NORMAL GENOMIC

• Fujimoto K et al.
• Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins.
• Biochem Biophys Res Commun. 2001; 280: 164-71
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• DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal-q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development. Copyright 2001 Academic Press.

• Nakakura EK et al.
• Mammalian Scratch: a neural-specific Snail family transcriptional repressor.
• Proc Natl Acad Sci U S A. 2001; 98: 4010-5
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• Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix--loop--helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

• Trott RL, Kalive M, Paroush Z, Bidwai AP
• Drosophila melanogaster casein kinase II interacts with and phosphorylates the basic helix-loop-helix proteins m5, m7, and m8 derived from the Enhancer of split complex.
• J Biol Chem. 2001; 276: 2159-67
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• Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a D. melanogaster embryo cDNA library for proteins that interact with DmCKIIalpha. One of the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex (E(spl)C), which regulates neurogenesis. m7 interacts with DmCKIIalpha but not with DmCKIIbeta, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, we demonstrate that DmCKIIalpha also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKIIalpha with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKIIalpha, as well as by the alpha2beta2 holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser(159) of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. We also demonstrate that m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Finally, substitution of Ser(159) of m8 with Ala attenuates interaction with DmCKIIalpha, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis.

• Vasiliauskas D, Stern CD
• Expression of mouse HES-6, a new member of the Hairy/Enhancer of split family of bHLH transcription factors.
• Mech Dev. 2000; 98: 133-7
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• We studied the expression of mouse HES-6, a new member of the Hairy/Enhancer of split family of basic helix-loop-helix transcription factors. HES-6 is expressed in all neurogenic placodes and their derivatives and in the brain, where it is patterned along both the anteroposterior and dorsoventral axes. HES-6 is also expressed in the trunk, in the dorsal root ganglia and in the myotomes. In the limb buds HES-6 is expressed in skeletal muscle and presumptive tendons.

• Ryoo HD, Marty T, Casares F, Affolter M, Mann RS
• Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex.
• Development. 1999; 126: 5137-48
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• To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

• Nellesen DT, Lai EC, Posakony JW
• Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators.
• Dev Biol. 1999; 213: 33-53
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• In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while 4 others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Here we present strong evidence from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes were known previously to be direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Our extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. We next show that the very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. We also demonstrate that the characteristic inactivity of the E(spl)mgamma enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity of the N receptor. We conclude that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative.

• Leimeister C, Externbrink A, Klamt B, Gessler M
• Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis.
• Mech Dev. 1999; 85: 173-7
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• We have identified a novel subfamily of mammalian hairy/Enhancer of split (E(spl))-related basic helix-loop-helix (bHLH) genes together with a putative Drosophila homologue. While hairy/E(spl) proteins are characterized by an invariant proline residue in the basic domain and a carboxyterminal groucho-binding WRPW motif, our genes encode a carboxyterminal KPYRPWG sequence and were thus designated as Hey genes (Hairy/E(spl)-related with YRPW motif). Furthermore, they bear a unique C-terminal TE(I/V)GAF motif and the characteristic proline is changed in all Hey family members to glycine. RNA in situ hybridization analysis revealed specific expression of Hey1 during development of the nervous system, the somites, the heart and the craniofacial region. Hey2 is similarly expressed in the somites whereas it shows a complementary expression in the heart, the craniofacial region and the nervous system. The diversity of expression patterns implies unique functions in neurogenesis, somitogenesis and organogenesis.

• Ashraf SI, Hu X, Roote J, Ip YT
• The mesoderm determinant snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis.
• EMBO J. 1999; 18: 6426-38
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• The Snail protein functions as a transcriptional regulator to establish early mesodermal cell fate. Later, in germ band-extended embryos, Snail is also expressed in most neuroblasts. Here we present evidence that this expression of Snail is required for central nervous system (CNS) development. The neural function of snail is masked by two closely linked genes, escargot and worniu. Both Escargot and Worniu contain zinc-finger domains that are highly homologous to that of Snail. Although not affecting expression of early neuroblast markers, the deletion of the region containing all three genes correlates with loss of expression of CNS determinants including fushi tarazu, pdm-2 and even-skipped. Transgenic expression of each of the three Snail family proteins can rescue efficiently the fushi tarazu defects, and partially the pdm-2 and even-skipped CNS patterns. These results demonstrate that the Snail family proteins have essential functions during embryonic CNS development, around the time of ganglion mother cell formation.

• Nagel AC, Yu Y, Preiss A
• Enhancer of split [E(spl)(D)] is a gro-independent, hypermorphic mutation in Drosophila.
• Dev Genet. 1999; 25: 168-79
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• Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway. The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)). The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed. In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level. In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype. This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity. Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed. These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl).

