InteractiveFly: GeneBrief

Hairless : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - Hairless

Synonyms -

Cytological map position - 92E14--92E14

Function - transcription factor

Keywords - CNS Development, Notch pathway

Symbol - H

FlyBase ID:FBgn0001169

Genetic map position - 3-69.5

Classification - novel basic protein

Cellular location - cytoplasmic and nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Praxenthaler, H., Smylla, T. K., Nagel, A. C., Preiss, A. and Maier, D. (2015). Generation of new hairless alleles by genomic engineering at the Hairless locus in Drosophila melanogaster.. PLoS One 10: e0140007. PubMed ID: 26448463
Summary:
Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way a complete H knock out allele HattP was generated, reintroduced a wild type H genomic and a cDNA-construct (Hgwt, Hcwt) as well as two constructs encoding H proteins defective of Su(H) binding (HLD, HiD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N5419) and Delta (DlB2) mutants were addressed. Overall, phenotypes were largely as expected: both HLD and HiD were similar to the HattP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs Hgwt and Hcwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably Hcwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, Hgwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.

Yuan, Z., Praxenthaler, H., Tabaja, N., Torella, R., Preiss, A., Maier, D. and Kovall, R. A. (2016). Structure and function of the Su(H)-Hairless repressor complex, the major antagonist of Notch signaling in Drosophila melanogaster. PLoS Biol 14: e1002509. PubMed ID: 27404588
Summary:
Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. This study determined the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, mutants in Hairless and Su(H) were designed that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed characterization the repressor function of Su(H) in vivo.
Smylla, T.K., Preiss, A. and Maier, D. (2016). In vivo analysis of internal ribosome entry at the Hairless locus by genome engineering in Drosophila. Sci Rep 6: 34881. PubMed ID: 27713501
Summary:
Cell communication in metazoans requires the Notch signaling pathway, which is subjected to strict regulation of both activation and silencing. In Drosophila, silencing involves the assembly of a repressor complex by Hairless (H) on Notch target gene promoters. An in-frame internal ribosome entry site in the full length H transcript results in two H protein isoforms (Hp120 and Hp150). Hence, H may repress Notch signalling activity in situations where cap-dependent translation is inhibited. This study demonstrates the in vivo importance of both H isoforms for proper fly development. To this end, the endogenous H locus was replaced by constructs specifically affecting translation of either Hp150 or Hp120 isoforms using genome engineering. Findings indicate the functional relevance of both H proteins. Based on bristle phenotypes, the predominant isoform Hp150 appears to be of particular importance. In contrast, growth regulation and venation of the wing require the concomitant activity of both isoforms. Finally, the IRES dependent production of Hp120 during mitosis was verified in vivo. Together these data confirm IRES mediated translation of H protein in vivo, supporting strict regulation of Notch in different cellular settings.

Praxenthaler, H., Nagel, A. C., Schulz, A., Zimmermann, M., Meier, M., Schmid, H., Preiss, A. and Maier, D. (2017). Hairless-binding deficient Suppressor of Hairless alleles reveal Su(H) protein levels are dependent on complex formation with Hairless. PLoS Genet 13(5): e1006774. PubMed ID: 28475577
Summary:
Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, these mutations were introduced into the native Su(H) locus by genome engineering. The three H-binding deficient Su(H) alleles were shown to behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, it was found that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, it is proposed that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.
BIOLOGICAL OVERVIEW

The Hairless gene was first described in 1923 by Bridges and Morgan as a haplo-insufficient mutation in Drosophila. In heterozygous flies, a large number of bristles on the head and thorax are lost, and the wing veins, mostly the fourth and fifth longitudinals, are shortened. Cloning of H reveals it is a large, rather novel basic serine/threonine rich protein that lacks structural similarities to other proteins of known function (Bang, 1992 and Maier, 1992). Hairless acts as an antagonist of Notch-signaling activity. The H protein is thought to inhibit Notch signaling by sequestering Suppressor of Hairless [Su(H)], a DNA-binding protein that mediates Notch signaling. Hairless binds directly to Su(H) in vitro, inhibits its DNA-binding activity and blocks transcriptional activation mediated by Su(H) in transfected cells (Brou, 1994).

Experiments were performed to determine if Hairless is essential during embryonic development or only later, during imaginal disc development. Animals that are genotypically null for H frequently survive embryogenesis, indicating that there is no obligatory embryonic requirement for zygotic H activity (Bang, 1991). Nevertheless, expression of H during embryonic development is to be expected, based on mutational studies, because H mutations have been reported to suppress the embryonic neural hyperplasia caused by loss-of-function alleles of the neurogenic genes Notch, Delta, mastermind, and neuralized (Vassin, 1985 and de la Conca, 1988). H transcripts are present in the developing CNS at the time of neuroblast segregation. The observation that H is expressed maternally raises the possibility that H may have an important embryonic function but that maternally supplied H+ activity is sufficient to allow the development of zygotically null embryos (Bang, 1994). To eliminate the maternal contribution of H to the embryo, germ-line clones homozygous for a mutant H allele were generated. Mutant embryos derived from null oocytes survive to the larval stage. It is concluded that H activity is not essential for embryonic viability (Schweisguth, 1998).

A complete lack of zygotic H function results in a loss of bristle phenotype associated with a failure to specify bristle precursor cells, due to an excess of lateral inhibition (Bang, 1991). The latter may result either from an up-regulation in signal transduction, i. e. in cells receiving an inhibitory signal, or from an increased level of inhibition produced by cells sending the signal. To distinguish between these two alternatives, the ability of H mutant cells to inhibit neighboring wild-type cells was measured. The prediction that H acts cell-autonomously cannot be experimentally tested in the pupal notum during bristle development, since presumptive neural cells failing to differentiate cannot be identified from epidermal cells. However, the cell-autonomous behaviour of H mutant cells may be studied in the wing. H is required for vein differentiation: no veins form in homozygous H mutant pharate adults. H mutant clones that interrupt vein differentiation were analyzed at the cell level. Mutant cells do not participate in the formation of veins, but instead appear to interrupt vein differentiation in a cell-autonomous manner. This cell-autonomous behaviour of H mutant cells shows that H acts in cells receiving the lateral inhibitory signal to down-regulate N signaling (Schweisguth, 1998).

Suppressor of Hairless mutant alleles exhibit dose-sensitive interactions with H loss-of-function mutations. Genetic interactions with H loss-of-function alleles has led to the definition of two classes of Su(H) mutant alleles: 'loss-of-function' alleles that, like deficiencies, suppress the haplo-insufficient H phenotype, and 'gain-of-function' alleles that, like duplications, enhance it. These gain-of-function alleles were thought to increase N signaling. However, somatic clones of cells mutant for such gain-of-function alleles, produce typical loss-of-function phenotypes. Further genetic analysis shows that gain-of-function alleles are actually partial loss-of-function alleles. It is suggested that the mutant proteins encoded by gain-of-function Su(H) alleles are defective for N-signaling activity but retain their ability to bind H: This binding results in a titration of H, hence in an enhancement of the haplo-insufficient H phenotype. These results provide a simple solution to a paradox that arose from classifying Su(H) mutation alleles using an interaction assay. More importantly, they provide strong genetic evidence that Su(H) is a direct target of H (Schweisguth, 1998).

In summary, Hairless plays an important role as the major antagonist in the Notch signaling pathway in Drosophila. It appears to be a direct inhibitor of the signal transducer Su(H). Hairless encodes a pioneer protein that has been dissected in a structure-function analysis: a series of deletion constructs was tested for wild type and gain of function activity in the fly as well as for Su(H) binding. In this way, the Hairless protein was subdivided into the absolutely essential Su(H)-binding domain, important N- and C-terminal domains and a central antimorphic domain. A construct C2 that deletes the Su(H) binding domain has some activity during wing development, suggesting that Hairless has additional functions apart from Su(H) binding. For example, overexpression of the C2 deleted protein causes a novel, net-like wing phenomenon that cannot be explained by Su(H) inhibition. The central acidic domain may mark a repression domain of the Hairless protein required for silencing Hairless function, e.g. for releasing Su(H) from a H/Su(H) complex. It is speculated that the C-terminal region comprises an interactive surface for additional components involved in H function. Therefore, Hairless protein might have additional functions apart from Su(H) binding and may antagonize Notch mediated cell-cell communication in a more complex way than currently anticipated (Maier, 1997).

Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless

Newer work suggests that Hairless suppresses the function of Su(H) by recruiting corepressor proteins. Su(H) functions as an activator during Notch pathway signaling, but can act as a repressor in the absence of signaling. Hairless, 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 in just the opposite manner as 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).

Potent inhibition of Su(H)'s DNA binding activity by H, as proposed by Brou (1994), is clearly incompatible with the proposition that a H/Su(H) complex directly represses N/Su(H) target genes. The mechanosensory organ lineage offers a particularly clear experimental example of this conflict. Proper specification of the shaft cell fate requires autorepression by Su(H); loss of this repression causes the shaft cell to transform its fate at substantial frequency to that of its sister, the socket cell. If H functions primarily to antagonize DNA binding by Su(H), then reduction of H activity should if anything lead to an increase in Su(H) autorepression, which should in turn stabilize the shaft fate. Instead, the shaft cell is highly sensitive to decreased H function, which readily causes its transformation to a socket cell. Thus, the genetic data on H's role in preserving the bristle shaft cell fate are irreconcilable with a simple Su(H)-DNA-binding-inhibition model for H function (Barolo, 2002b).

More generally, it has become apparent that repression of N targets by Su(H) is just as general and important a mechanism in Drosophila as it is in vertebrates. If Su(H)-mediated repression of N target genes is essential for proper specification of N-independent cell fates, as has been shown for the shaft cell, then preventing Su(H)-DNA interaction by overexpressing H would have the same effect as deleting Su(H) binding sites in N target genes: namely, their derepression, leading to N gain-of-function phenotypes (such as shaft-to-socket conversions). Instead, however, overexpression of H has been shown to repress the activity of N-regulated genes, and the phenotypic effects of gain and loss of H function suggest a strictly antagonistic relationship between H and N signaling. Therefore, it is believed that the accumulated genetic data point to a role for H in facilitating Su(H)-mediated repression of N target genes (Barolo, 2002b).

Purified Su(H) can bind efficiently to both purified H and DNA simultaneously, allowing the possibility that a H/Su(H) complex may function as a transcription factor. A weak supershift of Su(H)/DNA complexes by full-length H has been reported, and a stronger supershift by an N-terminal H fragment (H1-293). S robust supershift of Su(H)/DNA complexes occurs with full-length H, strongly supporting the notion that H/Su(H)/DNA complexes may form efficiently in vivo. The discrepancy between these results and those of previous studies may reflect different experimental conditions, such as buffer composition or protein purification protocols, or may be due to the relatively low H:Su(H) molar ratios used in these experiments. Further work will be needed to reject or confirm the possibility that H antagonizes the Su(H)-DNA interaction under physiological conditions. However, given the genetic arguments outlined above, and the consistency of the results with a view of H as a transcriptional repressor, it seems likely that DNA-binding inhibition is not the primary mechanism by which H contributes to the specification of N-independent cell fates (Barolo, 2002b).

Misexpression of wild-type and modified forms of H in the adult bristle lineage has led to three conclusions about the function of the H protein in vivo. (1) The replacement of the putative repression domains of H with a transcriptional activation domain results in a dominant-negative form of H that elicits N gain-of-function phenotypes, a result consistent with normal repression of N target genes by H. Conversion to an antimorphic form by the addition of an activation domain is a common property of transcriptional repressor proteins (Barolo, 2002b).

(2) The significant, but somewhat weakened, effects of HDeltaC relative to wild-type H suggest that the C-terminal region of H including a dCtBP-interaction domain is important for some, but not all, of H's activity in vivo. This conclusion is supported by analysis of the H22 allele, which produces mutant H protein lacking its C-terminal 69 aa, including the PLNLS motif. H22, unlike H null alleles, is a homozygous viable mutation, and its effects on N-mediated cell fate decisions, though strong, are milder than those of H null mutations. The fact that the H22/H22 phenotype, unlike the H null heterozygote phenotype, is not enhanced by loss of dCtBP, as well as the absence of any other consensus CtBP binding sites in the H protein, are consistent with the idea that H interacts with dCtBP solely via this C-terminal motif. If this is indeed the case, then both the mildness of the H22 mutant phenotype and the potency of misexpressed HDeltaC protein indicate that the dCtBP corepressor contributes some, but not all, of the repressive activity of the H protein (Barolo, 2002b).

(3) The dominant-negative activity of H-VP16 depends on the removal of the region of H containing the Gro-binding domain, suggesting that this region contributes some of the wild-type repression function of the H protein. These last two conclusions conflict with the assertion that the C-terminal dCtBP binding site is wholly responsible for H-mediated repression. Overall, the misexpression experiments provide evidence that both the region of H that binds to dCtBP and the region that binds to Gro contribute to the function of H in vivo. This is consistent with the strong phenotypic interaction between H and the genes encoding these two corepressors (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).

