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ã Oncogene (2001) 20, 8342 ± 8357 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular dierentiation and embryonic patterning Robert L Davis1 and David L Turner*,2 1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, MA 02115, USA; 2Mental Health Research Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, MI 48104-1687, USA The basic-helix-loop-helix (bHLH) proteins are a superfamily of DNA-binding transcription factors that regulate numerous biological processes in both invertebrates and vertebrates. One family of bHLH transcriptional repressors is related to the Drosophila hairy and Enhancer-of-split proteins. These repressors contain a tandem arrangement of the bHLH domain and an adjacent sequence known as the Orange domain, so we refer to these proteins as bHLH-Orange or bHLH-O proteins. Phylogenetic analysis reveals the existence of four bHLH-O subfamilies, with distinct, evolutionarily conserved features. A principal function of bHLH-O proteins is to bind to speci®c DNA sequences and recruit transcriptional corepressors to inhibit target gene expression. However, it is likely that bHLH-O proteins repress transcription by additional mechanisms as well. Many vertebrate bHLH-O proteins are eectors of the Notch signaling pathway, and bHLH-O proteins are involved in regulating neurogenesis, vasculogenesis, mesoderm segmentation, myogenesis, and T lymphocyte development. In this review, we discuss mechanisms of action and biological roles for the vertebrate bHLH-O proteins, as well as some of the unresolved questions about the functions and regulation of these proteins during development and in human disease. Oncogene (2001) 20, 8342 ± 8357 Keywords: basic-helix-loop-helix; transcription; Notch; corepressor; hairy Introduction Over the past decade, numerous vertebrate proteins structurally related to the Drosophila hairy and Enhancer of split [E(spl)] basic helix-loop-helix (bHLH) proteins have been identi®ed. The ®rst of these were the rat HES1/Hairy-like protein and several related proteins designated HES2 ± 5 (Akazawa et al., 1992; Sasai et al., 1992; Feder et al., 1993). Subsequently, additional related proteins have been identi®ed in humans and other mammals, as well as chickens, *Correspondence: DL Turner, Neuroscience Laboratory Building, 1103 East Huron, Ann Arbor, Michigan, MI 48104-1687, USA; E-mail: [email protected] frogs, and zebra®sh. As we discuss below, the vertebrate hairy and E(spl) related proteins can be grouped into distinct subfamilies based on their primary structures. However, all proteins in these subfamilies contain a conserved amino acid sequence known as the Orange domain located just C-terminal to the bHLH domain. The tandem arrangement of the bHLH and Orange domains is the major structural feature shared among these proteins, so for convenience we refer to all hairy and E(spl) related proteins collectively as bHLH-Orange (bHLH-O) proteins. In both vertebrates and invertebrates, bHLH-O proteins function as DNA-binding transcriptional repressors, and regulate a wide variety of biological processes. These include negative control of dierentiation (Fisher and Caudy, 1998a; Kageyama et al., 2000), anteroposterior segmentation in both invertebrates and vertebrates (probably by distinct mechanisms; Palmeirim et al., 1997; Jen et al., 1999; Damen et al., 2000 and references therein), and sex determination in ¯ies (Parkhurst et al., 1990; Younger-Shepherd et al., 1992). In many but not all of these processes, bHLHO proteins function as eectors of the Notch signaling pathway (Artavanis-Tsakonas et al., 1999). As might be expect from their diverse roles, both structural and functional analyses suggest that dierences exist between the bHLH-O subfamilies, and even among members of the same subfamily. Here we review the bHLH-O protein subfamilies and domain structures, known and proposed mechanisms of bHLH-Omediated repression, and our current knowledge about the roles and regulation of bHLH-O proteins in vertebrates. We also consider some unresolved questions about these proteins and their functions in vertebrates. Vertebrate bHLH-O proteins can be divided into four distinct subfamilies Most sequence comparisons and derived phylogenetic relationships among the bHLH-O proteins have been based on the bHLH domain alone, while other conserved domains have received less attention in comparisons of the family as a whole. Figure 1 shows a phylogenetic tree derived from pairwise comparisons of