• Wurmbach E, Wech I, Preiss A
• The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes.
• Mech Dev. 1999; 80: 171-80
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• Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su(H)) mediated transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex (E(spl)-C), encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. Here we show that the E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and ma are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide.

• Jennings BH, Tyler DM, Bray SJ
• Target specificities of Drosophila enhancer of split basic helix-loop-helix proteins.
• Mol Cell Biol. 1999; 19: 4600-10
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• Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.

• Poortinga G, Watanabe M, Parkhurst SM
• Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression.
• EMBO J. 1998; 17: 2067-78
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• hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

• Fisher A, Caudy M
• The function of hairy-related bHLH repressor proteins in cell fate decisions.
• Bioessays. 1998; 20: 298-306
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• Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.

• Corbo JC, Fujiwara S, Levine M, Di Gregorio A
• Suppressor of hairless activates brachyury expression in the Ciona embryo.
• Dev Biol. 1998; 203: 358-68
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• The Ciona Brachyury gene (Ci-Bra) is regulated, in part, by a 434-bp enhancer that mediates restricted expression in the notochord. Here we present evidence that a Ciona Suppressor of Hairless ?Ci-Su(H)? protein functions as an activator of this enhancer. Point mutations that reduce the binding of a GST/Ci-Su(H) fusion protein in vitro diminish the expression of mutagenized Ci-Bra/lacZ transgenes in electroporated embryos. Overexpression of a Ci-Su(H) fusion protein containing the Drosophila Hairy repression domain interferes with notochord differentiation, producing mutant tadpoles with shortened tails. Expression of a constitutively activated Xotch receptor in the notochord, endoderm, and CNS also alters tail morphogenesis. These results suggest that a Notch-Su(H) pathway might participate in notochord differentiation in Ciona.

• Jimenez G, Ish-Horowicz D
• A chimeric enhancer-of-split transcriptional activator drives neural development and achaete-scute expression.
• Mol Cell Biol. 1997; 17: 4355-62
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• Drosophila melanogaster neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Here, we test if E(spl) proteins function as promoter-bound repressors by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. Our results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway.

• Hanna-Rose W, Licht JD, Hansen U
• Two evolutionarily conserved repression domains in the Drosophila Kruppel protein differ in activator specificity.
• Mol Cell Biol. 1997; 17: 4820-9
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• To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, we cloned the Kruppel homolog from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that was most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encoded a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region was able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibited transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development.

• Giebel B, Campos-Ortega JA
• Functional dissection of the Drosophila enhancer of split protein, a suppressor of neurogenesis.
• Proc Natl Acad Sci U S A. 1997; 94: 6250-4
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• The Enhancer of split [E(spl)] gene complex of Drosophila comprises seven related genes encoding a special type of basic helix-loop-helix proteins, the function of which is to suppress the neural developmental fate. One of these proteins is E(spl) itself. To gain insight into the structural requirements for E(spl) function, we have expressed a large number of deletion variants in transgenic flies. Three protein domains were identified as essential for suppression of bristle development: the carboxyl-terminal tetrapeptide WRPW, the region comprising the putative helix III and helix IV, and the region between helix IV and the WRPW motif. Lack of the basic helix-loop-helix domain, helix III or IV, only partially inhibits the suppressor activity of the protein. Truncated variants that lack all the regions carboxyl-terminal to helix IV elicit the development of additional neural progenitors, and thus act as dominant-negative variants. All these results suggest that E(spl) suppresses neural development by direct interaction with other proteins, such as groucho and the proneural proteins.

• Barolo S, Levine M
• hairy mediates dominant repression in the Drosophila embryo.
• EMBO J. 1997; 16: 2883-91
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• hairy encodes a bHLH repressor that regulates several developmental processes in Drosophila, including embryonic segmentation and neurogenesis. Segmentation repressors such as Kruppel and knirps have been shown to function over short distances, less than 50-100 bp, to inhibit or quench closely linked upstream activators. This mode of repression permits multiple enhancers to work independently of one another within a modular promoter. Here, we employ a transgenic embryo assay to present evidence that hairy acts as a dominant repressor, which can function over long distances to block multiple enhancers. hairy is shown to repress a heterologous enhancer, the rhomboid NEE, when bound 1 kb from the nearest upstream activator. Moreover, the binding of hairy to a modified NEE leads to the repression of both the NEE and a distantly linked mesoderm-specific enhancer within a synthetic modular promoter. Additional evidence that hairy is distinct from previously characterized embryonic repressors stems from the analysis of the gypsy insulator DNA. This insulator selectively blocks the hairy repressor, but not the linked activators, within a modified NEE. We compare hairy with previously characterized repressors and discuss the consequences of short-range and long-range repression in development.