If protein complexes containing H are important for Su(H)-mediated repression, why is H not found in vertebrates? Several possible explanations are apparent. First, vertebrate homologs of Su(H) may not make use of an adaptor/corepressor complex, but rather may recruit all corepressors (possibly including Gro and CtBP) directly, as in the case of SMRT and CIR. A second, related possibility is that vertebrate versions of Su(H) do not utilize Gro and CtBP as corepressors; in this view, H may have appeared exclusively in the protostome lineage to add to Su(H)'s corepressor repertoire. Third, it is possible that vertebrates employ not a homolog, but an analog, of H, one evolved independently after the divergence of protostomes and deuterostomes. Finally, the fact that the predicted D. melanogaster H protein is only 33% identical to its apparent ortholog in A. gambiae suggests that the vast majority of the H sequence is not under selective constraint. Thus, an ortholog of H may indeed exist in vertebrates, but be so highly diverged as to be unrecognizable by typical sequence analyses. This possibility seems less likely, since at least the Su(H)-interaction domain might be expected to be well conserved, given the strong evolutionary conservation of Su(H) itself (Barolo, 2002b).

In contrast to a DNA-binding inhibition model for H function, an adaptor/corepressor model explains why H counters NIC/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 NIC-containing activation complexes and H/Gro/dCtBP repression complexes. NIC 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)/NIC/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 NIC-containing activation complexes by an excess of H-containing repression complexes, thus repressing NIC/Su(H) target genes in cells that respond to the N signal (Barolo, 2002b).

Similarly, it is proposed that the Su(H) overexpression phenotype, which resembles a gain of N function, is caused by a titration of H repression complexes by excess Su(H), and the subsequent derepression of Su(H) target genes. The fact that overexpression of Su(H) strongly enhances the effect of H overexpression on lateral inhibition supports this view, but is very much at odds with a DNA-binding-inhibition model for H function (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).

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).


REGULATION

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 Hp150 isoform from the second M2, and less efficiently, from the first start codon M1 and the short Hp120 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 Hp120 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 Hp120 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 Hp120 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 Hp120 isoform, which is initiated from the IRES, is specifically enriched in mitotic cells that largely exclude the Hp150 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 Hp120 is translated during mitotic phases when Hp150 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).

Transcriptional control of stem cell maintenance in the Drosophila intestine

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. This study found that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and genes of the Enhancer of split complex [E(spl)-C] were identified as the major targets of this repression. In addition, it was found that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. It is proposed that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation (Bardin, 2010).

Adult stem cells self-renew and, at the same time, give rise to progeny that eventually differentiate. This work provides evidence that one of the strategies used to maintain the identity of ISCs in Drosophila is to repress the expression of Notch target genes. Consistent with this finding, the loss of a general regulator of transcriptional repression, the Histone H2B ubiquitin protease Scrawny, gives a similar phenotype to Hairless (Buszczak, 2009). Additionally, several recent studies indicate that transcriptional repression of differentiation genes may be a central hallmark of stem cells in general (Bardin, 2010).

Two models have been proposed for Hairless activity. One proposes that Hairless competes with NICD for interaction with Su(H), thereby preventing transcriptional activation of Notch target genes by low-level Notch receptor activation. A second, non-exclusive, model proposes that Hairless antagonizes the transcriptional activation of Notch target genes by tissue-specific transcription factors other than Notch. Since the loss of Su(H) can suppress the phenotype of Hairless on ISC clone growth, it is proposed that Hairless promotes ISC maintenance by repressing the transcription of genes that would otherwise be activated by Notch signaling in ISCs. Thus, Hairless appears to set a threshold level to buffer Notch signaling in ISCs. In the absence of this repression, the expression of E(spl)-C genes and other Notch targets would lead to loss of the ISC fate (see Model for ISC maintenance). Importantly, these findings suggest a mechanism for how the transcriptionally repressed state is turned off and activation of the differentiation program is initiated: high activation of Notch in enteroblasts (EBs) displaces Hairless from Su(H) and leads to expression of the E(spl)-C genes (Bardin, 2010).

It is proposed that Hairless prevents ISC loss by repressing expression of Notch target genes, including the E(spl)-C genes. It is further proposed that Da-dependent bHLH activity promotes ISC identity, including the ability to self-renew and to express Delta. Delta, in turn, activates Notch in the adjacent EB, releasing the intracellular domain of Notch (NICD). It is speculated that, in response to Notch activation, the E(spl)-bHLH repressors downregulate Da-dependent bHLH activity in EBs as described in other systems, thereby shutting off ISC identity and promoting differentiation (Bardin, 2010).

E(spl)-C bHLH repressors act in part through their ability to inhibit bHLH activators. The data demonstrate that Da is also essential to maintain ISC fate and that E-box Da-binding sites are required to promote ISC-specific enhancer activity. Thus, it is proposed that activation of E(spl)-C genes by Notch in EBs downregulates Da bHLH activity and thereby contributes to turning off ISC identity in the differentiating cell (see Model for ISC maintenance). The specificity of ISC-specific E-box expression might be due to the ISC-specific expression of a bHLH family member. Although an array analysis raised the possibility that Scute may be specifically expressed in ISCs, genetic analysis indicates that scute function is not essential for ISC maintenance. Alternatively, specificity of gene expression might result from inhibition of bHLH activity in the EB and differentiating daughters, possibly by E(spl)-bHLH factors, rather than by the ISC-specific expression of a Da partner. It is also possible that a non-bHLH, ISC-specific factor restricts the Da-dependent bHLH activity to ISCs in a manner similar to the synergism observed in wing margin sensory organ precursors (SOPs) between the Zn-finger transcription factor Senseless and Da (Bardin, 2010).

Recently, a role for the Da homologs E2A (Tcf3) and HEB (Tcf12) has been found in mammalian ISCs marked by the expression of Lgr5 and, in this context, E2A and HEB are thought to heterodimerize with achaete-scute like 2 (Ascl2), which is essential for the maintenance and/or identity of Lgr5+ ISCs (van der Flier, 2009). In Drosophila, however, AS-C genes are not essential for ISC maintenance, but appear to play a role in enteroendocrine fate specification. The observation that Da bHLH activity is required for the identity of both Drosophila ISCs and mammalian Lgr5+ ISCs suggests that there might be conservation at the level of the gene expression program. Additionally, the bHLH genes Atoh1 (Math1) and Neurog3 are both important for differentiation of secretory cells in the mammalian intestine. Clearly, further analysis of the control of Da/E2A bHLH activity, as well as of the gene networks downstream of Da/E2A, will be of great interest (Bardin, 2010).

The data suggest that ISC fate is promoted both by inhibition of Notch target genes through Hairless/Su(H) repression and by activation of ISC-specific genes through bHLH activity. How then is asymmetry in Notch activity eventually established between the two ISC daughters to allow one cell to remain an ISC and one cell to differentiate? Three types of mechanisms can be envisioned that would allow for asymmetry of Notch signaling (Bardin, 2010).

First, the binary decision between the ISC and EB fates might result from a competition process akin to lateral inhibition for the selection of SOPs. In this process, feedback loops establish directionality by amplifying stochastic fluctuations in signaling between equivalent cells into a robust unidirectional signal. The finding that the Da activator and E(spl)-bHLH repressors are important to properly resolve ISC/EB fate is consistent with this type of model. Activation of the Notch pathway in one of the daughter cells may then lead to the changes in nuclear position (Bardin, 2010).

Second, the asymmetric segregation of determinants could bias Notch-mediated cell fate decisions. The cell fate determinants Numb and Neur are asymmetrically segregated in neural progenitor cells to control Notch signaling. However, no evidence was found for the asymmetric segregation of these proteins in dividing ISCs. Additionally, the data indicate that Numb is not important to maintain ISC fate. It cannot be excluded, however, that another, unknown Notch regulator is asymmetrically segregated to regulate the fate of the two ISC daughters (Bardin, 2010).

A third possibility is that after ISC division, one of the two daughter cells receives a signal that promotes differential regulation of Notch. Indeed, it has been noted that the axis of ISC division is tilted relative to the basement membrane, resulting in one of the progeny maintaining greater basal contact than the other. An extracellular signal coming either basally or apically could bias the Notch-mediated ISC versus EB fate decision. For instance, Wg secreted by muscle cells could act as a basal signal to counteract Notch receptor signaling activity in presumptive ISCs. This could be accomplished by Wg promoting bHLH activity or gene expression. Indeed, Wg has been demonstrated to promote proneural bHLH activity in Drosophila (Bardin, 2010 and references therein).

These models are not mutually exclusive, however, and proper control of ISC and differentiated cell fates during tissue homeostasis might involve multiple mechanisms (Bardin, 2010).

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 HRP1 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).

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

The UAS/GAL4 two component system was used to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi it was demonstrated that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi (Negeri, 2002).

Phenotypically, the consequences of Bx42RNAi often resemble effects obtained by interference with components of the Notch pathway. Studies of protein interaction in vitro suggest an involvement of Bx42 and its human homolog Skip in the Notch signal transduction. Both Skip and Bx42 were found to interact with Notch-IC, CBF1 and components of the CBF1 corepressor complex like SMRT, N-CoR, CIR, Sin3a and HDAC2 proteins (Zhou, 2000a; Zhou, 2000b; Zhang, 2001). By yeast two hybrid interaction and coimmunoprecipitation it was found that Bx42 physically interacts with the Drosophila CBF1-homolog Su(H) and with its antagonist Hairless, for which so far no vertebrate counterpart is known. The current study presents evidence that these interactions are biologically meaningful. Ubiquitous early induction of Bx42 RNAi results in embryos with dorsal cuticle only, a phenotype similar to Notch mutations. Induction of Bx42 RNAi in the eye disc results in an eye to antenna transformation as is observed following overexpression of dominant negative forms of Notch in the same tissue. Both effects could be interpreted that Bx42 normally functions as a coactivator of the Notch pathway. However, Other studies have demonstrated that overexpression of Notch-IC in the eye-antennal discs results in the formation of ectopic antennae too, but only if the eyeless function is reduced by a hypomorphic mutation. eyeless is one of the master regulators for eye development and functions in a cross-regulatory circuitry together with six other master regulatory genes. One of them is dachshund, whose human homolog Ski is a known interactor for the Bx42 human homolog Skip (Dahl, 1998). Thus, the observed eye antennal transformation by Bx42 RNAi could also be interpreted as a downregulation of eye master regulatory genes via diminished dachshund activity and a simultaneous derepression of the Notch pathway. Negative interference of Bx42 with Notch signaling is consistent with the results of ptc-GAL4 driven induction of Bx42 RNAi. A similar loss of scutellar bristles is observed on ptc-GAL4-driven overexpression of the Notch-ankyrin repeats, a part of Notch-IC involved in active signal transductio. However, this is not fully understood, since overexpression of Notch extracellular domains missing certain EGF repeats results in a similar antineurogenic phenotype. Moreover, Notch mutant clones result in a loss of bristles as well. A negative role of Bx42 in Notch signaling is suggested by the wing phenotype of dpp-GAL4/Bx42RNAi. Following reduction of Bx42 the veins are thinner or missing consistent with a Notch gain of function (Negeri, 2002).

Ectopic expression of Su(H) prevents sensory organ development in a similar manner to activated Notch. This may reflect an excess of lateral inhibition or it may be due to interference with the establishment of the correct fates in the progeny of the sensory organ precursor cells. The failure of sensory organ formation in the scutellum and the wing following local Bx42 RNAi may be related to a gain of Su(H) function. The enhancement of the Su(H) overexpression phenotype by Bx42 RNAi and the similar effects of Bx42 RNAi and Su(H) overexpression on Notch target genes strongly support this argument and suggest a functional relationship between both proteins (Negeri, 2002).

Although the data suggest a negative role, it is not believed that Bx42 protein acts as a repressor within the Notch pathway. Earlier work, which suggested an activation function for Su(H) was at odds with data demonstrating a repressive role for its mammalian homolog CBF1. More recent work provides evidence for Su(H) acting as a switch between repression and activation of Notch target genes. It is proposed that Bx42 contributes to the switch provided by the Su(H) protein. How this is accomplished can only be speculated at the moment. By its direct interaction with Su(H) Bx42 may stabilize a switching complex. By its direct interaction Bx42 could recruit Hairless into the complex contributing to its repressive function. Modification or removal of Bx42 protein (as by RNAi) would result in a destabilization of this repressive complex allowing to switch to the active state. Similarly, due to squelching by a large excess of overexpressed Su(H), only a reduced amount of Bx42 protein would be available to stabilize the repression complex. It is important to emphasize that Bx42 is able to physically interact with Notch-IC (Zhou, 2000a) and may act as a switching protein in the recruitment of activators as well (Negeri, 2002).

An important role in Notch signaling and functional relation between Su(H) and Bx42 were also suggested when the effects of reducing Bx42 on Notch target gene expression were studied. Both cut and E(spl)m8 were suppressed by Bx42 RNAi as was the vestigial quadrant element (vgQE) enhancer, indicating a Bx42 activating function for these genes in wild type. wingless expression was not affected under these conditions, excluding Bx42 RNAi induced cell death as an explanation for the observed effects. The effects on Notch target gene expression are consistent with the proposed role for Bx42 as part of a switch. Following Su(H) overexpression repression of cut, E(spl)m8 and the vgQE enhancer was demonstrated. wingless, on the other hand, was not affected. The similar effects of Su(H) overexpression and Bx42 RNAi on Notch target gene expression underscores the close relationship in the function of both proteins (Negeri, 2002).

