whole area of the wing, confirming the clonal analysis. vgBE is also active in several Notch mutant clones near the DV and anteroposterior (AP) boundary, but the activity is not maintained in all clones. One explanation for this might be again the requirement of other so far unidentified factors emanating from the two compartment boundaries. In agreement with this, the vgBE enhancer has a late expression domain along the AP boundary, suggesting an input from these areas for its proper expression. However this domain is also dependent on Notch during normal development. The removal of the Su(H)-binding site in the enhancer leads to the loss of all expression domains in the wing pouch, suggesting that Su(H) is required (Klein, 2000).

Therefore, the fact that the cells of several mutant clones do express the vgBE suggest that the vgBE can be activated in the complete absence of Notch activity and that the inactivation of H is sufficient to activate Su(H). No activation of the vgBE was ever found in Notch mutant clones induced in wild-type wing pouches, suggesting that during wild-type development, the activity of Notch is required to activate the vgBE. Hence, Notch probably activates Su(H) through inactivation of H. An examination was performed to see whether the degree of endogenous Su(H) activation that results from the removal of H is sufficient to elicit a biological effect. To assay this, it was asked whether or not removal of H activity can induce Su(H)-dependent development of the pouch in wing discs in which Notch signaling is absent, such as apterous and Presenilin mutant wing discs. Loss of H function rescues the loss of wing development of ap mutants: whereas ap mutants have no wing pouch, ap;H double mutants have large wing pouches with no margin structures. The enlarged pouch of the double mutant discs expresses spalt (sal) and the two vg reporters, vgQE and vgBE, all of which are expressed specifically in the wing pouch in a Notch/Su(H)-dependent manner and are not expressed in ap mutants. In contrast, no wg expression is induced in these double mutant discs, suggesting that the observed rescue is likely to be due to the activation of Su(H) in the double mutants. This is strongly supported by the fact that Su(H);H double mutants exhibit a small wing rudiment identical to that of Su(H) mutants. Expression of UAS-vg by dpp-Gal4 in ap mutant discs can recover the pouch-specific expression domain of sal, suggesting that the activation of vg expression by Su(H) is responsible for the recovered sal expression in the ap;H double mutant wing discs. Similar to overexpression of UAS-Su(H) in ap mutant wing discs, the pouch in ap;H mutant discs develops near the residual wg expression in the remaining hinge. As expected from the analysis of the wing discs, the pharate adult ap;H double mutants have large wing pouches, which are devoid of any margin like structure such as innervated bristles (Klein, 2000).

The effects on wing development of removing H in Psn mutants were examined. As in the case of ap, loss of function of H effects a strong rescue of the wing pouch in the Psn;H mutant discs in comparison to the Psn mutant discs. However, in this case, the morphology of the discs is more like wild type and, in contrast to ap;H mutant discs, the pouch develops at its normal place. Closer monitoring of double mutant discs reveals some expression of wg and the vgBE along the DV boundary. This suggests that, in contrast to the situation of ap mutants, in Psn mutants, there is some activation of Notch and it seems that the lack of H activity can enhance this residual signaling of Notch at the DV boundary. This is remarkable considering that the wing phenotype caused by the loss of Psn is stronger than that caused by loss of Su(H) function. Taken together, these results provide further evidence for a positive transcriptional activity of Su(H). They further show that H is an antagonist of Su(H) during early wing development and that it suppresses the activity of Su(H) in the absence of Notch signaling. The results also suggest that the inactivation of H is sufficient to activate Su(H) and that the activity of Notch is required to inactivate H during normal development (Klein, 2000).

Overexpression of Su(H) leads to three different responses: (1) activation, as is the case for vg, some E(spl) genes, Dl and Ser; (2) inactivation, as shown for cut and E(spl)m8; or (3) no effect, as is the case for wg. This differential behavior is, at least in some cases, a consequence of direct binding of Su(H) to the promoters: the vgBE as well as the E(spl) genes contain Su(H)-binding sites to which Su(H) binds; such sites are necessary for the activation of these genes in vivo. Despite that, they react differently towards Su(H) overexpression. Since E(spl)m8, which is suppressed by Su(H) overexpression, can be activated by expression of Su(H)VP16 or Nintra, it is concluded that Nintra is required in addition to Su(H) to activate E(spl)m8 expression. The results suggest that, in this case, Nintra probably acts as an activation domain of a dimeric transcription factor containing Su(H), as has been proposed. From this, it follows that Nintra might have two function during a Notch signaling event: first it inactivates H, which leads to the release of Su(H) and then, in some instances, it provides the transactivation domain for free Su(H) to activate the expression of target genes (Klein, 2000).

