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).

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 (H^1-293). 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 H^DeltaC 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 H^DeltaC 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 N^IC/Su(H)-mediated activation, but not Su(H)-mediated repression. Like previous views of H function, this model presumes competition between Su(H)-binding partners, in this case between N^IC-containing activation complexes and H/Gro/dCtBP repression complexes. N^IC activation complexes are likely to include the Mastermind (Mam) protein, and may also include the p300 coactivator. In the presence of N signaling, Su(H)/N^IC/Mam complexes presumably replace Su(H)/H/Gro/dCtBP complexes on target genes, and convert Su(H) from a repressor to an activator. Whether this occurs by simple affinity-based competition for binding to Su(H), or by a mechanism involving active impairment of the H/Su(H) interaction, is unknown. Under an adaptor/corepressor model, the H mutant phenotype results from derepression of Su(H)/N target genes in cells lacking N pathway activity, thus mimicking an increase in N signaling. The H overexpression phenotype may be explained by the displacement of N^IC-containing activation complexes by an excess of H-containing repression complexes, thus repressing N^IC/Su(H) target genes in cells that respond to the N signal (Barolo, 2002b).

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).

GENE STRUCTURE

Bases in 5' UTR - 267

Exons - 5

Bases in 3' UTR - There are multiple cDNA termini, with the longest 3' UTR consisting of 2127 bases.

PROTEIN STRUCTURE

Structural Domains

Hairless is a novel basic protein with a number of common motifs: Hairless is a serine/threonine-rich protein with a 'Paired box' domain protein (consisting of alternating histidine and proline residues, a KX-repeat and an NEDL-repeat region. The content of proline residues is higher than average. (Maier, 1992 and Bang, 1992).

date revised: 2 December 2002

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