Besides its involvement in Notch signaling the observed phenotypic effects of Bx42 RNAi suggest that the Bx42 protein is involved in other signaling pathways as well. Data from its vertebrate homolog, which indicate that Bx42 takes part in nuclear receptor pathways, are supported by observations on chromosomal binding to sites occupied by the ecdysone receptor complex. A possible interaction with Dachshund, one of the master regulators in eye development, which is widely expressed in the nervous system, has already been mentioned. It remains to be established how Bx42 is involved in these other pathways (Negeri, 2002).

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 NICD, 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 NICD and increase the stimulation seen in the presence of NICD. 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 NICD (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 vgBE-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 NICD activation. For example, CtBP interferes with recruitment of p300, a histone acetyltransferase that is reported to interact with mammalian NICD. However, the data suggest that CtBP and Gro are both needed to repress Grh even in the absence of NICD, 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 (N55e11) 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 (NMcd1) 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 (groE48) 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).

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

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

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

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

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

The physical interaction of dCtBP with H is implicated in sensory organ formation, wing formation, and embryonic patterning. H acts as an adaptor protein to bridge the Groucho and dCtBP corepressors to the DNA-binding factor Su(H), to ultimately inhibit Notch target genes. Vertebrate Notch target genes are similarly repressed by a complex consisting of CtBP with RBP-Jkappa (the mammalian counterpart to Su(H)) and the SHARP/CtIP corepressors. This study demonstrates that the bristles that are affected in dCtBP mutants also show defects in H loss-of-function mutants, although the effect of H is stronger than that of dCtBP. H mutations induce two distinct phenotypes associated with loss of bristles; one is the bald phenotype (a complete loss of both sockets and bristles) due to lack of SOP cells, and the other is the double-socket phenotype (also lack of bristles). A similar bald phenotype was observed in dCtBP mutant backgrounds, such as dCtBP RNAi, the dCtBP87De-10/dCtBP03463 transheterozygote, the dCtBP87De-10 clonal backgrounds. Although compared to what is seen in dCtBP mutants, reduction of H activity interferes more uniformly with the formation of all 11 bristles that were analyzed, the bald phenotype further supports previous observations that dCtBP is involved in H-mediated repression. The double-socket phenotype seen in H loss-of-function mutants was never observed in dCtBP mutants. This distinct phenotype suggests that H may play a role independent of dCtBP, possibly by interacting with another corepressor Groucho. Interestingly, the bald phenotype was also induced by overexpression of dCtBP. The mechanism by which overexpression causes the bald phenotype in all regions except the DC region remains unclear, although one simple explanation could be that overproduction of dCtBP may disrupt the stoichiometric balance of the H/dCtBP/Groucho repression complex (Stern, 2009).

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

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

Molecular analysis of the notch repressor-complex in Drosophila: characterization of potential hairless binding sites on suppressor of hairless

The Notch signalling pathway mediates cell-cell communication in a wide variety of organisms. The major components, as well as the basic mechanisms of Notch signal transduction, are remarkably well conserved among vertebrates and invertebrates. Notch signalling results in transcriptional activation of Notch target genes, which is mediated by an activator complex composed of the DNA binding protein CSL, the intracellular domain of the Notch receptor, and the transcriptional coactivator Mastermind. In the absence of active signalling, CSL represses transcription from Notch target genes by the recruitment of corepressors. The Notch activator complex is extremely well conserved and has been studied in great detail. However, Notch repressor complexes are far less understood. In Drosophila, the CSL protein is termed Suppressor of Hairless [Su(H)]. Su(H) functions as a transcriptional repressor by binding Hairless, the major antagonist of Notch signalling in Drosophila, which in turn recruits two general corepressors--Groucho and C-terminal binding protein CtBP. Recently, the C-terminal domain (CTD) of Su(H) was shown to bind Hairless and a single site in Hairless was identified that is essential for contacting Su(H) (Maier, 2011). This study presents additional biochemical and in vivo studies aimed at mapping the residues in Su(H) that contact Hairless. Focusing on surface exposed residues in the CTD, two sites were identified that affect Hairless binding in biochemical assays. Mutation of these sites neither affects binding to DNA nor to Notch. Subsequently, these Su(H) mutants were found to function normally in cellular and in vivo assays using transgenic flies. However, these experiments rely on Su(H) overexpression, which does not allow for detection of quantitative or subtle differences in activity (Kurth, 2011; full text of article).

Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster

CSL is the nuclear effector of the Notch signaling pathway and is required for both repression and activation of transcription from Notch target genes. In the absence of a signal, CSL functions as a transcriptional repressor by interacting with corepressor proteins, such as SHARP, SMRT/NCoR, KyoT2, and CIR. CSL-corepressor interactions function to localize histone deacetylase and histone demethylase activity at Notch target genes, which converts the local chromatin into a condensed, transcriptionally silent state. On pathway activation, the ICN binds CSL, and together with Mam, forms a transcriptionally active ternary complex that ultimately displaces corepressors from CSL and upregulates transcription from Notch target genes. In mammals, a number of corepressors have been shown to interact with the BTD of CSL, similar to ICN, which provides a model in which ICN displaces or outcompetes corepressors for binding to CSL. Thus, there are potentially two modes of repression mediated by corepressors: 1) at the transcription or chromatin level, in which the recruitment of HDAC/HDM-containing complexes by corepressors silences gene expression -- this mode of transcriptional repression is independent of Notch; and 2) at the protein level, by which corepressors and ICN compete for binding to CSL (Maier, 2011).

Although several of the mammalian corepressors have fly orthologues, these molecules do not seem to be generally involved in repressing transcription from Notch target genes in flies. A complex involving the SMRT homologue SMRTER negatively regulates Notch signaling during the specification of a subset of nonneuronal cell types in the developing Drosophila retina. However, mammalian SMRT is believed to contact CBF1 directly, whereas SMRTER does not bind Su(H) on its own. The Drosophila orthologue of SHARP/MINT, termed Spen, which genetically inhibits Notch signaling in the context of eye development, is presumably not a transcriptional repressor of Notch target genes in this process. Recently it was shown that Spen is required for the activation rather than the repression of Notch target genes during the development of hemocytes. Moreover, the region of SHARP/MINT that has been defined to interact with CSL is not conserved in Spen. Although formally SHARP/MINT might act as a functional Hairless analogue in mammals, the role of the structurally related Spen proteins seems largely diverse in different organisms (Maier, 2011).

In flies, the major antagonist of Notch signaling is the transcriptional corepressor Hairless, which is ubiquitously expressed in all tissues. Hairless binds the transcription factor Su(H), as well as the corepressors Groucho and CtBP, which serves to localize the transcriptional repression machinery in the nucleus to Notch target genes, thereby repressing gene expression. Removal of the Groucho and CtBP-binding sites from Hairless does not completely eliminate its activity as a repressor, suggesting that, similar to other corepressors, Hairless might compet.e with ICN for binding Su(H). However, the molecular mechanism by which Su(H) is converted from a repressor to an activator complex is unclear (Maier, 2011).

This work investigated the molecular details of the Notch repressor complex in Drosophila. The analysis was multidisciplinary in nature, using biophysical, biochemical, cellular, and in vivo assays to characterize the protein-protein interface between Hairless and Su(H). Hairless was shown to forms a high-affinity 1:1 complex with Su(H) (~1 nM Kd) but only interacts with the CTD, which is in stark contrast to mammalian CSL-corepressor interactions, which are largely mediated through BTD contacts. Previous ITC binding studies of the mammalian Notch components Notch1 (ICN) and RBP-J showed that the Kd of the ICN/RBP-J complex is ~10 nM, suggesting that Su(H)-Hairless and Su(H)-Notch interactions are likely of comparable affinity (Maier, 2011).

Given the similar affinities of ICN and Hairless for Su(H), the question arises whether ICN and Hairless compete for binding to CSL. On one hand, gel-shift assays with purified protein components showed that ICN can displace Hairless from Su(H) independent of Mastermind. On the other hand, it was shown that residues on Su(H) that are important for Notch ANK and Mam binding to CTD do not affect interactions with Hairless. These data suggest that the ICN- and Hairless-binding sites on Su(H) do not overlap. If the ANK domain of ICN and Hairless are competing for binding to the CTD of Su(H), then there is an additional factor to consider: based on binding studies of the mammalian proteins, the vast majority of the binding energy for the Su(H)-ICN complex comes from the RAM domain interaction with the BTD of Su(H), whereas the isolated CTD-ANK interaction is of very low affinity. This represents at least a 10,000-fold difference in the affinities of ANK and Hairless for CTD, which suggests that the ANK domain of ICN would seem to be a very poor competitor for removing Hairless from Su(H) (Maier, 2011).

How then is ICN able to supplant Hairless from Su(H) in order to activate transcription from Notch target genes? Certainly additional experiments will be required to fully address this question; however, at present the following hypothesis is favored: the binding of ICN to Su(H), that is, the RAM-BTD interaction, results in allosteric changes in Su(H) that decreases its overall affinity for Hairless, thereby making ANK a more effective competitor for CTD. Consistent with this notion, gel-shift experiments showed that Hairless was far less effective at removing ICN from Su(H), even when Hairless was present in vast excess (Maier, 2011).

The studies also analyzed two absolutely conserved residues in Hairless (L235 and F243) for their contribution to binding Su(H). Whereas F243 was dispensable for binding, L235 was absolutely required for binding Su(H) in vitro. Mutation of this site to aspartate abrogated binding but did not change the secondary structure content of Hairless, which suggests that L235 lies at the Su(H)-Hairless interface. Given the conservation of F243 but its dispensability for Su(H) binding, this perhaps suggests that this residue is important for interacting with other nuclear factors. Consistent with in vitro binding results, the Hairless mutant L235D failed to assemble a repressor complex with Su(H) in cellular and in vivo assays in the fly. In fact, the L235D mutant was as deficient in repression as the Hairless deletion mutant ΔNT, which removes residues 232-270, emphasizing the importance of this contact in forming the Su(H)-Hairless complex (Maier, 2011).

In conclusion, the fly Notch repressor complex shows similarities and differences compared with the mammalian complex. Despite the high degree of sequence and likely structural conservation, Su(H) in Drosophila differs from mammalian CBF1/RBP-J in that it has no repressor activity on its own; overexpression of Su(H) in cell culture and in vivo results in a Notch gain-of-function phenotype. It is not until the binding of Hairless that Su(H) is transformed into a repressor. Of interest, this study showed that Hairless bound the mammalian CSL orthologue CBF1 nearly as avidly as Su(H), which suggests that the Hairless-binding site on the CTD has been conserved in mammals. In accordance, a potent repression of Notch transcriptional activity was found in cultured mammalian cells by the Hairless NT construct. This raises the possibility of identifying Hairless homologues in other organisms or potentially other transcriptional coregulators that use the Hairless-binding site on CTD, which may be indicative of an as-yet-unidentified mode in mammals to repress Notch signaling. Nonetheless, detailed knowledge of Su(H)-Hairless interactions can now be used to develop molecules that target Notch transcription complexes and either enforce or disrupt their activity, thereby opening new therapeutic avenues (Maier, 2011).

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

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

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

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

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

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

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

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

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

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

To further understand the function of the Drosophila CoREST in Notch pathway regulation, identification of other CoREST essential and specific binding partners would be useful. One previously identified partner for CoREST is Chn (Tsuda, 2006). Given that wild-type expression of Hnt and Cut was observed in chn mutant cells, this factor does not appear to partner CoREST in regulation of Notch signaling in FCs. Using yeast two-hybrid analyses and an embryonic cDNA fusion protein library, it was shown that all three splice variants of Drosophila CoREST interact with the unique C-terminus of Tramtrack88 (Ttk88), a known repressor without homology to REST. In addition, a Ttk69 splice variant can form a complex with CoREST and Ttk88. However, Ttk88 was not detected in the ovary by immunofluorescence or western blot analysis, and disruption of Ttk88 does not have any impact on oogenesis. Conversely, Ttk69 is steadily expressed in FCs before stage 10 and it is required for the M/E transition. However, in contrast to CoREST, which acts upstream of Hnt, Hnt expression is not affected in ttk1e11 mutant FCs, indicating a role of Ttk69 downstream of Hnt in the control of the M/E switch. Additionally, Ttk69 is not required for cell differentiation, as expression of FasIII, a cell fate marker for immature follicle cells, is normal in ttk1e11 mutant FCs. From these important phenotypic differences between Ttk69, Ttk88 and CoREST, it appears that CoREST plays a Ttk-independent role in Notch pathway regulation in the FCs. Future work to identify transcription regulators that act as binding partners of CoREST will help in determining the precise biochemical role of CoREST in modulating Notch signaling (Domanitskaya, 2012).

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

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

Gain of function notch phenotypes associated with ectopic expression of the Su(H) C-terminal domain illustrate separability of Notch and hairless-mediated activities

The Notch signaling pathway is instrumental for cell fate decisions. Signals from the Notch receptor are transduced by CSL-type DNA-binding proteins. In Drosophila, this protein is named Suppressor of Hairless [Su(H)]. Together with the intracellular domain of the activated Notch receptor ICN, Su(H) assembles a transcriptional activator complex on Notch target genes. Hairless acts as the major antagonist of the Notch signaling pathway in Drosophila by means of the formation of a repressor complex together with Su(H) and several co-repressors. Su(H) is characterized by three domains, the N-terminal domain NTD, the beta-trefoil domain BTD and the C-terminal domain CTD. NTD and BTD bind to the DNA, whereas BTD and CTD bind to ICN. Hairless binds to the CTD, however, to sites different from ICN. This work addresses the question of competition and availability of Su(H) for ICN and Hairless binding in vivo. To this end, the CTD was overexpressed during fly development. A strong activation of Notch signaling processes was observed in various tissues, which may be explained by an interference of CTD with Hairless corepressor activity. Accordingly, a combined overexpression of CTD together with Hairless ameliorated the effects, unlike Su(H) which strongly enhances repression when overexpressed concomitantly with Hairless. Interestingly, in the combined overexpression CTD accumulated in the nucleus together with Hairless, whereas it is predominantly cytoplasmic on its own (Maier, 2013).