over 60 bHLH-O proteins. This analysis, based on Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8343 Figure 1 Phylogenetic tree for the bHLH-O protein family. Sequence alignment was performed with ClustalX (Jeanmougin et al., 1998). The plot was bootstrapped 1000 times, and the same sequence relationships obtained. The entire protein sequence was used for each family member. A BLOSUM62 matrix was used for pairwise alignment, while a threshold BLOSUM matrix series was used for multiple alignment. Since no ancestral relationship is assumed in initial alignments, this tree demonstrates sequence relationships, but does not absolutely imply sequence ancestry. Genbank accession numbers, where available, are as follows: worm (C. elegans) lin22 (AF020555); red ¯our beetle (Tribolium castaneum) hairy (S29712); spider (Cupiennius salei) hairy (AJ252154); ¯y (Drosophila melanogaster) hairy (X15905), deadpan (S48025), E(spl)m3 (M96165), E(spl)m5 (X16552), E(spl)m7 (X16553), E(spl)m8 (X16553), E(spl)mbeta (X67047), E(spl)mdelta (X67048), E(spl)mgamma (X67049), Hesr1 (AF151523); zebra®sh (Danio rerio) hairy1 (AF301264), her1 (X97329), her2 (X97330), her3 (X97331), her4 (X97332), her5 (X95301), her6 (X97333), her7 (AF292032), gridlock (AF237948); frog (Xenopus laevis) hairy1 (U36194), hairy2a (AF383159), hairy2b (AF383160), ESR1 (AF383157), ESR2 (AF383158), ESR3/ESR7 (AF146088), ESR4 (AF137073), ESR5 (AF137072), ESR6e (AF146087), Hesr1 (AJ401271); chicken (Gallus gallus) hairy1 (AF032966), hairy2 (Jouve et al., 2000), Hey1 and Hey2 (Leimeister et al., 2000b) ; rat (Rattus norvegicus) HES1/Hairy-Like (NM_024360, L04527), HES2 (NM_019236), HES3 (NM_022687), HES5 (NM_024383), SHARP-1 (AF009329), SHARP-2 (AF009330); mouse (Mus musculus) HES1 (NM_008235), HES2 (NM_008236), HES3 (NM_008237), HES5 (NM_010419), HES6 (AB035178), HES7 (AB049065), Hey1/HRT1/Hesr1 (AJ243895/AF172286/AF151521), Hey2/HRT2 (AJ271867/AF172287), HeyL/HRT3 (AJ271868/AF172288), Stra13/CLAST5 (AF010305/AF364051), DEC2/BHLHB2 (AB044090, (Continued over page) Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8344 comparison of full-length protein sequences, suggests that there are four major subfamilies of these proteins. We refer to the four subfamilies by the names of the prototypic protein for each: hairy, E(spl), Hey, and Stra13. Except for Stra13, each of these subfamilies has members from Drosophila to humans. Although all of these proteins are transcriptional repressors, the conserved dierences in the primary structures imply that members of dierent subfamilies have distinct functions and/or post-translational regulation. Most bHLH-O proteins have been isolated based on sequence similarity within the bHLH domain. However, as noted above, a second domain, denoted either as the Orange domain (Dawson et al., 1995) or as helix III/IV (Knust et al., 1992) is conserved in every member of each subfamily. Figure 2a shows a schematic of the domains present in each bHLH-O subfamily, while Figure 2b shows sequence alignments of representative domains. The bHLH domains share typical features of the bHLH superfamily, although residues at certain positions are speci®c to bHLH-O proteins (see below). The intervening sequence between the bHLH and Orange domains ranges from seven to 41 amino acids. Although not shown in Figure 2, this intervening sequence is highly similar among members of the hairy, Hey, and Stra13 subfamilies, respectively, while in the E(spl) subfamily it shows more variation. The Orange domain is about 30 amino acids in length, with a well-conserved N-terminal boundary in most family members. The C-terminal boundary shows more variation in some of the vertebrate E(spl)-like proteins. Thus far, an Orange domain has not been identi®ed in any non-bHLH protein. Except for the Stra13 proteins, members of each subfamily also have a conserved C-terminal tetrapeptide motif, either WRPW for hairy and E(spl) subfamilies, or YXXW for the Hey subfamily. The Hairy subfamily proteins also have a short sequence between the Orange domain and the Cterminus that is conserved in all invertebrate and vertebrate members (HC domain, Figure 2b). Additional sequences conserved among subfamily members, though not necessarily conserved between invertebrates and vertebrates, can be discerned by alignments of the individual subfamilies (not shown). Taken together, all of these sequence features allow clear assignment of each bHLH-O protein to one of the subfamilies. The nomenclature for the bHLH-O family is complicated by the