Flies carrying reporter lacZ constructs with up to 12 Su(H)-binding sites do not display any activity in the wing disc. This suggests that Su(H) (even in association with Nintra) is not sufficient to activate transcription and requires other collaborating factors. It further suggests that, even in promoters that can be activated by Su(H) in the absence of Nintra, Su(H) probably interacts with other factors to promote gene expression. This is confirmed by a study of the vgBE. Although the Su(H)-binding site is absolutely necessary for its activity, other sites are equally important. So far the factors that bind to these sites are not identified. The dependence of Su(H) on these others factors is probably the reason for the differential expression of Notch target genes in H and H/N mutant clones that have been observed (Klein, 2000).

Recently it has been shown that Su(H) acts as a suppressor of single minded transcription during the formation of the midline cells in the embryonic central nervous system of Drosophila. This observation provides the first evidence that Su(H), like its mammalian counterpart CBF1, can act as a suppressor of transcription. The inactivation of the cut and E(spl)m8 expression in absence of Nintra suggests that Su(H) can act as a suppressor of gene expression also during adult development and provides further evidence for a suppressing activity of Su(H). However, this suppression is context dependent and not a general feature of Su(H). This context dependency might also exist for CBF1, since only the reaction of a small number of genes towards its activity has been tested so far and it is possible that some target genes can be activated by CBF1 in the absence of Nintra in a similar way, as has been shown for Su(H). In summary, these results suggest that the consequence of the binding of Su(H) to a promoter is dependent on its local architecture and, therefore, Su(H) can at the same time activate and suppress gene expression, like many other transcription factors. The removal of both the maternal and zygotic expression of H during embryogenesis seems to be of no consequence for the embryo. Since the overactivation of Notch/Su(H) signaling during embryogenesis has deleterious consequences, this observation contradicts the conclusion that H is required to inactivate Su(H). However, the context dependency and differential reaction of the target genes observed during wing development offer two explanations for this discrepancy, without having to postulate an unknown factor, which can functionally replace H. First, it is likely that the interacting factors, which are required for gene expression in concert with Su(H), are different during embryogenesis and this could modulate the responsiveness of the target promoters. This conclusion is supported by the observation that the genes of E(spl)C, although probably all requiring Su(H) for their expression, are all very similarly expressed in the embryo, but their expression pattern in the wing imaginal disc is very different. Another explanation is that the target promoters of binding Su(H) during embryogenesis might be all of the type that require the additional activity of Nintra. Therefore they would stay inactive even in the presence of free Su(H) until Notch is activated (Klein, 2000).

Transcriptional repression by Suppressor of Hairless involves the binding of a Hairless-dCtBP complex in Drosophila

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 sim gene is expressed in a single row of cells abutting the mesoderm in the Drosophila embryo at the cellular blastoderm stage. Sim confers to these cells a mesectodermal identity. Su(H) has a dual function in the regulation of sim expression: (1) Su(H) directly inhibits the expression of the sim gene in the neuroectoderm -- in Su(H) mutant embryos derived from germ-line clones (GLC), both endogenous sim and a sim-lacZ transgene that mimics the expression of sim are ectopically expressed in 2-3 rows of neuroectodermal cells; (2) Su(H) upregulates the expression of sim in the mesectoderm. Transcriptional activation by Su(H) depends on Notch signaling, but repression by Su(H) is independent of Notch activity (Morel, 2001).

Hairless is a nuclear protein that binds Su(H) and antagonizes Notch activity in numerous cell fate decisions. It is, however, unclear how Hairless inhibits transcriptional activation by Su(H). One hypothesis is that Hairless promotes repression by Su(H). Analysis of the sim promoter has allowed for a test of whether Hairless is required for Su(H)-dependent repression. The expression of the sim gene was analyzed in embryos that lack both the maternal and the zygotic contributions of Hairless. In these Hairless GLC embryos, sim-lacZ is ectopically expressed in cells located dorsally to the mesectoderm. This ectopic expression in the neuroectoderm is similar to the one observed in Su(H) mutant embryos. Thus, Hairless is required for the repression of sim in the same cells that also require Su(H) for repression (Morel, 2001).

Hairless binds Su(H) and inhibits transcriptional activation by Su(H). However, because Su(H)-Hairless complexes do not bind DNA in vitro, Hairless has been proposed to inhibit the DNA binding activity of Su(H). In this model, Hairless inhibits Notch signaling by titrating Su(H) and not by repressing Notch target genes. This model does not explain, however, why the loss of Hairless activity leads to the same phenotype as the loss of Su(H) activity, i.e., derepression of sim in neuroectodermal cells. It is therefore envisaged that Hairless and Su(H) act together to repress transcription. This implies that Hairless must bind to a DNA bound form of Su(H). Whether Hairless and Su(H) can form a DNA bound complex has been reexamined in a gel retardation assay. Hairless and a truncated version of Hairless that binds Su(H) (H[1-293]) are shown to supershift a Su(H)-oligonucleotide complex. It is concluded that Hairless associates with DNA via Su(H) (Morel, 2001).