Mutation of potential MAPK phosphorylation sites in the Notch antagonist Hairless

Cellular differentiation during eumetazoan development is based on highly conserved signalling pathways. Two of them, the Notch and the EGFR signalling pathways, are closely intertwined. This study has identified two potential target sites of the Mitogen activated kinase (MAPK): the downstream effector kinase of EGFR and Hairless (H), the major antagonist of Notch signalling in Drosophila. Assuming that phosphorylation of these sites modulates H activity, a direct influence of EGFR signalling on Notch pathway regulation might be possible. This hypothesis was tested by generating a phospho-deficient and a phospho-mimetic H isoform and by assaying for their biological activity. The binding was assessed of known H interaction partners Su(H), Gro, CtBP and Pros26.4; this binding was similar between mutant and wild type H. Next eye, wing and bristle development, which are strongly affected by the overexpression of H due to the inhibition of Notch signalling, was assayed. Overexpression of the mutant constructs resulted in phenotypes similar to wildtype H overexpression, yet with subtle differences in phenotypic severity. However, large variations suggest that the mutated residues may be critical for the overall structure or stability of H. Albeit of minor impact, EGFR may fine tune Notch signalling via MAPK dependent phosphorylation of H (Nagel, 2014).


DEVELOPMENTAL BIOLOGY

Embryonic

Initially, Hairless transcripts are detected in a uniform distribution in embryos until the blastoderm stage; this pattern most likely corresponds to maternal mRNA deposition in the egg. With the onset of gastrulation, zygotic expression appears in the entire germ band, where mRNA clearly accumulates basally in the cells. During germ band extension Hairless transcripts rapidly fade from the ectodermal cell layer and become enriched in the mesodermal sheath and in the invaginating anterior and posterior midgut. With the flattening of the mesoderm, this staining becomes less evident, but can be still detected. Midgut staining is most prominent particularly in older embryos where expression also in the central nervous system and body wall is discernible, but it is uncertain whether this body wall staining represents cells of ectodermal or mesodermal origin (Maier, 1992).

Maternal H transcripts accumulate in ovarian nurse cells. These transcripts are present in syncytial embryos and persist until the cellular blastoderm stage. The transcript that begins to accumulate during late gastrulation and early germ-band extension evidently represents the onset of zygotic H expression. This interpretation is consistent with the appearance at 4-6 hr of a novel 6.0 kb transcript that is not present earlier. These zygotic transcripts are broadly distributed in the embryo throughout germ-band extension and retraction, although initially they appear to accumulate at a somewhat higher level in the mesodermal layer, whereas lower levels are observed in parts of the head region, especially the procephalic lobe and the clypeolabrum. H transcripts are present in the developing CNS at the time the zygotic neurogenic genes become active, consistent with the suppression by H mutations of the neural hyperplasia caused by loss of neurogenic gene function (Bang, 1992 and references).

Larval

There is a widespread, apparently uniform distribution of H transcripts at the time of macrochaete SOP determination. Within the first 16 hours after puparium formation, H transcripts appear to be uniformly distributed in the notum epithelium, except for a persistently higher level of accumulation in two cells of the developing macrochaetes. By 16 hr after puparium formation, in a background of generalized expression, higher levels of transcript are detectable in single cells and, possibly, pairs of cells in the positions of the future microchaetes. These cells may represent the secondary precursors that will generate the trichogen and tormogen: such precursors are present and known to be about to commence their division at this time (Bang, 1992).


EFFECTS OF MUTATION

A number of genetic loci, called neurogenic, have been found to be involved in directing the segregation of neural and epidermal lineages within the ectodermal germ layer of Drosophila. With the aim of understanding the regulation of this developmental function, interactions among the loci N, Dl and E(spl) were studied, as were interactions with another locus (Hairless), by means of increasing and decreasing the number of wild-type copies of any one of these genes in the presence of mutations in any other one. The results reveal a functional community that exists among these neurogenic loci. E(spl) overlaps functionally with both N and Dl because genotypes involving only one copy of E(spl)+ and either an N or Dl mutation are lethal. Furthermore the normal H+ allele behaves as if it represses the activity of the 3 neurogenic loci, and whereas E(spl) seems to be a close target of H repressive action, the influence of H on the other two seems to be indirect (Vassin, 1985).

Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either 'lost' (i.e. the shaft and socket fail to appear) or they exhibit a 'double socket' phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the 'bristle loss' phenotype results from greater loss of H+ function than the 'double socket' phenotype. The systematic study of H allelic combinations enabled the identification of genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted an investigation of the cellular development of sensilla in these same positions in larvae and pupae and thereby established the developmental basis for the mutant phenotypes. H is required for at least two steps of adult sensillum development. In positions where 'double socket' microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting 'bristle loss' in adult H mutants, at the appropriate stages of development to detect sensillum-specific cell types, neither the precursor cell divisions that generate them, nor the primary precursor cells themselves could be detected. Thus, the H 'bristle loss' phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development (Bang, 1991).

Overexpression of H produces two bristle shafts in the position of mechanosensory bristles, with no socket. This defects is interpreted as a tormogen-to-tricogen cell transformation, which is the opposite transformation from that which underlies the H hypomorphic double socket phenotype. In addition, adult flies developed from heat-shocked H overexpression exhibit a high frequency of multiplication and/or loss of microchaetes or macrochaetes. These phenotypes strongly mimic those caused by loss-of-function mutations of the neurogenic genes (Bang, 1992).

The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are the progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. The H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. These results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H (Bang, 1992).

Successive alternative cell fate choices in the imaginal disc epithelium lead to the differentiation of a relatively invariant pattern of multicellular adult sensory organs in Drosophila. The activity of Suppressor of Hairless is required for both the sensory organ precursor (SOP) versus epidermal cell fate decision, and for the trichogen (shaft) versus tormogen (socket) cell fate choice. Complete loss of Suppressor of Hairless function causes most proneural cluster cells to accumulate high levels of the Achaete and Delta proteins and to adopt the SOP fate. Late or partial reduction in Suppressor of Hairless activity leads to the apparent transformation of the tormogen (socket) cell into a second trichogen (shaft) cell, producing a 'double shaft' phenotype. Overexpression of Suppressor of Hairless has the opposite phenotypic effects. SOP determination is prevented by an early excess of Suppressor of Hairless activity, while at a later stage, the trichogen (shaft) cell is transformed into a second tormogen (socket) cell, resulting in 'double socket' bristles. It is concluded that, for two different cell fate decisions in adult sensory organ development, decreasing or increasing the level of Suppressor of Hairless function confers mutant phenotypes that closely resemble those associated with gain and loss of Hairless activity, respectively. These results, along with the intermediate SOP phenotype observed in Suppressor of Hairless;Hairless double mutant imaginal discs, suggest that the two genes act antagonistically to stably commit imaginal disc cells to alternative fates (Schweisguth, 1994).

In Drosophila imaginal discs, the function of the Hairless (H) gene is required at multiple steps during the development of adult sensory organs. Reported is a series of experiments designed to investigate the in vivo role of H in sensory organ precursor (SOP) cell specification. The proneural cluster pattern of proneural gene expression and of transcriptional activation by proneural proteins is established normally in the absence of H activity. By contrast, single cells with the high levels of achaete, scabrous, and neuralized expression characteristic of SOPs almost always fail to appear in H mutant proneural clusters. These results indicate that H is required for a relatively late step in the development of the proneural cluster, namely, the stable commitment of a single cell to the SOP cell fate. Expression of an activated form of the Notch receptor leads to bristle loss with the same cellular basis (failure of SOP determination) as loss of H function. Simultaneous overexpression of H suppresses this effect. Epistasis experiments demonstrated that the failure of stable commitment to the SOP fate in H null mutants requires the activity of the genes of the Enhancer of split complex, including groucho. These results indicate that H promotes SOP determination by antagonizing the activity of the Notch pathway in this cell, thereby protecting it from inhibitory signaling by its neighbors in the proneural cluster. A simple threshold model is proposed in which the principal role of H in SOP specification is to translate a quantitative difference in the activity of the Notch pathway (in the SOP versus the non-SOP cells) into a stable binary cell fate decision (Bang, 1995).

A single copy of Hairless, is able to suppress the wing defects of heterozygous strawberry notch, suggesting that Hairless and sno exhibit related antagonistic activities downstream of the Notch pathway. In a similar fashion, a single copy of Suppressor of Hairless and a single copy of sno show enhanced defects, indicating that Su(H) and Sno cooperate closely in patterning the wing. As Su(H) and Sno have not been shown to physically interact, this may mean that the two proteins work in parallel or that the interaction is too weak to be detected (Majumdar, 1997).

Hairless full length cDNA constructs give a robust rescue of H haplo-insufficiency phenotypes. In order to determine the respective wild type Hairless activity, deletion constructs were tested for their ability to restore loss of macrochaetae on head and notum and the wild type wing venation. Hairless derivatives are heavily impaired in their rescue capacity. Even a small deletion (the C4 deletion in the C-terminal protein of the protein) causes a dramatic drop in performance. Reduction of H activity is also observed in either N- or C-terminally truncated constructs, C1 and C6. C1, lacking the N-terminal amino acids has nearly lost all H wild type activity, while C6, lacking the 3' untranslated region and the 15 C-terminal amino acids, gives measurable rescue. Unexpectedly, the C3 construct in which the central acidic domain is deleted restores the number of bristles even better than full length H. This central domain, termed the A-domain, may mark a repression domain of the Hairless protein required for silencing Hairless function, e.g. for releasing Su(H) from a H/Su(H) complex. Loss of the N-terminal domain results in a construct that no longer rescues Hairless loss of function mutants yet is still able to induce gain of function phenotypes and, in addition, shows normal Su(H) binding in an in vitro assay. Apparently, the N-domain harbors sequences crucial for H wild type function, separable from the adjacent S-domain, required for binding of Su(H). It is conceivable that the N-domain is involved in the inhibition of Su(H) DNA binding and/or trans-activation, whilst the S-domain might represent mainly the H-Su(H) interactive surface (Maier, 1997).

Drosophila Hairless (H) encodes a negative regulator of Notch signaling. H activity antagonizes Notch (N) signaling during bristle development at the pupal stage. Clonal analysis reveals that H acts by inhibiting signal transduction rather than by promoting signal production, during both selection of microchaete precursors in the notum and vein cell differentiation in the wing. Allele-specific interactions further suggest that H inhibits Notch signal transduction by interacting directly with Suppressor of Hairless. Unexpectedly, this regulatory function of H appears to be essential only during imaginal development. Using a null allele of H that corresponds to a deletion of the H coding sequence, it has been shown that embryos devoid of both maternal and zygotic gene products develop similar to wild-type embryos. Thus, H activity is not strictly required to regulate N-mediated cell fate choices in the embryo (Schweisguth, 1998).

Quantitative trait loci (QTL) affecting response to short-term selection for abdominal bristle number have been mapped to seven suggestive regions that contain loci involved in bristle development and/or that have adult bristle number mutant phenotypes, and are thus candidates for bristle number QTL in natural populations. To test the hypothesis that the factors contributing to selection response genetically interact with these candidate loci, high and low chromosomes from selection lines were crossed to chromosomes containing wild-type or mutant alleles at the candidate loci, and the numbers of bristles were recorded in trans heterozygotes. Quantitative failure to complement, detected as a significant selection line*cross effect by analysis of variance, can be interpreted as evidence for allelism or epistasis between the factors on selected chromosomes and the candidate loci. Mutations at some candidate loci (bb, emc, h, Dl, Hairless) show strong interactions with selected chromosomes, whereas others interact weakly (ASC, abd, Scr) or not at all (N, mab, E(spl)). These results support the hypothesis that some candidate loci, initially identified through mutations of large effect on bristle number, either harbor or are close members in the same genetic pathway as variants that contribute to standing variation in bristle number (Long, 1996).

Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical Notch pathway in the process of lateral inhibition. The subsequent cell type specifications rely on distinct subsets of Notch signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act upon Su(H), strongly influences the entire cell lineage, demonstrating that it functions through targets other than Su(H) within the inner lineage. Therefore, Hairless and Numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications involve different sets of mediators depending on the cell type considered (Nagel, 2000a).