common problem of independent isolation in multiple laboratories, as well as by the subfamily structure. In both frogs and chickens, names for hairylike and E(spl)-like proteins have maintained the distinction between these subfamilies. However, the hairy-like and E(spl)-like proteins have been named HES (hairy and enhancer of split) or her (hairy and enhancer of split related) in mammals and zebra®sh respectively, and numbered by the order of isolation. Hey proteins have also been named HRT or Hesr, or in the case of Hey2, Gridlock or CHF1, while Stra13 proteins have also been named SHARP, DEC, CLAST, or BHLHB2 (see Figure 1 legend). For brevity, we generally use only a single name for a bHLH-O protein with multiple names in a given species. The recent completion of sequencing of the Drosophila and human genomes permits a comparison of bHLH-O family complexity between a highly evolved invertebrate and humans. While many transcription factor families have expanded in number in humans relative to ¯ies (e.g. homeobox and activator bHLH proteins), the bHLH-O family has not signi®cantly increased in size. Drosophila has 13 known bHLH-O proteins (Moore et al., 2000), including three hairy proteins (hairy, deadpan and side), eight E(spl) bHLH-O proteins (seven m-type, and her), and two Hey-like proteins (Hesr-1 and sticky/ch1). Humans appear to have 12 bHLH-O proteins. These include two hairy-like proteins (HES1 and HES4). Zebra®sh and chickens also have two hairy-like proteins, suggesting that this is the common number for vertebrates. Humans have ®ve E(spl) bHLH-O proteins (HES2, 3, 5, 6, 7), three Hey proteins (1, 2, L), and 2 Stra13 proteins (Stra13/DEC1, DEC2). In Table 1, we show the current human protein set, as well as the probable orthologs in other animal models, when such assignments can be made. The surprisingly similar size of the bHLH-O families in ¯ies and humans suggests that the increase in the number of vertebrate transcriptional activator proteins has not required a parallel increase in the number of bHLH-O repressors. Molecular basis of bHLH-O protein function DNA binding In contrast to the Id/emc HLH repressor proteins described in the accompanying articles, the bHLH-O proteins bind to DNA. As for other bHLH proteins, bHLH-O DNA-binding is mediated by a region of basic amino acids immediately N-terminal to the HLH dimerization domain. The basic regions of proteins in the hairy and E(spl) subfamilies dier from other bHLH proteins by the presence of a proline residue at a conserved position. The Hey proteins have a NM_024469); Human (Homo sapiens) HES1/HRY (L19314), HES2 (AL031848), HES3 (located on human chromosome 1, AL031847), HES4 (AB048791), HES5 (located on human chromosome 1, NT_004350), HES6 (AB035179), HES7 (AB049064), Hey1/HRT1/Hesr1 (NM_012258/AF311883/AF151522), Hey2/HRT2/CHF1/gridlock (NM_012259/AF311884/AF173901/ AF237949), HeyL/HRT3 (NM_014571/AF311885), DEC1/Stra13/BHLHB2 (AB043885), DEC2 (AB044088). Not included in this tree are very recent additions or proteins with distant relationships to the family as a whole: worm ref-1 (AF358857), ¯y sticky ch1/ stich1 (AF203477), ¯y side (GadFly accession CG10446), ¯y her (GadFly accession CG5927, and zebra®sh her8a/b (AY007990, AY007991). The Genbank cDNA sequence used for rat SHARP-1 encodes a C-terminal truncated protein when compared to human and mouse DEC2 proteins. The rat SHARP-1 is likely to be an ortholog of human and mouse DEC2 genes since the rat cDNA encodes a longer C-terminus, similar to human and mouse DEC2, in a dierent reading frame (consistent with a cloning or sequencing error for the rat cDNA,). Also, Xenopus ESR3 and ESR7 are the same gene Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8345 Figure 2 Schematic of bHLH-O protein domains and domain alignments. (a) Schematic of bHLH-O subfamily domain structures. The bHLH and Orange domains are present in all family members. The HC domain is speci®c to the hairy subfamily. Unlabeled regions also contain one or a few motifs shared within a subfamily, and sometimes between subfamilies. Hairy-like proteins end with WRPW, and in some cases two additional amino acids. E(spl)-like proteins usually end with WRPW, but a small subset ends with an additional proline (zebra®sh her7, frog ESR4 and ESR5, mouse and human HES7). Frog ESR6e is unusual, ending in WRPWQVLSPP. All Hey-like proteins have a YXXW motif near the C-terminus, with a generally conserved 6 ± 10 amino acid extension. (b) Alignments of the bHLH, Orange, and carboxy termini