To test whether Hairless is able to cooperate with Su(H) in vivo, an assay based on the expression of Hairless and Su(H) during pupal development was used. Lateral inhibition mediated by Notch signaling controls the spacing of bristle sensory organs on the dorsal thorax of the fly. Increasing the level of Notch signaling results in the determination of a reduced number of sense organs. Conversely, decreasing the level of Notch signaling leads to an increased density of sense organs. Similarly, overexpression of Su(H) under heat-shock control decreases sense organ density, while overexpression of Hairless has the opposite effect. Control flies in which the expression of either Su(H) or Hairless was induced under mild heat shock conditions (30 min at 37°C) display only a weakly decreased or increased bristle density, respectively. The titration model for Hairless predicts that when Su(H) and Hairless are simultaneously overexpressed, they should counteract each other's activity to produce an intermediate phenotype. In contrast to this prediction, low-level expression of Hairless and Su(H), under the same mild heat shock conditions, leads to a dramatic increase in sense organ density on the dorsal thorax. This shows that Su(H) and Hairless strongly synergize to inhibit Notch signaling in this experimental situation. This synergy between Hairless and Su(H) was also seen for the regulation of sim expression in the mesectoderm, as well as during wing development. These findings suggest that this synergy represents a general feature of the function of these two genes. These observations are not consistent with the titration model described above, but rather they support the hypothesis that Hairless acts in a Su(H)-dependent manner to antagonize Notch signaling activity (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).

Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by Suppressor of Hairless

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).

Hairless-mediated repression of Notch target genes requires the combined activity of Groucho and CtBP corepressors

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 has been shown in this study (Nagel, 2005).

A molecular link between Hairless and Pros26.4, a member of the AAA-ATPase subunits of the proteasome 19S regulatory particle in Drosophila

The proteasome is the major degradation machinery of the cell that regulates multiple cellular processes as diverse as cell cycle, signal transduction and gene expression. Recognition and unfolding of target proteins involves the regulatory cap whose base contains six AAA-ATPases that display reverse chaperone activity. One of them, Rpt2 (also known as S4), has an essential role in gating the degradative central core. The orthologous gene Pros26.4 has been isolated from Drosophila melanogaster as a molecular interaction partner of Hairless. Hairless plays a major role as antagonist of Notch signalling in Drosophila, prompting an interest in the Hairless-Pros26.4 interaction. Pros26.4 negatively regulates Hairless at the genetic and molecular level. Depletion of Pros26.4 by using tissue-specific RNA interference (RNAi) results in a specific stabilization of the Hairless protein, but not in stabilization of the intracellular domain of Notch or the effector protein Suppressor of Hairless. Thus, the Hairless-Pros26.4 interaction provides a novel mechanism of positive regulation of Notch signalling (Muller, 2006).

The proteasome is a highly conserved multi-enzyme complex destined to degrade poly-ubiquitylated proteins in eukaryotic cells. The proteasome consists of a 20S central core that confers proteolytic activity, whereas the 19S regulatory caps are involved in substrate recognition and unfolding. The cap can be divided into a lid and a base, and contains six different AAA-ATPases displaying reverse chaperone activity. ATPase activity is required at several steps of protein degradation, e.g., for proteasome assembly and for effective substrate recognition and unfolding. One of the six AAA-ATPases, Rpt5 (or S6'), has been shown to contact poly-ubiquitin chains on substrate proteins only, however, when in a complex with the other five AAA-ATPases. This suggests an intimate interaction among the six AAA-ATPases and, furthermore, a general involvement of the base in substrate recognition. Thereby, ATPase activity may be contributed from any of these proteins. The AAA-ATPase Rpt2 (or S4) stands out in some respects. It belongs to the four proteins of the cap most conserved in the evolution from yeast to human. It is indispensable for yeast vitality and is highly susceptible to mutation. It enhances the peptidase activity of the proteasome, presumably by gating the central core of the proteasome's hydrolytic 20S unit. The orthologous Pros26.4 gene has been isolated from Drosophila in a yeast two-hybrid screen for interaction partners of Hairless. Hairless plays a major role as an antagonist in the Notch signalling pathway in Drosophila (Muller, 2006).

In a yeast two-hybrid screen for potential molecular partners of Hairless, clone pJG7-9 was the most frequently isolated and also corresponds to Pros26.4. Pros26.4 has a single transcript and associated protein. In comparison to the published sequence, clone pJG7-9 lacked 13 codons (K₄₇-L₅₉) and had N-terminally an extension of seven codons that were derived from untranslated leader sequences. To exclude artificial interactions, the Pros26.4 coding sequence was recloned from a full-length cDNA clone to generate pJG-S4. In yeast two-hybrid assays, pJG-S4 behaved identical to pJG7-9. Using several deletion constructs of Hairless, the Pros26.4-interacting domain was mapped within the C-terminal third of the Hairless protein (CX). Moreover, the Hairless-interacting domain was mapped to the N-terminal half of Pros26.4, excluding an involvement of the AAA-ATPase domain. The molecular interactions were confirmed in vivo: anti-Hairless immunoprecipitates from embryonic protein extracts indeed contained Pros26.4 protein (Muller, 2006).