Hairless is an important antagonist of Notch signaling throughout the mechano-sensory organ lineage As suggested by the enrichment of H protein within all four cells of an emerging bristle, it is a potent antagonist of Notch signaling within this entire cell lineage. H protects the sensory organ precursor from a Notch signal. In the absence of H nearly all of the SOPs are forced into a non-neuronal fate. The consequence is a pronounced bristle loss. H is absolutely essential within shaft cells and the neuron and is important for the pIIb (precursor of a glial cell and of the pIIIa, the precursor of the sheath and neuron cells) as well, to allow for proper cell fate choices. Changing the dose of H affects all three levels of sensory organ development. However, achievement of the first level, emergence of the sensory organ precursor, is a prerequisite for the development of its progeny. Thus, the number of descendants that can be assessed for requirement of H activity is naturally small in a strong H compromised background. In accounting for this obstacle, a phenotypic series of H mutants has been analyzed and different sensitivities of external and internal cell fate decisions have been found: whereas the socket/shaft transformation is always complete, that of neuron to sheath appears gradual when looking in the hypomorphic condition. Only in a strong loss of function condition like in H amorphs, is the transformation complete. In the gain of function situation, the transformation is always complete in a wild-type background, where extra neurons arise at the expense of sheath cells, whereas a gradual transition is seen when deltex activity is elevated simultaneously. This finding suggests that the two internal cells, sheath cell and neuron, are not equivalent, in that the neuronal fate appears to be more stable over the thecogen fate dependent on deltex activity. Another example of such a bias is found in the decision between internal and external cell fate, where pIIb is clearly preferred over pIIa. In both cases, Notch signaling might be rather weak between the two cells, thus favoring the ground state (Nagel, 2000a).

The decision between the tormogen (socket) versus trichogen (shaft) fate of the pIIa progeny seems to depend strictly on the balanced doses of H and Su(H). Changes in the dose of either one pushes the equilibrium completely towards the opposite fate. Accordingly, Su(H) protein accumulates to very high levels in the future tormogen, and can thus override the elevated levels of H protein within this cell. The epistasis of H over dx regarding outer bristle cell fates can be easily explained by the dominating activity of Su(H) within the pIIa progeny. The choice between neuron and thecogen (sheath) fate is based on a quite different mechanism, because unlike H, Su(H) is not necessary for the emergence of the two opposing cell types. The default state of pIIIb descendants is neuronal. The Notch signal redirects one of these cells into thecogen fate. Although both Su(H) and dx, positively influence Notch signaling in the presumptive thecogen, none of the two is required for the generation of this cell type. Thus, the Notch signal in the thecogen might be transduced by a molecular mechanism independent of Su(H) or dx involving as yet unknown factor(s). In the absence of H, the presumptive neuron gains thecogen fate. Therefore, H has an important role in protecting the neuron from the Notch signal. Since this signal does not emanate from Su(H), H must act through unknown component(s). This is the first unambiguous example of a Su(H)-independent function of H (Nagel, 2000a).

Earlier work has identified numb as an important factor during binary cell fate decisions within the sensory organ lineage, where it is partitioned into those cells in which it acts as intrinsic inhibitor of the Notch signal. Both loss and gain of numb activity causes cell transformations very similar to H except for the selection of the SOP during lateral inhibition, which is controlled by H and not by numb. It is noted, however, that phenotypes are always much more penetrant when H is involved in comparison to numb. H mutations act as dominant enhancers of numb mutations, and the two proteins might physically interact with each other. Both activities are required for normal bristle development because loss of function of either gene gives a similar phenotype regarding cell type specification. To what degree do these activities overlap? Close inspection of the phenotypes shows that numb has a very strong influence on the pIIa/pIIb fate selection but less on the subsequent binary decisions because cell type transformations are always partial. Although this might be a quantitative difference, it is noted that H activity is strictly required to guard both shaft cell and neuron from a faulty Notch signal and that complete cell type transformations are observed as a consequence of H activity loss. Furthermore, H activity is also required during the process of lateral inhibition, suggesting that H is the more general antagonist in Notch-dependent processes (Nagel, 2000a).

Both in loss of function and gain of function combinations numb is epistatic to Su(H) within the pII cells, indicating that Su(H) acts downstream of numb. This is in agreement with a model, whereby numb antagonizes Notch signaling through direct interference with the Notch receptor. Within the pIIa progeny, however, Su(H) can override the inhibiting activity of ectopic numb protein. This interference might again be direct, because preliminary evidence from yeast interaction trap experiments shows that Numb and Su(H) physically interact. Furthermore, by inhibiting Notch signaling, numb might indirectly modulate the levels of Su(H) transcriptional activity. Thus, the conflicting epistasis data might reveal once more different sensitivities of the sensory organ cell lineage regarding Notch signaling, especially the preference of the pIIb over the pIIa fate (Nagel, 2000a).

During the development of mechanosensory organs, Notch is required at two distinct steps: the singling out of the sensory organ precursor, SOP, and the correct specification of cell fates within the sensory organ lineage, SOL. Apparently, different subsets of Notch signaling components are used for these two processes. Whereas SOP selection in the process of lateral inhibition requires the 'classical' battery of Notch signaling components, namely Su(H), dx, mam, E(spl) bHLH and H, the subsequent asymmetric cell divisions require only certain Notch components plus the intrinsic activity of numb. Numb plays a major role in the distinction between the pII siblings. In the pIIa progeny, socket cell fate is enforced with the help of Su(H) and, to a lesser degree, dx, and the role of H is to protect the shaft from this fate. In the sub-epidermal progeny, Notch signaling determines sheath cell fate, promoted to some degree by dx and Su(H). However, since neither of the components, Su(H), dx nor E(spl) bHLH are strictly required for the selection of thecogen fate, the Notch signal is transduced by other factor(s), X. The role of mam in this process is as yet undecided, since the mutant cell clones are rather uninformative and appropriate overexpression constructs are unavailable. The neuron has to be protected from the Notch signal, and both numb and H play a pivotal role in this process. Apparently, the target of numb is the Notch receptor itself. It is not clear whether H acts at the same level, or whether it acts on a different target, maybe directly involving the presumptive signal transducer X. Although both mam and dx might be targets of H, no physical interactions were observed in the yeast interaction trap assay. Overall, H represents a key player in antagonizing the Notch signal and thus assures, that in the end all four different cell types of the mechano-sensory organ arise (Nagel, 2000a).

A summary of Notch signaling during mechano-sensory organ development is presented. Notch signaling is required in the entire cell lineage, as is the antagonist Hairless. Whereas the lateral inhibition process uses the classical battery of Notch signaling components, the subsequent binary cell fate specifications rely only on a subset of these components and involve in addition the intrinsic antagonist Numb. In a first step SOP is singled out by lateral inhibition from a proneural cluster, protected through the activity of H. The surrounding cells are forced by the SOP into epidermal fate through the activation of the Notch receptor, implemented with the help of Su(H), dx, mam and E(spl) bHLH proteins (classical pathway). In a second step, Notch signal, promoted by Su(H) and dx, forces one SOP daughter cell into pIIa fate from which the pIIb cell is protected by the antagonists numb and H. The pIIb gives rise to the pIIIb and a glial cell. In a third step, the progeny of pIIa are socket and shaft cell. The socket cell receives the Notch signal via Su(H) and dx, the effector genes are unknown. The shaft cell is protected by H and numb from the Notch signal. In a fourth step, the progeny of the pIIIb are sheath cell and neuron. The sheath cell receives a Notch signal promoted by unknown factor X, whereas the neuron is protected by H and numb (Nagel, 2000a).

Overexpression of m4/alpha (see E(spl) region transcript m4) blocks lateral inhibition. The SOP pattern is a result of an interplay between proneural proteins, which promote neural fate, and Notch signaling, which inhibits it. The fact that supernumerary chaetael/SOPs arise in close apposition to each other suggests that the contact-dependent Notch signaling that normally counters SOP fate may be compromised. To locally block lateral inhibition for comparison purposes, a well characterized negative regulator of Notch signaling, Hairless (H) was overexpressed. H is known to negatively modulate Notch signaling by interfering with the activity of the transcriptional activator Su(H), by which at least part of the Notch signal is transduced to the nucleus (Apidianakis, 1999).

Generally, the effect of H overexpression is similar to that of m4/alpha. At the ACV (anterior cross-vein campaniform sensillum), L3 (third longitudinal vein campaniform sensillum) and wing margin clusters, the extent of SOP overcommitment is comparable to that caused by m4/alpha, whereas at the dorsal radius H gives much higher numbers of supernumerary SOPs. The effects of H differ from those of m4/alpha in two further respects. (1) H abolishes some wing margin sensilla, presumably by interfering with the Su(H)-dependent inductive Notch signaling that sets up the dorsoventral boundary, which subsequently induces margin SOPs. m4/alpha does not affect the process of dorsoventral wing patterning, consistent with the presence of a full complement of margin SOPs. (2) In the adult phenotype, whereas m4/alpha produces solely bristle tufting, H overexpression variably produces naked patches or double-shaft socketless bristles, consistent with its proposed role in the SOP lineage cell fate decisions. Ectopic expression of m4/alpha gives neither of these phenotypes, suggesting that it affects only SOP singularization but not the SOP lineage. In order to test this hypothesis, pupal nota were stained with antibodies directed against Elav, a neuron specific marker, and Pros, specific to the sheath cell. There is a one-to-one correspondence between Elav positive and Pros positive cells. Therefore, overexpression of m4/alpha does not upset the Notch/Numb mediated asymmetric divisions in the SOP lineage. The only step in sensory organ development that m4/alpha seem to affect is that of lateral inhibition, which restricts the number of SOPs produced per proneural cluster (Apidianakis, 1999).

Experiments by Nagel (2000), suggest that the overexpression phenotype of E(spl) m4 and E(spl) malpha obtained by Apidianakis (1999) is likely to be due to a dominant negative effect and does not reflect the biological function of these two genes. In order to elucidate m4/malpha gene function directly, RNAi, which causes sequence-specific transcript degradation, was carried out by injecting either m4 or malpha double-stranded RNA or a mixture of both into pre-blastoderm embryos. In agreement with genetic data, RNAi causes a high incidence of lethality (~50%). Dead embryos develop intermediate to strong neurogenic phenotypes (too many neurons) typical of loss of E(spl) bHLH activity. Surviving embryos hatch into wild type appearing larvae that develop normally to adult flies. From this it is concluded that the m4/malpha genes are required to positively transduce the Notch signal during neurogenesis, and presumably during bristle development as well. Therefore, suppression of lateral inhibition observed after overexpression of either m4/malpha family member must be due to a dominant-negative effect, presumably by titrating out other important Notch pathway components (Nagel, 2000b).

roDom is a dominant allele of rough (ro) that results in reduced eye size due to premature arrest in morphogenetic furrow (MF) progression. The roDom stop-furrow phenotype is sensitive to the dosage of genes known to affect retinal differentiation, in particular members of the hedgehog (hh) signaling cascade. roDom interferes with Hh's ability to induce the retina-specific proneural gene atonal (ato) in the MF and normal eye size can be restored by providing excess Ato protein. roDom was used as a sensitive genetic background in which to identify mutations that affect hh signal transduction or regulation of ato expression. In addition to mutations in several unknown loci, multiple alleles of groucho (gro) and Hairless (H) were recovered. Analysis of their phenotypes in somatic clones suggests that both normally act to restrict neuronal cell fate in the retina, although they control different aspects of ato's complex expression pattern (Chanut, 2000).

Loss-of-function ro mutations cause eye roughness, due to mis-specification of photoreceptors R2 and R5, and the formation of ommatidia with more than one R8 photoreceptor. Repression of R8 cell fate has been attributed to inhibition of ato expression by the Ro homeodomain protein. In support of this proposal, Rough and Atonal proteins appear in complementary sets of cells behind the MF, and ato expression is expanded behind the MF in ro mutants. Generalized expression of ro under a heat-shock promoter (hs-ro) leads to loss of ato expression in the MF and eventually results in furrow arrest. Furrow arrest in roDom is also accompanied by loss of ato expression in the MF. By analogy to the hs-ro phenotype, it is proposed that roDom leads to excess Ro production, although that excess is not detectable by antibody staining (Chanut, 2000).

roDom is very sensitive to alterations of ato gene dosage, since it is enhanced by loss-of-function ato alleles and almost completely rescued when high levels of ato expression are restored ahead of the MF. The roDom phenotype therefore appears to result primarily from inhibition of ato expression due to excess Ro protein. On the basis of this understanding, the role of two of the strongest suppressors isolated in this screen, new alleles of gro and H, were analyzed on ato regulation and furrow progression (Chanut, 2000).

Hairless inhibits N signaling by preventing Su(H), a transcription factor, from translocating to the nucleus and activating transcription of N targets such as the E(spl) complex genes. In the absence of H, Su(H) is free to enter the nucleus upon activation of N. Su(H) mutant clones lead to expanded Ato expression behind the MF, consistent with a role for Su(H) in the N-mediated lateral inhibition that leads to the refinement of ato expression. In H clones are found in which the refinement of ato expression to single cells appears accelerated. This is consistent with a role for H as an inhibitor of N and Su(H) in lateral inhibition. Surprisingly, however, individual clusters of Ato-expressing cells often persist in H mutant tissue behind the MF, instead of resolving to single R8 precursors; in adults as well, mutant ommatidia often contain more than one R8. This would suggest that at later stages H is required to resolve ato expression to single R8 precursors, a role that is not expected for an inhibitor of the N pathway. Anterior H mutant clones show precocious ato expression anterior to the MF. This might explain the patterning defects behind the MF, if precocious and excessive accumulation of Ato protein in the MF interferes with the proper execution of lateral inhibition via N or with downregulation by Ro. In this regard, it is noted that excess Ato protein, as provided under heat-shock control, is found to perturb the resolution of ato expression to single R8 precursors (Chanut, 2000).