of selected ¯y, frog, and human bHLH-O proteins. Alignments are based on ClustalX analysis and visual inspection. The asterisk in the bHLH alignment indicates the conserved proline found in hairy-related and E(spl)-related subfamilies, and the conserved glycine at the same position in the Hey-related subfamily. The HC domain is conserved from ¯y to human (Zf=Zebra®sh). Beetle (T. castaneum) and spider (C. salei) hairy proteins also contain the HC domain (not shown) conserved glycine at the same position, while the basic region of the Stra13 proteins has a proline at a dierent position (Figure 2b). The functional signi®cance of these signature residues is not entirely clear. Based upon the crystal structure of the myogenic bHLH protein MyoD (Ma et al., 1994), the proline and glycine residues in the hairy/E(spl)/Hey proteins are on the face of the basic region pointing into the major groove. Intriguingly, a threonine residue in MyoD present at the same position as the proline in the hairy and E(spl) bHLH-O proteins is essential for myogenic speci®city (Davis et al., 1990). Perhaps a Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8346 Table 1 Human bHLH-O proteins and probable orthologs in other vertebrates Human Mouse Rat Chicken Frog HES1/HRY HES2 HES3 HES4 HES5 HES6 HES7 Hey1 Hey2 HeyL Stra13/DEC1 DEC2 HES1 HES2 HES3 HES1 HES2 HES3 hairy2 hairy1 SubZebrafish family her6 hairy E(spl) her3 E(spl) hairy1 hairy2a/b hairy1 hairy HES5 HES5 ESR1 E(spl) HES6 HES6 E(spl) HES7 ESR5 her1 E(spl) Hey1 Hey1 Hesr1/Hey1 Hey Hey2 Hey2 gridlock Hey HeyL Hey Stra13 SHARP2 Stra13 DEC2 SHARP1 Stra13 Identi®cation of probable orthologs in other organisms and bHLH-O subfamily assignments are based on phylogenetic analysis in Figure 1. Where no name is listed, no de®nite ortholog has been isolated proline at this position introduces a dierent local structure in the bound bHLH-O protein that contributes to its function. Alteration of the basic region proline to other residues in the Drosophila hairy or E(spl) proteins has not revealed a requirement for this residue (Tietze et al., 1992; Dawson et al., 1995), but its absolute evolutionary conservation indicates that it must have a signi®cant role under some circumstances. Most bHLH proteins bind as either hetero- or homodimers to a consensus DNA sequence of CANNTG, known as an E-box. Additional binding speci®city is derived from interactions between the basic regions and the middle two bases, as well as bases ¯anking the E-box (Blackwell and Weintraub, 1990). The ¯y E(spl) bHLH-O proteins, as well as mammalian HES1 and HES5, were found initially to bind as homodimers to an alternate sequence, CACNAG, known as an N-box (Akazawa et al., 1992; Sasai et al., 1992; Tietze et al., 1992). However, subsequent studies have found that the ¯y E(spl) and mammalian HES2 proteins preferentially bind to an E-box instead of the N-box, although they can bind to the N-box (Ishibashi et al., 1993; Jennings et al., 1999). In contrast, HES1 preferentially binds to the N-box (Sasai et al., 1992), although it also can bind to an E-box (Hirata et al., 2000), while Drosophila hairy homodimers prefer a sequence similar to the N-box, CACGCG (Ohsako et al., 1994; Van Doren et al., 1994). The Hey proteins have been observed to bind to an E-box, but not the N-box (Nakagawa et al., 2000). At present, a binding site for the Stra13 proteins has not been identi®ed, although it has been reported that they do not bind to either E-boxes or N-boxes (Boudjelal et al., 1997; Garriga-Canut et al., 2001). In Drosophila and in mammalian systems, both N-boxes and E-boxes have been shown to mediate repression by bHLH-O proteins (Sasai et al., 1992; Takebayashi et al., 1994; Van Doren et al., 1994; Jennings et al., 1999). The ability to bind an E-box raises the possibility that bHLH-O proteins could compete with bHLH activator proteins for binding to E-box target sequences (Figure 3c). Consistent with this, an optimal E-box binding site Oncogene Figure 3 Mechanisms of transcriptional repression by bHLH-O proteins. (a) The most common mechanism for bHLH-O protein repression is recruitment of corepressors (shown here as groucho/ TLE proteins) to target gene promoters by DNA binding of bHLH-O hetero- or homodimers at speci®c binding sites. (b) Vertebrate bHLH-O proteins (e.g. HES5) inhibit reporter activation by activator bHLH heterodimers, such as MASH1E47, and interact directly with bHLH activator proteins in vitro. This is consistent with either the formation of inactive heterodimers