Molecular interactions are relevant only when the respective proteins are expressed at the same time within the same tissues during development. The expression patterns of Pros26.4 mRNA and protein were analyzed. Pros26.4 mRNA is uniformly distributed in early eggs owing to maternal product, as has been described before for Hairless transcripts. Later in development, transcripts appeared enriched within the developing nervous system thus overlapping the Hairless expression domain. In third instar larvae, Pros26.4 mRNA levels are higher in dividing cells, e.g., in neuroblasts of the ventral chord or in eye imaginal discs anterior to the morphogenetic furrow. This pattern is very similar to that of Pros28.1, which encodes an alpha-subunit of the central core. Pros26.4 protein is ubiquitously distributed in all tissues; it is cytoplasmic and also, albeit to a lesser degree, nuclear. During oogenesis the protein is enriched in the germinal vesicle as was reported before for the alpha- and ß-subunits of the 20S central core. The general distribution of both Pros26.4 and Hairless proteins indicates extensive overlap in many tissues (Muller, 2006).

Pros26.4 was overexpressed in a variety of different tissues using the Gal4/UAS system. Despite a strong expression, the flies developed completely normally without any obvious phenotypes. Based on the close molecular contacts between the six AAA-ATPases forming a ring at the base of the 19S regulatory cap, one might expect that overexpression of any of them disrupts the stoichiometry of the ring and, therefore, proteasome activity. However, this is not the case because Drosophila is insensitive to a large increase in the amount of Pros26.4 (Muller, 2006).

Reduction of Pros26.4 activity in cultured cells by RNAi affects cell growth rates and results in an increased number of apoptotic cells. Presumably, Drosophila cells that completely lack Pros26.4 cannot survive because respective mutations in yeast are lethal. To study loss of Pros26.4 activity in Drosophila, transgenic flies were constructed containing an inverted Pros26.4 construct (UAS-dsS4) thus allowing tissue-specific RNAi. In the animal, ubiquitous knock-down with the da-Gal4 driver caused death at about second larval instar. Likewise, larval or pupal death was observed upon tissue-specific RNAi when using a number of different driver lines (e.g. ptc-Gal4, en-Gal4, sca-Gal4). Apparently, depletion of Pros26.4 causes cell death not only in Drosophila cell lines but also during fly development. For example, specific expression of UAS-dsS4 within the eye anlagen using ey-Gal4 allowed recovery of pharate adults from the pupal case that lacked the eyes and most of the head capsule. Closer inspection of the defects caused by loss of Pros26.4 was possible by using gmr-Gal4, which drives expression later in development within differentiating cells of the eye imaginal disc. In these cases, fusions of ommatidia were found in a posterior to anterior gradient in the adult eye. Frequently, necrotic tissue was attached to the very posterior end of the eye. The underlying defect was revealed in sections, which showed a similar graded loss of internal retinal tissue, reflecting the respective differentiation wave. Only cells behind the morphogenetic furrow, which sweeps from posterior to anterior over the eye imaginal discs during larval life, express gmr-Gal4. Thus, cells furthest anterior were the last to be exposed to RNAi and survived, whereas those more posterior were destroyed. A strong increase in cell death was already observed in third larval instar eye discs, and could be rescued by simultaneous overexpression of the anti-apoptotic baculoviral p35 protein. The defects seen in the adult, however, arose later in development, and only a marginal rescue was caused by the overexpression p35. Apparently, interference with Pros26.4 activity occurs with a pronounced time-lag, presumably due to a stable pool of Pros26.4 protein. As a consequence, cells eventually die and the retina degenerates with a late onset (Muller, 2006).

The 19S regulatory particle is indispensable to the recognition and unfolding of proteins destined for degradation by the 20S core particle. Thus, it is not surprising that interference with its activity results in cell death. In addition, it has been assigned a number of other, completely different, activities: in yeast it has been shown that the 19S regulatory particle plays an independent role in nucleotide excision repair of damaged DNA and, moreover, in transcription activation and elongation. To date it is unknown whether these multiple activities also apply to the fly. If this were the case RNAi with Pros26.4 might interfere with all these processes. Interestingly, impeding the activity of the central core subunits ß6 or ß2 causes superficially very similar phenotypes to depletion of Pros26.4. Respective dominant negative mutants DTS5 and DTS7 were mis-expressed in the photoreceptor cells. Necrosis was observed after a noticeable time-lag but the underlying defects were not analyzed. The stunning resemblance of phenotypes strongly suggests that, above all, RNAi of Pros26.4 interferes with proteasome activity proper. Apparently, the function of Pros26.4 within the proteasome degradation machinery is the most dose-sensitive, such that reduction in activity causes instant and obvious defects. By means of the tissue-specific RNAi assay it is now possible to identify and further analyse possible roles of Pros26.4 in Drosophila, e.g., in gene transcription or nucleotide-excision repair (Muller, 2006).