It has been suggested that early ato expression, ahead of the MF, is in part the result of an as yet unsuspected 'proneural' effect of N signaling. The anterior expansion of ato expression in H mutant clones is consistent with this model, assuming that H would act as an inhibitor of N there as well. However, the proneural function of N must not be mediated by Su(H), since removal of Su(H) function does not abolish ato expression ahead of the MF. The results presented here may indicate that H antagonizes the proneural function of N via a mechanism that does not involve Su(H). Alternatively, the role of H on early ato expression may be independent of N signaling. Regardless of the exact mechanism, the enhanced expression of ato ahead of the MF in H mutants is likely to explain suppression of the roDom phenotype by counteracting the effect of ectopic Ro on ato expression in the MF (Chanut, 2000).

Finding that similar levels of suppression can be achieved by loss-of-function mutations in H and gro (which act in opposite directions in the N pathway) is not unique. A similar situation was encountered in another study where mutations in gro and H were both found to enhance the wing and bristle phenotypes associated with loss-of-function mutations in Egfr. The observation that mutations in both genes elevate ato expression in the vicinity of the MF, but at different stages of the differentiation process, helps resolve this paradox. The results also indicate that the exact timing (or location) of ato expression might not be crucial to MF progression, provided adequate levels are reached. This conclusion is supported by the finding that Ato supplied anterior to its normal expression domain, in the h expression domain, restores normal eye size in a roDom background. Whether proper R8 spacing and ommatidial patterning can be achieved under these conditions remains to be shown (Chanut, 2000).

The Notch pathway is known to act during initiation and differentiation of wing veins to refine the adult vein pattern. Since nemo mutant, nmoadk, was identified as a modifier of Notch in the eye, the link between nmo and Notch signaling in the wing was investigated. Genetic interactions between nmoadk and mutations in several components of the Notch pathway were characterized. Mutations in the ligand Delta (Dl/+) cause a mild vein thickening phenotype. This mutation is synergistically enhanced by homozygosity for nmoadk. Conversely, mutations in the negative regulator Hairless (H/+), which normally exhibit shortening of LV, suppress the ectopic veins seen in nmoadk. In addition to interactions in wing veins, H and nmoadk show a synergistic interaction in the macrochaete bristles of the head and notum (Verheyen, 2001).

nmoadk flies have a mild bristle loss phenotype, and occasionally display bent bristles or duplicated bristles. H/+ flies display a characteristic dominant loss of macrochaetes. Homozgosity for nmoadk in a H/+ background leads to a dramatic enhancement of the H/+ bristle loss phenotype. Since nmoadk mutations are enhanced by Dl, and are suppressed in the wing by H, whether nmo acts upstream of Notch was examined. It was asked if the nmoadk extra vein defect could be rescued through ectopic activation of Notch signaling. Delta and E(spl)mß were ectopically expressed with the 32B-Gal4 driver, which is expressed in the wing blade. E(spl)mß is normally expressed in the cells flanking the presumptive veins and acts to suppress rhomboid expression to the narrow band of vein progenitors. Ectopic expression of UAS-E(spl)mß leads to mild vein thinning and a shortening of LV. Both UAS-Delta and UAS-E(spl)mß specifically suppress the extra veins associated with nmoadk mutations. Thus, both ectopic activation of the Notch pathway and loss of a negative regulator as seen with H1/+ can lead to suppression of ectopic veins caused by nmoadk. These results suggest that Nemo is upstream of Notch and acts in a common vein regulatory pathway (Verheyen, 2001).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose has also been identified in a genetic screen for modifiers of Hairless (H), a Notch pathway antagonist (Schreiber, 2002) and rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Hairless was identified as antagonist in the Notch signaling pathway based on genetic interactions. Molecularly, Hairless inhibits Notch target gene activation by directly binding to the Notch signal transducer Su(H). Additional functional domains apart from the Su(H) binding domain, however, suggest additional roles for the Hairless protein. To further understanding of Hairless functions, a genetic screen was performed for modifiers of a rough eye phenotype caused by overexpression of Hairless during eye development. A number of enhancers were identified that comprise mutations in components of Notch- and Egfr-signaling pathways, some unknown genes and the gene rugose. Mutant alleles of rugose display manifold genetic interactions with mutants in Notch and Egfr signaling pathway components. Accordingly, the rugose eye phenotype is rescued by Hairless and enhanced by Delta. Molecularly, interactions might occur at the protein level because rugose appears not to be a direct transcriptional target of Notch (Schreiber, 2002).

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development. Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling. Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions. Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Spencer, 2003 and references therein; Islam, 2003 and references therein).

Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).

In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).

The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).

H antagonizes Notch target gene activation by binding to the Notch signal transducer, Su(H). Accordingly, overexpression of H phenocopies reduction of Notch activity. Ectopic expression of H in the pnr domain results in the formation of multiple/split bristles and loss of epidermal tissue. This phenotype is enhanced in animals with suppressed fred activity in the pnr domain. Functional interactions between H and fred are also evident in the eye. UAS-H/GMR-GAL4 flies have eyes that are slightly smaller along the anterior-posterior axis and show ommatidial fusion and interommatidial bristle tufting, as well as bristle loss. When fred activity is suppressed in this genetic background, there is an enhanced disruption of the eye morphology. Ommatidia lack definition, bristle tufting is more severe, and loss of bristles is observed (Chandra, 2003).

The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).

Hairless interaction with the Egf pathway

The Drosophila epidermal growth factor receptor (Egfr) is a key component of a complex signaling pathway that participates in multiple developmental processes. Carried out was an F1 screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. In this screen, mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci were all recovered. All of the dominant enhancer mutations, termed E(Egfr)s, identified show dominant interactions in transheterozygous combinations with one another and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and Egfr signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro reveal that reductions in Egfr activity enhance both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles lead to the formation of ectopic compound eyes in a dosage sensitive manner. These findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context (Price, 1997).

Wing vein development in Drosophila is controlled by different morphogenetic pathways, including Notch. Hairless (H) antagonizes Notch target gene activation by binding to the Notch signal transducer Suppressor of Hairless [Su(H)]. Accordingly, overexpression of H phenocopies reduction of Notch activity. In the construct H-C2, the presumptive Su(H)-binding domain of Hairless has been removed. As a consequence, H-C2 protein has completely lost the ability to bind to Su(H) protein and to interfere with Su(H)-dependent developmental processes in vivo like bristle development, wing margin specification or vein width refinement. Apart from the internal deletion, the H-C2 protein compares to the wild type with respect to antibody recognition, apparent molecular weight, subcellular distribution, and stability as well as biochemical interactions with other H partner proteins. With regard to endogenous activity, the H-C2 lines are rather weak, compared to the full length H constructs; however, after heat shock induction, expressivity is similar. Surprisingly, overexpression of H-C2 induces lethality as does the wild type construct and in addition, leads to the induction of ectopic vein material in certain intervein regions of the wing (Johnnes, 2002).

Keeping in mind that H itself is not a transcriptional regulator but rather functions through protein-protein interactions with different protein targets, these phenotypes cannot be simply explained by altered activation of Notch target genes. Rather, they might uncover a currently unknown Su(H) independent activity of H involving different protein(s) maybe outside of the Notch signaling cascade. In order to understand this phenomenon, the involvement of H-C2 in the process of vein establishment was analyzed in comparison to wild type H. The data presented in this work are in agreement with a model whereby H, apart from antagonizing Notch signaling, positively regulates EGF signaling during the process of wing vein formation (Johnnes, 2002).

In a screen for genetic modifiers of the H-C2 phenotype, several genes involved in Notch and epidermal growth factor (EGF) signaling were identified. Most notably veinlet (rhomboid), an activator of EGF signaling, acts downstream of H-C2. H-C2 positively regulates veinlet maybe through inhibition of intervein determinants in agreement with a model, whereby Notch and EGF signaling pathways cross-regulate vein pre-patterning (Johnnes, 2002).

Overexpression of hs-H-C2 induces ectopic veins only between day 5 and 6 after egg deposition. Phenotypes vary significantly and were arranged into a phenotypic series of five classes. Using precisely synchronized cultures, the pheno-critical period was restricted to pre-pupal and early pupal developmental stage, starting approximately at the larval-prepupal transition. Induction of H-C2 during mid- to late-third instar larval stages did not result in ectopic vein formation even with prolonged and, in their consequence, semi-lethal heat shocks. Ectopic venation was not randomly distributed and certain regions of the wing blade were more sensitive than others. In order to distinguish between temporal and/or sensitivity differences, the wing was partitioned into distinct intervein sectors A-F and the appearance of extra veinlets over time for each sector was scored independently. As became apparent, the six different sectors respond with a similar temporal profile, but with different sensitivities. The less sensitive sectors A and C revealed two pheno-critical periods, which might, however, be a consequence of the time convolution of the data. In summary, the main impact of H-C2 on the wing venation process occurs in the pre-pupal and early pupal stages of development (Johnnes, 2002).

The H-C2 protein is unable to bind to Su(H) and has lost basically all of H wild type activity: H-C2 is unable to rescue the haplo-insufficient H loss-of-function phenotype and does not cause the typical H gain-of-function bristle phenotypes. Thus, H-C2 venation phenotypes might either uncover a Su(H) independent function of H or an unrelated, novel activity. Although overexpression of H wild type constructs causes only little ectopic vein material, H and H-C2 proteins are able to synergize upon overexpression. It is concluded that the vein inductive property of H-C2 is a native function of H, and that H is able to partially substitute for H-C2 in this process (Johnnes, 2002).

If indeed the vein inductive property of H-C2 uncovers a Su(H) independent activity of H, the question arises as to what other targets H might act upon. In order to identify such putative targets, a candidate screen was set up for dominant modifiers, concentrating on the three main signaling pathways which normally contribute to wing vein development: Notch, Egf and Dpp signaling cascades. In the double heterozygotes, H-C2 was induced during the pheno-critical period with double heat shocks to make up for the weak phenotypes caused by a single hs-H-C2 copy (Johnnes, 2002).

In a first set of experiments, combinations with Notch-family members and relatives were analyzed. These included mutations in the Notch receptor itself and in Notch ligands, mutations affecting the signal transduction machinery as well as Notch target genes. Furthermore, other neurogenic mutations as well as several proneural members were included in the screen. Reduction of single doses of Dl or components of the E(spl) complex including E(spl)mß, enhance the H-C2 wing phenotype in a dominant manner. The behavior of most of the other components of the Notch signaling pathway were largely neutral in this screen. Interestingly, N loss of function alleles act as strong negative modifiers, whereas NAx alleles enhance the response to H-C2 overexpression (Johnnes, 2002).

Components of the EGF signaling pathway, screened for dominant interactions with H-C2, included ligand and its processing, the receptor itself, components of the signal transduction cascade as well as related genes. As expected, ve and to a lesser degree Star (S) mutations dominantly reduce the amount of extra vein material. Both genes are essential for vein formation, and thus, reduction of their activity was expected to antagonize the H-C2 vein-promoting effect. By lowering the ve gene dose, the suppression is almost complete except for some ectopic vein material in the region of the anterior cross-vein, a region which is not affected by the homozygous ve1. Only weak, interactions were found with Dpp pathway mutations (Johnnes, 2002).

In agreement with the essential role of ve in the establishment of vein fate, data indicated that the homozygous ve1 mutant completely suppresses ectopic vein induction through hs-H-C2, except for some small veinlets. No ectopic veins were visible in the distal wing blade, where the ve1 phenotype is apparent, even after strong overexpression. This demonstrates that H-C2 strictly depends on ve for the induction of veins and suggests that overexpression of H-C2 might somehow result in the ectopic activation of ve (Johnnes, 2002).

In accordance with the proposed role of net as negative regulator of ve, net1 mutations cause extensive extra veins. This phenotype comprises nearly all aspects of hs-H-C2 overexpression in a wild type background. Unexpectedly, overexpression of H-C2 enhances the net phenotype considerably: not only do the heterozygous net1 mutants resemble the homozygotes, but the homozygotes developed massive patches of vein tissue and extensive blistering of the wing. Moreover, net1; hs-H-C2 homozygotes were semi-lethal at 25°C and the stock was only viable at 18°C. Because net1 is a complete null allele, this result excludes the simple model that H-C2 promotes vein development by inhibition of net activity. Instead, H-C2 acts independent of net either as a vein-promoting factor, e.g., by activation of ve, or by repression of other negative regulators of ve that act in addition to net. The latter seems more plausible with regard to normal H function. In the heterozygous net1 background, which is phenotypically wild type but sensitized for ectopic vein formation, the vein-promoting activity of H is revealed: overexpression of full length H in net1 heterozygotes results in ectopic venation that is a perfect phenocopy of the H-C2 effects. Apparently, wild type H has a vein-promoting activity, which also can be explained by antagonizing a negative regulator of ve (Johnnes, 2002).