• Fisher AL, Ohsako S, Caudy M
• The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.
• Mol Cell Biol. 1996; 16: 2670-7
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• Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

• de Celis JF, de Celis J, Ligoxygakis P, Preiss A, Delidakis C, Bray S
• Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development.
• Development. 1996; 122: 2719-28
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• The basic helix-loop-helix proteins of the Enhancer of split complex constitute a link between activation of the transmembrane receptor Notch and the resulting effects on transcription of downstream genes. The Suppressor of Hairless protein is the intermediary between Notch activation and expression of all Enhancer of split genes even though individual genes have distinct patterns of expression in imaginal discs. A comparison between the phenotypes produced by Notch, Suppressor of Hairless and Enhancer of split mutations in the wing and thorax indicate that Suppressor of Hairless and Notch requirements are indistinguishable, but that Enhancer of split activity is only essential for a subset of developmental processes involving Notch function. Likewise, the ectopic expression of Enhancer of split proteins does not reproduce all the consequences typical of ectopic Notch activation. We suggest that the Notch pathway bifurcates after the activation of Suppressor of Hairless and that Enhancer of split activity is not required when the consequence of Notch function is the transcriptional activation of downstream genes. Transcriptional activation mediated by Suppressor of Hairless and transcriptional repression mediated by Enhancer of split could provide greater diversity in the response of individual genes to Notch activity.

• Gigliani F, Longo F, Gaddini L, Battaglia PA
• Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila.
• Mol Gen Genet. 1996; 251: 628-34
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• The Enhancer of split and achaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis in Drosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.

• Bailey AM, Posakony JW
• Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity.
• Genes Dev. 1995; 9: 2609-22
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• We have investigated the functional relationships among three loci that are required for multiple alternative cell fate decisions during adult peripheral neurogenesis in Drosophila: Notch (N), which encodes a transmembrane receptor protein, Suppressor of Hairless [Su(H)], which encodes a DNA-binding transcription factor, and the Enhancer of split gene complex [E(spl)-C], which includes seven transcription units that encode basic helix-loop-helix (bHLH) repressor proteins. We describe several lines of evidence establishing that Su(H) directly activates transcription of E(spl)-C genes in response to N receptor activity. Expression of an activated form of the N receptor leads to elevated and ectopic E(spl)-C transcript accumulation and promoter activity in imaginal discs. We show that the proximal upstream regions of three E(spl)-C genes contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, are required not only for normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters, but also for their transcriptional response to hyperactivity of the N receptor. Our results establish Su(H) as a direct regulatory link between N receptor activity and the expression of E(spl)-C genes, extending the known linear structure of the N cell-cell signaling pathway.

• Van Doren M, Bailey AM, Esnayra J, Ede K, Posakony JW
• Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete.
• Genes Dev. 1994; 8: 2729-42
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• hairy (h) acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development in Drosophila. Here, we demonstrate that h, a basic-helix-loop-helix (bHLH) protein, is a sequence-specific DNA-binding protein and transcriptional repressor. We identify the proneural gene achaete (ac) as a direct downstream target of h regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity h binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, in a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by h plays an essential role in pattern formation in the PNS. Our results demonstrate that HLH proteins negatively regulate ac transcription by at least two distinct mechanisms.

• Paroush Z et al.
• Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins.
• Cell. 1994; 79: 805-15
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• We have used the interaction trap, a yeast two-hybrid system, to identify proteins interacting with hairy, a basic-helix-loop-helix (bHLH) protein that represses transcription during Drosophila embryonic segmentation. We find that the groucho (gro) protein binds specifically to hairy and also to hairy-related bHLH proteins encoded by deadpan and the Enhancer of split complex. The C-terminal WRPW motif present in all these bHLH proteins is essential for this interaction. We demonstrate that these associations reflect in vivo maternal requirements for gro during neurogenesis, segmentation, and sex determination, three processes regulated by the above bHLH proteins, and we propose that gro is a transcriptional corepressor recruited to specific target promoters by hairy-related bHLH proteins.