In the last few years, an intimate relationship between the proteasome and the regulation of gene expression has been established. In principle, the proteasome might control transcriptional regulators at the level of availability (i.e., their translocation into the nucleus or into nuclear bodies) or at the level of activity (i.e., by delimiting co-activators or alike) and finally at the level of abundance. Indeed, it has been reported that Notch-mediated gene activation is negatively regulated by targeting the intracellular domain of Notch for proteasomal destruction. Transcription-coupled proteolysis seems common to unstable transcriptional activators, in which trans-activation domains and degradation signals often overlap. Thereby, activation of the transcription machinery is directly linked to the degradation of the trans-activator, a process coined the 'black widow' model. In the process of activation, the transcriptional regulator is marked for degradation. Indeed, many transcriptional regulators are phosphorylated by components of the basal transcription machinery. This renders them susceptible to ubiquitylation and degradation by the proteasome. In the case of Notch signalling the process has been analysed in detail. The transcription-activation complex affecting Notch signals includes the intracellular domain of Notch, a CSL-type DNA-binding protein and also the co-activator Mastermind (Mam). Mam is essential for transcription initiation and promotes hyperphosphorylation of Notch by coupling to the CycC:Cdk8 kinase. This results in PEST-dependent degradation of active Notch (Muller, 2006).

Besides coupling transcriptional activation to destruction, a non-proteolytic role for the 19S regulatory particle has been stated in transcriptional activation and elongation. Notably, the proteasomal AAA-ATPases Rpt6/Sug1 and Rpt4/Sug2 are located at promoters where they direct chromatin remodelling. They are recruited by mono-ubiquitylated histone H2B, which participates in the activation of transcription and relief of telomeric silencing in yeast. H2B ubiquitylation is mediated by the Rad6 complex, which includes the E3 ubiquitin ligase Bre1. It is a first and mandatory step for subsequent histone H3 methylation at lysine residues 4 and 79, which also depends on the presence of Rpt6/Sug1 and Rpt4/Sug2. Whether similar mechanisms apply to Drosophila as well has not yet been studied in detail. However, very recently it has been shown that a mutation in Drosophila Bre1 particularly affects Notch target gene expression (Bray, 2005), arguing that, in Drosophila, Notch target genes are particularly susceptible to the effects of histone H2B ubiquitylation. Albeit all six AAA-ATPases quickly associate with active promoters in yeast, there is little evidence for a specific role of Rpt2/S4 in transcriptional regulation. In this case, knock-down of Pros26.4 by RNAi would result in a downregulation of Notch target genes, similar to mutations in Bre1 (Muller, 2006).

The results demonstrate that Pros26.4 positively influences Notch signalling by specifically destabilizing the Notch antagonist Hairless. The data reveal a remarkable, Pros26.4-dependent preference of the proteasome for Hairless compared with other potential substrates such as Notch. It is proposed that this is based on a direct molecular interaction of Pros26.4 and Hairless. It is conceivable that protein-protein interactions of proteasomal AAA-ATPase subunits with candidate substrates occur independently of ubiquitylation, which regulates proteasomal access. Hairless interferes with Notch activity in two ways. (1) It interferes with the assembly of the transcription-activator complex by competing for CSL/Su(H) and (2) it forms, directly on Notch-responsive promoters, a repressor complex together with Su(H), and the co-repressors Groucho and CtBP. Thus, by decreasing the availability of Hairless protein, Pros26.4 promotes Notch target gene activation (Muller, 2006).

In conclusion, transcriptional regulation of Notch target genes is influenced in several ways by the ubiquitin-proteasome system. (1) Activated Notch undergoes proteasomal destruction in a typical transcriptional activation-destruction process. (2) Notch target genes are particularly susceptible to ubiquitin-dependent chromatin activation. (3) By means of Hairless destabilization, the AAA-ATPase Pros26.4 promotes Notch signalling (Muller, 2006).

Gene-specific targeting of the histone chaperone asf1 to mediate silencing: Asf1 can be coprecipitated with Su(H), the corepressor Hairless, and Groucho

The histone chaperone Asf1 assists in chromatin assembly and remodeling during replication, transcription activation, and gene silencing. However, it has been unclear to what extent Asf1 could be targeted to specific loci via interactions with sequence-specific DNA-binding proteins. This study shows that Asf1 contributes to the repression of Notch target genes, as depletion of Asf1 in cells by RNAi causes derepression of the E(spl) Notch-inducible genes. Conversely, overexpression of Asf1 in vivo results in decreased expression of target genes and produces phenotypes that are strongly modified (enhanced and suppressed) by mutations affecting the Notch pathway, but not by mutations in other signaling pathways. Asf1 can be coprecipitated with the DNA-binding protein Su(H) and the corepressor Hairless and interacts directly with two components of this complex, Hairless and SKIP. Thus, in addition to playing more general roles in chromatin dynamics, Asf1 is directed via interactions with sequence-specific complexes to mediate silencing of specific target genes (Goodfellow, 2007).