Since induction of ectopic vein material by H-C2 is extremely sensitive to developmental time, the epistatic relationship with ve1 was reassessed by continuously overexpressing H-C2 or H with the aid of the Gal4/UAS system in a wild type and a ve1 background. Prolonged overexpression of H-C2 with en-Gal4 or BxMS1096-Gal4 driver lines results in conversion of most of the distal intervein areas to vein tissue. Only the region between L3 and L4 proved resistant. Furthermore, induction of microchaetae in this area was observed. Apart from this conversion, extensive tissue loss was noted while the wing margin itself remained intact. Overexpression of H-C2 in the ve1 background shows both tissue loss and ectopic microchaetae on the wing blade. Again, no induction of ectopic vein material was observed in the more distal regions in support of the notion that H-C2 acts upstream of ve. It is proposed that H-C2 promotes vein induction by up-regulating ve activity. Moreover, processes independent of ve are influenced by H-C2, which finally lead to the induction of ectopic bristles on the wing blade and to tissue loss. Overexpression of H in the same experimental set-up was unable to convert intervein tissue into vein material, apart from few ectopic veinlets. At the same time, H causes wing tissue and margin loss accompanied by broadened wing veins. These are the known hallmarks of an impaired Notch signaling (Johnnes, 2002).

Since vein determination depends on the balance between ve and bs activity, the influence of H or H-C2 on wild type bs or ve gene expression was examined. Full length H and H-C2 expression was ectopically induced from UAS-constructs in the posterior compartment via the en-Gal4 driver. ve expression was monitored with either enhancer trap line, verho-lac1 or veX81. Expression of ve and bs is mutually exclusive in vein and intervein tissue, respectively. Overexpression of H does not alter this complementary expression pattern, however, overexpression does cause strong ve expression within and confined to pro-vein areas. This is in contrast to the effects of H-C2 overexpression where ectopic expression of verho-lac1 is very prominent in pupal wings already in early pupal stages and remains strongly activated at least until 36 h after pupal formation (APF). Earlier wing development was analyzed using the veX81-lacZ reporter. In wing discs from late third instar larvae, no patterning defects apart from tissue loss are observed. However, deviations from wild type become apparent already in the pre-pupal wing discs as early as 4 h APF. At 6 h APF, a strong and reliable ectopic staining near the wing margin, where pro-vein 5 develops, is observed that later spreads into the adjacent intervein field. Overall, ectopicve expression reliably predicts the pattern of ectopic venation caused by overexpression of H-C2. This is in agreement with the hypothesis that ve regulation is a target of H-C2 activity (Johnnes, 2002).

The simplest model to explain the differential effects of H and H-C2 on the regulation of ve expression would be the assumption of a vein inductive role of Notch in pre-pupal and early pupal development in agreement with the biphasic developmental role of Notch, e.g., during eye development, where, in the course of lateral inhibition, Notch first promotes proneural fate before restricting this fate to single photoreceptor precursor cells. By binding to Su(H), H would limit such a vein-promoting activity of Notch at an early inductive phase. Because H-C2 is unable to bind to Su(H), an assumed inductive Notch signal would be able to pass and thus, set the stage for ectopic veins. To test this assumption, an activated Notch receptor (Nintra) was expressed under heat shock control during the H-C2 pheno-critical period. Since hs-Nintra overexpression at 37° proved extremely lethal to larvae and pupae alike, the induction was performed at lower temperatures of 34-35°. Under these conditions, hs-Nintra is able to induce ectopic veinlets, mostly of cross-vein character, in all the regions where H-C2 is also able to induce ectopic vein material. These results demonstrate that Notch signaling is able to exert a positive influence on wing vein specification during early pupal stages, closely followed by the well characterized vein suppressing activity of Notch signaling (Johnnes, 2002).

The onset of pupariation is a major developmental switch, where expression of many genes as well as their developmental effects changes dramatically. This is, for example, observed in the regulation of ve and bs from third instar larval stage to early pupal stage: whereas the activation of both genes early on depends on pre-pattern genes like net, their regulation becomes inter-dependent and mutually exclusive about 4 h after puparium formation. The abrupt onset of H-C2 vein-promoting activity is interpreted accordingly. Maybe, H-C2 responds to or influences the activity of other factors which only become available at that time and play a role in the promotion or repression of vein development. This is reflected by the onset of the positive influence of H-C2 on ve expression at around 4 h after pupariation. Thus, the H-C2 pheno-critical period might reflect a developmental switch for a requirement of H activity for vein fate decisions (Johnnes, 2002).

Involvement of the Notch signaling pathway in the refinement of proper vein width is well established. Current models suggest that during this lateral inhibition process, Dl acts as an inhibitor of vein formation by directing cells, neighboring presumptive vein cells, into the intervein fate. This model is in line with the observation that Dl mutants act as enhancers of H-C2 vein promotion. In Dl mutants, the threshold for vein fate is lowered as determined vein cells are less likely to be driven back into intervein fate. The interrelationship of H-C2 and Notch signaling during vein formation is, however, not restricted to the process of vein width refinement. Overexpression of Nintra promotes early vein formation and may thus be setting the stage for pro-vein development within intervein areas. Despite the fact that Notch activity is not necessary for pro-vein specification itself, it is required for the vein-promoting activity of H-C2 because Notch mutations act as strong dominant suppressors of H-C2 effects. In agreement, NAx-E2, a hyper-activated allele of Notch, acts as a weak enhancer of H-C2 and it is even possible that this effect is initially stronger but then obliterated through enhanced lateral inhibition (Johnnes, 2002).

Reduction of the ve gene dose results in a very pronounced, dominant repression of the H-C2 phenotype despite the fact that the allele ve1 has no dominant visible phenotype. This suggests that ve plays a crucial role for H-C2 to exert its inductive effects. Interestingly, dosage reduction of either the Drosophila EGF receptor or the MAPK rolled (rl) has no dominant influence on the H-C2 ectopic venation phenotype. The former result was unexpected and suggests that the Drosophila EGF-receptor itself is not rate limiting in this process. This notion is in line with the observation that also ve1 is fully recessive in combination with loss-of-function alleles for the Drosophila EGF-receptor. The allele rl1 is a mild hypomorph and the reduction of MAPK activity might not be strong enough to influence the H-C2 phenotype. Together with the results of full epistasis of ve over H-C2, these data suggest that neither the Drosophila EGF-receptor nor the EGF signal transduction cascade are influenced by overexpression of H-C2 (Johnnes, 2002).

Overexpression of H results in the extension of ve expression all over the pro-vein area, whereas overexpression of H-C2 induces, in addition, ve outside the pro-veins, also within the intervein fields. Thus, both act positively on ve regulation but H-C2 is clearly different from H with respect to the apparent conversion of presumptive intervein- to pro-vein cell fates. Pro-vein activity is a normal aspect of H wild type function that is uncovered in a sensitized background: halving the gene dose of net or bs might result in a subtle increase of ve activity which can then be pushed by H above the threshold for pro-vein fate. Although the results clearly demonstrate that H and H-C2 act positively on the regulation of ve, it cannot be concluded that this regulation is direct. Rather H might act negatively on the output of vein repressing factors. Since H has the capability to interact with a number of different proteins, overexpression of either H or H-C2 could influence stoichiometry of complexes or availability of factors involved in ve regulation. Two such factors that have overlapping activity with regard to the negative regulation of ve are encoded by net and bs, however, they show remarkably different temporal activity profiles in that net acts during larval stage and bs at the transition of larval to pre-pupal stage. Thus, bs appears the more likely target of H activity; this is supported by the fact that H-C2 can promote vein induction even in the absence of net and that bs repression is observed at the anterior cross-vein without simultaneous ve induction. Whether this inhibition is direct or via as yet unknown factors and pathways requires further study (Johnnes, 2002).

Thus, both H and H-C2 have the ability to up-regulate ve. However, in contrast to H, H-C2 overexpression is capable of overriding intervein fate and thus inducing ve expression within intervein territories. Several lines of evidence suggest that this is a normal facet of H function. (1) Synergistic effects of combined overexpression of H and H-C2 were found. (2) In a sensitized genetic background of reduced copies of intervein specifying genes like bs and net, H itself possesses vein-promoting activity just like H-C2. These results can be explained if one assumes a dual, independent activity for H: in one case, H might up-regulate ve, for example, by interfering with intervein specifying factors, an activity retained by H-C2. In another case, H, by virtue of binding to Su(H), antagonizes Notch-dependent processes such as an early vein fate-promoting activity and subsequently vein width refinement. This activity is presumably lost in H-C2 due to the lack of the Su(H)-binding domain. Unfortunately, attempts to directly test this hypothesis failed because Su(H) mutant clones could not be generated in the background of H-C2 (Johnnes, 2002).

A dual role of H in suppressing inductive Notch signaling and enhancing ve activity would explain why the wild type protein is unable to induce ectopic venation except in a sensitized background, where ve activity has already reached a critical threshold by the reduction of its negative regulators. In contrast, H-C2 can no longer antagonize Notch activity, but might still promote vein formation by interfering with ve suppression. Altogether, the genetic data may be taken as an example for a link between Notch and EGF signaling, which in the context of vein formation appears to involve the activity of H influencing both Notch signaling and, via ve, EGF signaling as well (Johnnes, 2002).

Genetic modifier screens on Hairless gain-of-function phenotypes reveal genes involved in cell differentiation, cell growth and apoptosis in Drosophila melanogaster

Overexpression of Hairless (H) causes a remarkable degree of tissue loss and apoptosis during imaginal development. H functions as antagonist in the Notch (N) signaling pathway in Drosophila, and the link to growth and apoptosis is poorly understood. To gain further insight into H-mediated apoptosis, two large scale screens were performed for modifiers of a small rough eye phenotype caused by H overexpression. Both, loss- as well as gain-of-function screens revealed known and new genetic interactors representing diverse cellular functions. Many of them do not cause eye phenotypes on their own, emphasizing a specific genetic interaction with H. As expected, the components of different signaling pathways identified were supposed to be involved in the regulation of cell growth and cell death. Accordingly, some of them also act as modifiers of proapoptotic genes, suggesting a more general involvement in the regulation of apoptosis. Overall, these screens highlight the importance of H and the N pathway in mediating cell death in response to developmental and environmental cues and emphasize their role in maintaining developmental cellular homeostasis (Muller, 2005).

Programmed cell death is used to remove damaged or supernumerary cells and serves as a substantial patterning mechanism during the development of complex animal structures. In Drosophila, apoptosis has been shown to be required, e. g., for shaping of the nervous system, patterning of the pupal eye, metamorphosis or proper development of germ cells. Crosstalk between different signaling pathways fuels differentiation and apoptosis alike. The N signaling pathway is one example of a cell-cell communication pathway involved in a large number of cell fate decisions that is associated with apoptotic processes as well. This study aimed at finding factors that modify apoptotic phenotypes resulting from overexpression of H in the eye. Both a misexpression and a loss-of-function screen were performed based on chromosomal deficiencies. This twofold approach allowed the strengths of one to be played off the weaknesses of the other. While a deficiency-based screen can quickly map loci interacting with H, it can be difficult to subsequently identify specific mutations that account for this interaction. In addition, since only a fraction of mutations results in visible phenotypes, modifiers may go unnoticed especially in cases of gene duplication and redundancy. Therefore, a complementary overexpression screen may identify genes that are missed otherwise. In the past, gain-of-function genetics has been successful in identifying genes crucial to different developmental processes like oogenesis, tissue growth, sensory organ development or thorax formation. The gain-of-function screen identified a total of 86 factors including 57 enhancers and 29 suppressors. Effects arising from the misexpression of these factors on their own are a potential drawback of this screen: roughly 40% of the enhancers and 60% of the suppressors displayed phenotypes on their own when overexpressed in the eye. However, more than 50% of them (44 out of 86) showed the opposite effect on GMR (a glass promoter construct) driven H when tested in the respective loss-of-function mutant background, arguing for a specific connection to H. Moreover, some of the genes identified in this screen may regulate the glass gene itself. This was taken into account by testing all identified suppressors for their rescue ability of tissue loss and apoptosis caused by H overexpression during wing development. In support of specific interactions with H, the majority (23 of 29) ameliorated these effects arguing against an involvement of glass (Muller, 2005).

In the loss-of-function screen, 41 deficiencies were recovered and 36 different loci were subsequently mapped, 22 acting as suppressor and 14 as enhancer. 10 of them were also recovered in the gain-of-function screen. One explanation might be, that altogether the deficiencies uncovered just 75% of the genome, leaving a quarter uninspected. Moreover, the collection of EP lines used accounts for roughly 10% of the genes in the entire genome. These numbers illustrate the benefit of taking various genetic approaches and emphasize that no single screen will identify all or even most potential interactors (Muller, 2005).

The N signaling pathway regulates a plethora of developmental processes including various differentiation steps and cell death during eye development. Since H acts as general antagonist of N, one might expect a variety of diverse factors to modify phenotypes caused by H overexpression. For this reason, the isolated modifiers were subjected to further analyses with regard to their own phenotypes and their general involvement in apoptosis (Muller, 2005).

A majority of the 57 enhancers [33 or 58%] caused no phenotype or even bigger eyes upon overexpression, indicating that they do not induce tissue loss on their own. Interestingly, 15 of them were also identified in screens conducted to find factors involved in thorax formation, bristle development, mesoderm development, cell growth in the eye or synapse formation. Since N signaling regulates various aspects in the development of these different tissues and organs, one might speculate that this group of enhancers affects N activity primarily during differentiation processes. Although not identified in the other screens, the remaining 14 factors, belonging to functional categories as diverse as growth regulators, transcription factors or protein kinases and enzymes might be connected to the N signaling pathway as well, thus reflecting the manifold N dependent processes in the development of Drosophila (Muller, 2005 and references therein).