between bHLH-O proteins and activator bHLH proteins, or inhibition through other protein ± protein interactions. (c) It has been proposed that bHLH-O proteins can compete with activator bHLH proteins for the same DNAbinding sites (see text). In this hypothetical example, bHLH-O homodimers compete with MASH1-E47 heterodimers for DNAbinding to an E-box; the bHLH-O proteins also may recruit corepressors to inhibit promoter function for the Drosophila E(spl) bHLH-O proteins is also an optimal binding site for heterodimers of the Drosophila daughterless and lethal of scute bHLH activators, and these proteins can compete for this site in in vitro DNA binding assays (Jennings et al., 1999). Competition for binding sites has not yet been demonstrated for Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner vertebrate bHLH-O proteins, but the ability of HES2 and other vertebrate proteins to bind to E-boxes suggests that this may occur. Interactions between the bHLH-O proteins and transcriptional corepressors A key discovery for understanding the mechanism of bHLH-O protein function was made by Ish-Horowicz and colleagues. Using a two-hybrid screen, they determined that the Drosophila hairy protein binds to the transcriptional corepressor groucho, and they showed that this interaction requires the conserved Cterminal tetrapeptide WRPW motif (Paroush et al., 1994). Since the WRPW motif or a related sequence is present at the C-terminus of most bHLH-O proteins, this immediately suggested a common mechanism of action for these proteins in both Drosophila and vertebrates (Figure 3a). Subsequently it was shown that attaching the WRPW motif to the C-terminus of the yeast gal-4 DNA-binding domain is sucient to recruit groucho to a promoter with gal-4 binding sites and thus repress transcription (Fisher et al., 1996). Groucho has several vertebrate homologs, known as TLE proteins, and the TLE proteins interact with mammalian HES1 via its WRPW motif (Grbavec and Stifani, 1996; Grbavec et al., 1998). Groucho/TLE proteins are complex transcriptional corepressors that do not bind directly to DNA, but instead are recruited to target genes by a variety of DNA bound repressors (Fisher and Caudy, 1998b). Groucho/TLE proteins appear to function at least in part by recruiting histone deacetylases (HDACs) to repress target genes (reviewed by Chen and Courey, 2000). Recently it has been reported that TLE proteins can mediate an interaction between HES1 and the winged-helix repressor BF-1, and this interaction potentiates HES-1 repression (Yao et al., 2001). This raises the possibility that groucho/TLE proteins could mediate cooperative interactions between bHLH-O proteins and multiple types of repressors. Such cooperative repression would parallel the cooperative activation permitted by coactivator-mediated interactions between transcriptional activators, and might be expected to permit the evolution of more complex control of transcriptional repression. While Drosophila hairy and E(spl) proteins end precisely with the WRPW tetrapeptide, some vertebrate bHLH-O proteins have a short extension to this sequence (see Figure 2b). For example, the mammalian HES1 protein ends with WRPWRN. The signi®cance, if any, of the two additional amino acids is unclear, and these extra amino acids still permit HES1 to interact with the TLE1 protein (Grbavec and Stifani, 1996). Since vertebrates have multiple TLE proteins, one intriguing possibility is that variations on the WRPW motif might restrict which TLE proteins bind to a speci®c bHLH-O protein. Proteins in the Hey subfamily do not contain the WRPW tetrapeptide, but they do contain a related motif, YXXW, located near their C-termini. It is not yet known if this motif can recruit groucho/TLE proteins. Expression of Hey proteins can repress transcription of a reporter based on the mouse Hey2 promoter (Nakagawa et al., 2000). Unlike groucho/ TLE-mediated repression, inhibition of the Hey2 promoter by Hey proteins does not require HDAC function. However, it also does not require the YXXW motif, leaving open the question of whether this motif may recruit TLE proteins or other HDAC associated corepressors in another context. The Stra13 proteins do not contain any sequences related to the WRPW or YXXW motifs. However, the Stra13 and SHARP1 proteins can repress transcription by an HDACdependent mechanism and the sequences required for this repression map to near their C-termini (Sun and Taneja, 2000; Garriga-Canut et al., 2001). Whether TLE proteins or other