Modulation of the chromatin structure is a key feature in transcriptional regulation. Chromatin remodeling by ATP-dependent enzymes and posttranslational histone modifications are two important mechanisms that affect transcriptional activity, by influencing the accessibility of upstream regions and promoters. A third mechanism involves the breakdown and reassembly of nucleosomes on the DNA, a process that also allows for the incorporation of histone variants, such as H3.3. Histone chaperones, which bind to histone heterodimers, are required both for nucleosome assembly and for their disassembly. They include the H3/H4 chaperone Anti-silencing factor 1 (Asf1), which has roles in replication-dependent and replication-independent chromatin dynamics (Goodfellow, 2007).

In yeast, extensive Asf1-mediated exchange of histones that is independent of replication and of transcription has been detected at gene promoters and is likely to be highly significant in maintaining the balance between induction and silencing of genes. Indeed, there are now several examples of yeast Asf1 contributing to chromatin disassembly at promoters to facilitate binding of the RNA-polymerase complex. Conversely, Asf1 also plays important roles in gene silencing when the reassembly of nucleosomes accompanies transcriptional repression. For example, in the absence of Asf1, there is a delay in promoter closure at the PHO5 gene. However, it remains unclear whether Asf1-mediated nucleosome reassembly occurs via a targeted mechanism, involving sequence-specific DNA-binding proteins, or whether it occurs constitutively by default (Goodfellow, 2007).

A strong correlation between histone loss and gene activation has emerged from genome-wide studies in Drosophila, as it has in yeast, suggesting that transcription in higher eukaryotes is also likely to be regulated by histone loss and replacement at the promoter. However, thus far, the contribution of Asf1 to dynamic gene regulation during cell signaling in multicellular organisms has not been examined. One cell-signaling pathway with very direct effects on transcription is the highly conserved Notch pathway. Activation of the receptor results in the release of a nuclear-targeted intracellular fragment (Nicd), which binds directly to the CSL DNA-binding protein (Suppressor of Hairless, Su(H), in Drosophila) and recruits the coactivator Mastermind, resulting in the activation of target genes. CSL proteins also contribute to the silencing of target genes in the absence of Nicd, through adaptor-mediated recruitment of corepressors such as Groucho (Gro), CtBP, and SMRT. Previous analysis indicates that the activity of Notch target genes correlates with a reduction in histone H3 density, suggesting that nucleosome disassembly and reassembly is likely to be involved in their regulation, and prompting an investigatation of whether Asf1 could play a role (Goodfellow, 2007).

This study shows that Asf1 contributes to the repression of Notch target genes, and that it is recruited to the DNA through interactions with the Su(H)/H complex. Thus, Asf1 is targeted to specific loci by binding to sequence-specific DNA-binding complexes, where it can promote gene silencing during development (Goodfellow, 2007).

To investigate whether Asf1 contributes to the regulation of inducible genes in Drosophila, RNA interference (RNAi) was used to deplete S2-N cells and the levels of transcription were analyzed from the 11 well-characterized Notch target genes clustered in the E(spl) complex. Conditions were established for activating Notch in these cells and it was shown that activation results in Su(H)-dependent stimulation of E(spl) gene transcription (Goodfellow, 2007).

Unlike knockdown of the other chromatin regulators tested, depletion of Asf1 led to a 4-fold increase in E(spl)m7 mRNA levels, but it had no effect on the housekeeping genes rp49 and EF2B. More extensive analysis revealed that mRNA levels for all E(spl) genes were increased after Asf1 depletion in the absence of Notch activation; some showed a greater than 10-fold change in expression, suggesting that these Notch targets are derepressed as they are when the corepressor Hairless is depleted. In contrast, there was little effect of Asf1 depletion on several other repressed genes, including a phagocytosis receptor gene, nimrod. In addition to the derepression observed in resting cells, Asf1 depletion also altered the responsiveness to Notch activation. Many more of the E(spl) genes were susceptible to Notch activation in Asf1-depleted cells; for example, 5 of the 11 genes were expressed at greater than 20-fold higher levels after Asf1 RNAi. There was comparatively little change at the genes, such as E(spl)m3, which normally has the most robust response to Notch and is depleted for histones. Thus, it appears that Asf1 makes important contributions to the silencing of Notch target genes (Goodfellow, 2007).

Previous studies showed that overexpression of Asf1 in the Drosophila eye (ey::Gal4 UAS::asf1/+) causes a 'small-eye' phenotype in which the eye is reduced in size and ommatidia are disorganized. If these small-eye phenotypes are a consequence of Asf1 altering the transcription of Notch targets, they may be modified when combined with mutations in the Notch pathway. To investigate this possibility, flies overexpressing Asf1 were crossed to alleles affecting genes central to Notch or to other signaling pathways, and the eye size was analyzed in the heterozygous progeny (Goodfellow, 2007).