• Oellers N, Dehio M, Knust E
• bHLH proteins encoded by the Enhancer of split complex of Drosophila negatively interfere with transcriptional activation mediated by proneural genes.
• Mol Gen Genet. 1994; 244: 465-73
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• The Enhancer of split complex [E(SPL)-C] of Drosophila participates in the control of cell fate choice by uncommitted neuroectodermal cells in the embryo. It encodes seven proteins that belong to the basic helix-loop-helix (bHLH) family, six of which are expressed in very similar patterns in the neuroectoderm. Here we describe experiments aimed at unravelling the molecular basis of their function. We found that two products of the complex, HLH-M5 and ENHANCER OF SPLIT, are capable of binding as homo-and heterodimers to a sequence in the promoters of the Enhancer of split and achaete genes, called the N-box, which differs slightly from the consensus binding site (the E-box) for other bHLH proteins. In transient expression assays in cell culture, both proteins were found to attenuate the transcriptional activation mediated by the proneural bHLH proteins LETHAL OF SCUTE and DAUGHTERLESS at the Enhancer of split promoter.

• Harrison DA, Gdula DA, Coyne RS, Corces VG
• A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function.
• Genes Dev. 1993; 7: 1966-78
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• The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers controlling the expression of the mutant gene. A structural and functional analysis of su(Hw) was carried out to identify domains of the protein responsible for its negative effect on enhancer action. Sequence comparison among the su(Hw) proteins from three different species allows the identification of evolutionarily conserved domains with possible functional significance. An acidic domain located in the carboxy-terminal end of the Drosophila melanogaster protein is not present in su(Hw) from other species, suggesting a nonessential role for this part of the protein. A second acidic domain located in the amino-terminal region of su(Hw) is present in all species analyzed. This domain is dispensable in the D. melanogaster protein when the carboxy-terminal acidic domain is present, but the protein is nonfunctional when both regions are simultaneously deleted. Mutations in the zinc fingers result in su(Hw) protein unable to interact with DNA in vivo, indicating a functional role for this region of the protein in DNA binding. Finally, a region of su(Hw) homologous to the leucine zipper motif is necessary for the negative effect of this protein on enhancer function, suggesting that su(Hw) might exert this effect by interacting, directly or indirectly, with transcription factors bound to these enhancers.

• Ishibashi M, Sasai Y, Nakanishi S, Kageyama R
• Molecular characterization of HES-2, a mammalian helix-loop-helix factor structurally related to Drosophila hairy and Enhancer of split.
• Eur J Biochem. 1993; 215: 645-52
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• Drosophila hairy (h) plays a crucial role in early development as a pair-rule segmentation gene. h and its structurally related gene Enhancer of split [E(spl)] are also required for normal sensory neurogenesis in late development. To analyze the molecular mechanisms of mammalian development, we recently characterized three rat helix-loop-helix (HLH) factors that show structural homology to the Drosophila h and E(spl) gene products, and found that rat factors exhibit distinct spatiotemporal expression patterns and act as a negative regulator. Here, we report the molecular characterization of another member of this family, designated HES-2. Rat HES-2 protein has a basic HLH domain homologous to h and E(spl) as well as the carboxy-terminal Trp-Arg-Pro-Trp sequence conserved among this family. The HES-2 mRNA is present as early as embryonic day 9.5 and is detected in a variety of tissues of both embryos and adults. DNase-I-footprinting analyses indicate that HES-2 binds to all E box sequences (CANNTG) we tested as well as to the N-box sequences (CACNAG). Further studies of gel-mobility-shift assays show that HES-2 has a higher affinity for the E box than for the N box. Transient transfection analyses suggest that HES-2 decreases the transcription originating from the promoters containing either the E box or the N box. These results indicate that HES-2 acts as a negative regulator through interaction with both E-box and N-box sequences.

• Sasai Y, Kageyama R, Tagawa Y, Shigemoto R, Nakanishi S
• Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split.
• Genes Dev. 1992; 6: 2620-34
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• We report the molecular characterization of two novel rat helix-loop-helix (HLH) proteins, designated HES-1 and HES-3, that show structural homology to the Drosophila hairy and Enhancer of split [E(spl)] proteins, both of which are required for normal neurogenesis. HES-1 mRNA, expressed in various tissues of both embryos and adults, is present at a high level in the epithelial cells, including the embryonal neuroepithelial cells, as well as in the mesoderm-derived tissues such as the embryonal muscle. In contrast, HES-3 mRNA is produced exclusively in cerebellar Purkinje cells. HES-1 represses transcription by binding to the N box, which is a recognition sequence of E(spl) proteins. Interestingly, neither HES-1 nor HES-3 alone interacts efficiently with the E box, but each protein decreases the transcription induced by E-box-binding HLH activators such as E47. Furthermore, HES-1 also inhibits the functions of MyoD and MASH1 and effectively diminishes the myogenic conversion of C3H10T1/2 cells induced by MyoD. These results suggest that HES-1 may play an important role in mammalian development by negatively acting on the two different sequences while HES-3 acts as a repressor in a specific type of neurons.