A total of 24 factors showed no apparent effect in cell death assays. They comprise several N pathway components (emc, sd, tom), the novel factor CG8788 and also smrter, which functions as a co-repressor and might mediate transcriptional repression of N target genes also in Drosophila. However, most of the genes in this category have functions related to cell division and cell growth. For example, Dap overexpression reduces growth and proliferation in the eye imaginal disc and causes lethality upon combined misexpression with H. Consistent with the notion that levels of dMyc determine growth and cell proliferation, mutants in this gene are also lethal in trans with GMR driven Hairlesws. dMyc activity is regulated by several morphogens and the results suggest that N may also be involved at least for some aspects of dMyc regulation. Another group of genes, including bazooka, fat, inflated or Rok, functions in cell adhesion and cell polarity. The incorrect establishment of epithelial polarity is accompanied by hyperplastic growth which can be synergistically enlarged by an ectopic N signal. The finding that mutations in any of these loci behave as suppressors of H overexpression raises the possibility of a rather direct connection to the N signaling pathway. Thus, this screen uncovered several genes, which influence H and N activity during growth and proliferation, raising the question of their molecular role in the N signaling pathway (Muller, 2005).

Exactly 50% of all different modifiers (56 of 112) were either rescued by DIAP1 or influenced cell death inducers themselves. The recovery of factors known to be generally involved in apoptosis, like reaper, thread and bantam or more specifically during eye development like klumpfuss, was not only expected but was demanded by the screening approach. Interestingly, two of these genes (Rac2 and Eip78C) were in a data set collected in course of a genome-wide analysis of steroid-triggered cell death response in Drosophila. A further connection between N and the ecdysone regulatory network was recently established during metamorphosis of the midgut. The regulatory input of EGFR-signaling as well as crosstalk with N-signaling in the control of cell death has been shown at different stages of Drosophila development, most notably in the eye. In agreement with these earlier findings, several EGFR-pathway members were identified as modifiers of H and cell death inducers alike (e.g. aop, DER, lilli, pnt and rho). More interestingly, several members were identified of the JNK pathway (e.g. bsk, hep, msn, puc), which has been involved earlier in morphogenetic as well as stress induced apoptosis. Genetic analyses have demonstrated that JNK signaling is an effector of larval and pupal apoptosis. During embryogenesis N signaling has a negative effect on JNK signaling in the process of dorsal closure. However, this seems to involve non-canonical N signaling. Besides this influence on patterning processes, this work points to an involvement of canonical N signaling in JNK mediated morphological cell death. In this context it is interesting to note that the screen also identified a phosphatase subunit: overexpression of PP2A-beta' (B56) encoded by widerborst strongly suppresses H, p53 and grim induced cell death in the eye, whereas wdb mutants act as enhancers of H. In agreement, knockdown of B56 PP2A during embryogenesis results in caspase activation. Genetically, it has been placed in the p53-regulated path of apoptosis (Muller, 2005).

A strong correlation was found between H induced cell death and p53 mediated apoptosis. For example, cell death induced by overexpression of p53 can be rescued by increasing N signals [N- or Su(H) gain- or H-loss-of-function]. Further studies will determine the molecular mechanisms underlying these genetic interactions (Muller, 2005).

Many of the identified interactors have been previously implicated in different aspects of development but not in apoptotic processes. One example is the IMP (IGF-II mRNA-binding protein). IMP is ecdysone inducible and was suggested to be involved in the regulation of translation, maybe during metamorphosis, arguing for a role of IMP in regulating cell death in this context. Interestingly, several genes involved in chromatin remodeling were identified as strong interactors of H and the other tested cell death inducers. The Drosophila Brahma complex plays an important role during G1 phase of the cell cycle. In the current study, brm2 mutants behaved as enhancer of H and the proapoptotic genes hid, rpr and grim, but not of the stress-induced p53 apoptotic pathway. This argues for an additional role of brm in the coordination of cell death, besides its well defined function in the regulation of cell growth. Another example is DspI that was identified in these screens as general repressor of apoptosis. DspI encodes a transcriptional corepressor that binds to Dorsal and Relish (Rel) proteins. Like their mammalian counterparts, Rel proteins mediate immune-response via JNK signaling. It is tempting to speculate that Rel proteins together with DspI might likewise protect against apoptosis by limiting the JNK signal. In this case, the effects of Dsp1 on H mediated apoptosis can be easily explained and provide a further link for a crosstalk between JNK and N pathway. A third factor in this group is the Drosophila CREB binding protein, encoded by the nej locus, and belonging to the CBP/p300 family. nej is required at successive stages of eye development and overexpression caused severe retina degeneration. Since nej mutants have anti-apoptotic effects on H and most cell death inducers alike, one might assume a 'manager' function in the control of cellular homeostasis and apoptosis (Muller, 2005).

Last, seven of 15 interactors with hitherto unknown function were shown to interfere with apoptosis. The future challenge will be to determine the molecular and functional relationship between these new genes and cell death induction by H (Muller, 2005).

The screens provided a wealth of new information regarding cell death induction observed after overexpression of H. The results are compatible with the notion that changes in N activity have an effect on cell death in response to abnormal or imbalanced developmental signals within a cell. In agreement, the identified modifiers include factors and signaling components like p53, JNK-signaling and hormone triggered factors, all known to be involved in the coordination of a wide range of biological responses, including growth, differentiation and programmed cell death. Apparently, N signaling is required for the correct interpretation of such developmental signals and for the crosstalk between different signaling pathways that is essential for cell survival and differentiation (Muller, 2005).

putzig is required for cell proliferation and regulates notch activity in Drosophila

The gene putzig (pzg) is a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that exists within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. This study provides in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. These findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation (Kugler, 2007; full text of article).

In a developing organism, cell proliferation and apoptosis must be strictly coordinated with patterning processes to correctly shape the organs. Thus, it is not surprising that all major morphogenetic and developmental signaling pathways have been involved in the regulation of cell proliferation and apoptosis and that they have been linked to numerous cases of cancer formation in mammals. In Drosophila, a large body of work shows that several of these pathways act in concert in the coordination of cell survival and death. For example, overexpression of Notch causes vast overgrowth, whereas inhibition of Notch activity by overexpression of its antagonist Hairless results in tissue loss and apoptosis. Indeed, the combined activity of Hedgehog (Hh), Decapentaplegic (Dpp), and Notch is required to promote reentry into the cell cycle after a developmentally regulated G1 arrest in the eye anlagen of Drosophila larvae. Moreover, it was shown that Hh signaling promotes cellular growth by transcriptional activation of G1 cyclins Cyclin D and Cyclin E. However, to this end, the understanding of the molecular principles that connect these pathways to either control of cell cycle or apoptosis remains largely fragmentary (Kugler, 2007).

Cell cycle entry requires the activity of G1-S cyclins that eventually activate dE2F1, a transcription factor that induces transcription of downstream genes required, e.g., for DNA replication. In Drosophila, transcriptional activation of replication-related genes encoding, for example, proliferating cell nuclear antigen (PCNA) or DNA-polymerase alpha subunit involves also DNA replication-related element factor (DREF) that recognizes DNA replication-related element (DRE) response elements. DREF can be part of a macromolecular complex including TRF2, a TATA-binding protein-related factor that binds to a subset of selected promoters, including one promoter in the PCNA gene. TRF2 has been isolated from several different organisms, where it is required for transcription of replication-related genes and key developmental genes as well. The TRF2/DREF complex consists of more than a dozen proteins, including several known chromatin-remodeling components. Three of them confer chromatin activation, whereas two others, including p160, resemble regulators of insulator function. Interestingly, p160 was recently found to enhance position effect variegation and hence chromatin silencing and to be associated with interband regions of polytene chromosomes (Eggert, 2004). To this end, the biochemical activity and functional specificity of most of the proteins within the TRF2-complex, i.e., their role in transcriptional activation or in chromatin remodeling, however, remain elusive (Kugler, 2007).

This study isolated the Zn-finger protein p160 as a genetic suppressor of Hairless activity, prompting an interest in its role during Drosophila development and especially during Notch signaling. In vivo RNA interference resulted in tiny larvae and developmental delay, which is why the corresponding gene was named putzig (pzg). This study presents in vivo evidence that pzg is essential for fly survival by regulating cell cycle entry and progression. In addition, pzg encodes a key regulator of the Notch signaling pathway and that it is involved in histone modification and chromatin activation. Interestingly, this activity is independent of DREF, suggesting context dependence of Pzg activity (Kugler, 2007).

EP756 was identified in a genetic screen as suppressor of tissue loss caused by an overexpression of the Notch antagonist Hairless (H) during eye development. This positive effect was not restricted to the eye, because it was likewise observed during wing development. Moreover, cell growth and proliferation induced by an enforced Notch signal was significantly enhanced (~20%) by a combined overexpression with EP756. Tissue specific overexpression of EP756 caused a very mild enlargement of the respective tissues on its own. These data suggest a more general role of EP756 in the control of cell proliferation as well as an intimate interaction with Notch signaling (Kugler, 2007).

Pzg is one component of a multiprotein complex that contains TRF2 and DREF. TRF2 allows transcription initiation from selected promoters independently of TFIID. DREF is a positive transcriptional regulator of cell cycle and replication-related genes, and it may guide TRF2 to the PCNA and DNA-polymerase alpha promoters. Assuming promoter recognition or binding requires Pzg contained within the TRF2/DREF complex, depletion of Pzg might destroy the complex or reduce its activity, easily explaining the dramatic proliferation defects. However, it is noted that only a subset of promoters containing DREF binding sites involves activation through TRF2, suggesting that DREF can act independently of TRF2. Moreover, Pzg activity is found independently of DREF, indicating that TRF2/DREF complex components can act either alone or in conjunction with other factors (Kugler, 2007).

The TRF2/DREF complex contains several proteins involved in chromatin remodeling. Notably, Pzg and one other TRF2/DREF component p190 are reminiscent of factors implicated in insulator function. In accordance, Pzg activity has been associated with position effect variegation and chromatin silencing. In contrast, assays reveal an essential function of Pzg in retaining robust K4-trimethylation of histone H3, which is directly associated with open chromatin structures. In accordance with these findings, EP756 was recently identified as a suppressor of the cut allele ctK. This cut mutation is caused by the insulator activity of a gypsy retrotransposon, which can be relieved by EP756 overexpression. EP756 is shown in this stody to drive Pzg expression, in support of the notion that Pzg's epigenetic activity overcomes gypsy insulator function (Kugler, 2007).

Three of the proteins found in the TRF2/DREF complex have been identified previously in the nucleosome-remodeling factor NURF (see NURF301), which consists in total of four subunits. NURF is associated with chromatin activation by facilitating transcription of chromatin in vivo. In fact, mutations in Drosophila ISWI, the catalytic subunit of NURF, and other nucleosome remodeling complexes caused phenotypes that are very reminiscent of pzg-RNAi-induced defects. Because DREF down-regulation has no effect on trimethylation of H3K4, it seems unlikely that the TRF2/DREF complex as a whole is involved in chromatin activation. Instead, Pzg may be part of a NURF-like chromatin-remodeling complex, depending on the developmental context (Kugler, 2007).

Apart from a role in proliferation, this study has uncovered an important role for Pzg as positive regulator of Notch signaling. Interestingly, it was found that Pzg binds to chromatin in the regulatory region of the Notch target genes E(spl)m8 and vg. This regulation is independent of DREF: albeit DREF binding sites are common to Drosophila promoters, neither Notch nor Notch target genes that were investigated are transcriptional targets of DREF. Thus, reduced transcriptional activity of Notch target genes in pzg-RNAi mutant cells is due to a DREF-independent role of Pzg. Alternatively, Pzg could facilitate formation of the transcriptional activator complex that is assembled on Notch target promoters involving intracellular Notch itself. By using the yeast two-hybrid system, several Notch pathway members were tested; however, no binding to Pzg was detected. It is proposed that Pzg has a dual function that is effected differently. On one hand, it activates proliferation-related genes in conjunction with TRF2/DREF, and on the other hand, it activates Notch signaling by chromatin activation independently of DREF (Kugler, 2007).

Several lines of evidence support the idea that Notch signaling is particularly susceptible to chromatin remodeling. For example, Notch transcriptional activity requires the histone-modifying enzyme dBre1 that is indirectly required for K4-methylation of histone H3. Moreover, chromatin-modifiers were also shown to potentiate Notch activity during Drosophila wing development. Finally, general transcriptional regulators and chromatin remodeling factors were found in several independent genetic screens to influence Notch signaling, indicating to a role of pzg in linking Notch to chromatin remodeling. The bimodal activity of Pzg onto both cell cycle genes and Notch signaling provides further insight into the complex interplay between cell proliferation and differentiation in the fly (Kugler, 2007).

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).

Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).

Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).

The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).

This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).

The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).

The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).

Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).

Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).

It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).

It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).

The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for Nintra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).

Integrins regulate epithelial cell differentiation by modulating Notch activity

Coordinating exit from the cell cycle with differentiation is critical for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the cell cycle exit-to-differentiation switch. Signals downstream of two key developmental pathways, Notch and Salvador-Warts-Hippo (SWH), and of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. This study identified a fourth player, the integrin signaling pathway. Elimination of integrin function blocks mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), via regulation of the CDK inhibitor Dacapo. In addition, integrin mutant PFCs show defective Notch signalling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Altogether, these findings reveal a direct involvement of integrin signalling on the spatial and temporal regulation of epithelial cell differentiation during development (Gomez-Lamarca, 2014).


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Biological Overview

date revised: 15 November 2014

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