The first dramatic result was that the heterozygous combination of a Notch loss-of-function allele (N^55e11) and Asf1 overexpression caused a severe reduction in the eye/head capsule ('pin-head') and resulted in lethality. Thus, the effects of Asf1 overexpression were strongly enhanced by a decrease in Notch function. Significant enhancement of the Asf1 phenotype also occurred with Delta loss-of-function alleles, but not with alleles affecting Hedgehog (smo), EGF-R (Egfr), or Wingless (arm, arrow) pathways or with alleles affecting the SET domain protein Trithorax-related (trr), the histone exchange factor Domino (dom), or the cell adhesion protein Pawn (pwn). Complementary results were obtained by using mutant alleles that increase Notch signaling: both a loss-of-function Hairless (H) allele and a gain-of-function Notch allele (N^Mcd1) suppressed the small-eye defect caused by Asf1 overexpression. These findings are fully consistent with the results of RNAi-mediated Asf1 depletion, and they suggest that Asf1 is involved in repression of Notch target genes. As asf1 mutant cells failed to proliferate, it was not possible to obtain clones of homozygous mutant cells to test the effects of eliminating Asf1 on Notch target genes in the eye (Goodfellow, 2007).

To investigate whether interactions between Notch and Asf1 occur in other tissues, it was asked whether Asf1 overexpression also perturbed Notch function in the Drosophila wing. Expression of Asf1 in the developing wing pouch (sd::Gal4/+; UAS::asf1/+) resulted in margin loss/wing nicks and mild vein thickening, characteristics of reduced Notch function (Notch/+ heterozygous flies have mild wing nicks due to reduced signaling at the dorsal/ventral (d/v) organizer of the wing). The Asf1 overexpression phenotypes were strongly enhanced when the levels of Notch were reduced; thus, wings had extensive scalloping/margin loss and more extensive vein thickening. Wing phenotypes, similar to the eye phenotypes, produced by Asf1 expression were thus enhanced by reduced Notch (Goodfellow, 2007).

To further assess whether Asf1 affects expression of target genes regulated by Notch (e.g., cut) or by other pathways (e.g., spalt), the effects of overexpressing Asf1 in wing discs was analyzed. In wild-type discs, Notch-dependent expression of Cut is detected in a stripe along the d/v boundary. This was interrupted and reduced in discs in which Asf1 was overexpressed. In contrast, there was no visible effect on Spalt under these conditions. Similar results were obtained when Asf1 was expressed in a more limited domain (by using ptc::Gal4), where a local loss of Cut, but not Spalt, expression was seen. Stronger expression of Asf1 resulted in more pronounced Notch-like phenotypes and loss of Cut expression, which could be rescued by a reduction in Hairless function. Under these conditions, where Asf1 was expressed more strongly, some more generalized effects of Asf1 were sometimes detected, compatible with its proposed role as a histone chaperone during replication. The replication defects became more severe at even higher levels of expression (29°C). Similarly, clones of cells mutant for asf1 failed to proliferate. Thus, as in yeast, Asf1 appears to have roles in replication-dependent as well as replication-independent chromatin dynamics in Drosophila. By moderating the levels of Asf1 expression, it was possible to uncouple these requirements, revealing a contribution to repression of Notch target genes (Goodfellow, 2007).

Complexes implicated in repression at Notch targets are formed by the CSL/Su(H) DNA-binding protein in conjunction with adaptor proteins, such as SKIP and Hairless, which recruit general corepressors, including SMTR or Gro and CtBP. On polytene chromosomes from Drosophila salivary glands, Asf1 is detected at most Su(H)-enriched sites, suggesting that these proteins are present at the same loci. Asf1 is also bound at many other loci, and it is strongly enriched at centromeres and telomeres, reflecting its multiple roles in chromatin dynamics (Goodfellow, 2007).

The colocalization of Su(H) and Asf1 on polytene chromosomes prompted a test of whether Su(H) and/or associated factors could copurify with Asf1 in immunoprecipitation (IP) experiments. For these experiments, extracts prepared from Drosophila embryos, and Su(H) or Asf1 was immunoprecipitated by using moderate salt conditions. Under these conditions, Asf1 was detected in Su(H) IP experiments, and, conversely, Su(H) was precipitated with Asf1, as was the corepressor Gro, but not CtBP. To exclude the possibility that the interaction between Asf1 and the Su(H) complex was mediated by the independent binding of both protein complexes to DNA, IP experiments were performed in the presence of ethidium bromide (EtBr), a DNA-intercalating drug that dissociates proteins from DNA. This treatment did not affect the interaction of Asf1 with Su(H). Thus, these data suggest that Asf1 is present in protein complexes containing the sequence-specific DNA-binding protein Su(H) and the Gro corepressor. A significant suppression of the Asf1-induced small-eye phenotype was observed in flies that were also heterozygous for a strong gro allele (gro^E48) and an enhancement was seen by Hairless proteins that retained a Gro-binding domain, agreeing with a model linking Gro to Asf1-mediated repression. Therefore whether any of the proteins in the Su(H) repression complex are able to bind to bacterially produced Asf1 (fused to glutathione S-transferase, GST), was examined. Of those tested, both Hairless and the adaptor protein SKIP were bound to GST-Asf1, but not to GST alone or to GST-CAF1p55 (a component of chromatin assembly factor 1). Neither Gro nor Su(H) itself showed direct interactions with Asf1 in this assay (Goodfellow, 2007).

Finally, to test whether Hairless contributes to the recruitment of Asf1 in vivo, chromatin immunoprecipitation (ChIP) was performed with anti-Asf1 antibodies in cells with and without RNAi-mediated depletion of Hairless and association with two E(spl) genes, m3 and m7, was assayed. The E(spl)m7 gene is silenced in the S2 cells and is strongly affected by Asf1 depletion, whereas E(spl)m3 is expressed in S2 cells, is highly induced by Notch activation, and is more mildly affected by Asf1 depletion. Of the two genes, the greatest effects were seen for E(spl)m7; binding of Asf1 to both enhancer and ORF fragments strongly decreased in ChIP after Hairless depletion. A decrease was also seen at the E(spl)m3 ORF region, but not at the E(spl)m3 enhancer. This enhancer is found to have very low histone coverage in these cells, and it was found that it shows only small Asf1 occupancy levels. The decrease in Asf1 from ORFs of both E(spl)m3 and E(spl)m7 after Hairless depletion may indicate that Asf1 spreads from the site of recruitment. Binding of Asf1 to E(spl)m7 and E(spl)m3 regions was confirmed by using affinity-purified anti-Asf1 antibodies raised in a different species. Loss of Hairless does not affect the binding of Asf1 to other loci that do not require Su(H)/H for their regulation, such as eiger or snRNP69D. Similarly, there was no change in the levels of Polycomb protein associated with bxd-PRE after Hairless knockdown. Together, these data support the model that recruitment of Asf1 to Notch targets requires Hairless (Goodfellow, 2007).

The density and precise positioning of nucleosomes are important factors in determining the transcriptional activity of a locus. It is now evident that most nonnucleosomal histones in cells are likely to be complexed with chaperones. It is therefore not surprising that the histone chaperone Asf1 is important for chromatin dynamics and has been shown to have multiple roles in transcription as well as in the disassembly and reassembly of chromatin during replication. These include gene-specific roles in repression, activation, and transcription elongation. For example, Asf1 is required for nucleosome disassembly and transcription activation at the yeast PHO5, PHO8, ADY2, and ADH2 promoters. However, the mechanisms responsible for targeting Asf1 to these loci remain unclear. This study has demonstrated that Asf1 can be specifically recruited to target loci by interactions with sequence-specific DNA-binding transcription factors. Asf1 is present in a complex with Su(H), the central DNA-binding protein in the Notch pathway, and that it interacts directly with two proteins found in CSL complexes, Hairless and SKIP. Importantly, it was found that Asf1 plays a significant role in the repression of Notch target genes. Thus, contrary to effects at many of the inducible loci examined in yeast, these data demonstrate a requirement for Asf1 in silencing rather than in activation of these inducible genes (Goodfellow, 2007).

As the global corepressor Gro is also coprecipitated with Asf1 and is implicated in Asf1-mediated repression through genetic interactions, Gro and Asf1 may cooperate in the repression of Notch target genes. Gro has been postulated to exert long-range repressive effects by nucleating a transcriptionally silent chromatin state, in a similar manner to its yeast relative Tup1. For example, at the STE6 locus, Tup1 recruitment results in increased nucleosomal density and local nucleosome positioning. The recruitment of the histone chaperone Asf1 with Gro to Su(H)/H DNA-binding complexes could facilitate a similar localized increase in histone deposition and participate in the spreading of repressed chromatin. Furthermore, since (H)/H complexes engage in comparatively low-stability interactions with target loci, it is suggested that Asf1 could be critical for translating these transient interactions into stable silencing. However, thus far, the analysis has focused on relatively few targets and tissues; thus, it remains to be determined whether Asf1 is recruited to all targets regulated by Su(H)/H, or whether there are additional factors that influence its recruitment at specific loci. Similarly, it will be important to determine whether other sequence-specific complexes are able to bind directly to Asf1 (Goodfellow, 2007).

In conclusion, these results show that the histone H3/H4 chaperone Asf1 contributes to selective silencing of genes in Drosophila, through interactions with the Su(H)/H DNA-binding protein complexes. In this way, chaperones can act as gene-selective regulators that contribute to the control of gene expression by developmental signaling pathways (Goodfellow, 2007).

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