corepressors participate in this repression has not been determined. In addition, Stra13 can repress transcription by a second, HDACindependent, mechanism (Boudjelal et al., 1997; Sun and Taneja, 2000). The bHLH-O proteins may interact with corepressors other than groucho/TLE proteins. The CtBP corepressor protein has been shown to bind to the Cterminus of Drosophila hairy (Poortinga et al., 1998). This interaction maps to a PLSLV motif in Drosophila hairy, and CtBP can also bind to a similar motif in E(spl)mdelta. The PLSLV motif is not present in the vertebrate hairy-like proteins, although it remains possible that CtBP proteins interact with vertebrate bHLH-O proteins via a divergent motif. CtBP has been reported to function as a short-range corepressor in ¯ies (Nibu et al., 1998), suggesting that CtBP could participate in hairy-mediated repression. However, CtBP can interfere with groucho-mediated repression (Zhang and Levine, 1999; Phippen et al., 2000). Since CtBP can bind to hairy simultaneously with groucho, this suggests that CtBP may negatively regulate the function of a hairy/groucho complex, a model consistent with the inhibitory genetic interaction between CtBP and hairy in early Drosophila embryos (Poortinga et al., 1998; Phippen et al., 2000). It is also possible that CtBP inhibits hairy function in the early embryo, but cooperates with hairy to repress transcription in another context. The likelihood of additional corepressor interactions with bHLH-O proteins suggests that the assembly of a DNA bound, bHLH-O `repressor complex' may turn out to be as sophisticated as the interactions between transcriptional activator proteins and coactivators required to form a DNA bound `activator complex'. 8347 HLH dimerization Like other bHLH proteins, the bHLH-O proteins form heterodimers or homodimers via their HLH domain. Members of the hairy subfamily including HES1 have been reported to homodimerize in vitro (Sasai et al., 1992; Van Doren et al., 1994) and in two-hybrid assays (Alifragis et al., 1997), and the SHARP1 protein can homodimerize in vivo (GarrigaOncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8348 Oncogene Canut et al., 2001). Other vertebrate HES proteins and the ¯y E(spl) bHLH-O proteins can homodimerize and in some cases also heterodimerize, either with related bHLH-O proteins, or with activator bHLH proteins (Akazawa et al., 1992; Ishibashi et al., 1993; Alifragis et al., 1997; Hirata et al., 2000). HES1 can interfere with DNA binding by heterodimers of the myogenic activator MyoD and E47 and it can inhibit reporter activation by E47 heterodimerized with either MyoD or MASH1 (Sasai et al., 1992; Hirata et al., 2000). HES5 can interact with the MASH1 or E47 bHLH proteins in vitro, and it can inhibit E47 from binding to E-boxes in vitro, as well as attenuate reporter activation by E47 in cultured cells (Akazawa et al., 1992). This suggests that, in addition to acting as DNA-binding repressors, the bHLH-O proteins may titrate activator bHLH proteins by dimerization, analogous to the Id/emc proteins (Figure 3b). However, most experimental tests of activator titration by bHLH-O proteins have used large amounts of bacterially synthesized protein for in vitro binding assays, or overexpressed bHLH-O proteins for reporter assays. It is not certain that dimerization between bHLH-O proteins and activator bHLH proteins occurs in vivo at physiological expression levels. In addition, the domains required for interactions between bHLH-O proteins and activator proteins have rarely been tested, leaving open the possibility that some of these interactions may not be mediated by HLH dimerization. An interesting variation on inhibition by dimerization appears to result from alternate promoter usage in the mammalian HES3 gene. Hes3 protein is expressed in two forms, one with a bHLH domain and one that lacks part of the DNA binding basic region, but retains the HLH dimerization domain (Hirata et al., 2000). The two HES3 proteins are expressed at dierent times during development, suggesting distinct functional roles (Hirata et al., 2000). The truncated HES3 protein seems likely to titrate other bHLH proteins by dimerization, but whether its targets are other bHLH-O proteins or bHLH activator proteins is not known. It was recently observed that the Hey1 and Hey2 proteins interact with the ARNT bHLH-PAS protein using a two-hybrid screen (Chin et al., 2000). This suggests that these proteins may heterodimerize, although the speci®c domains mediating the interaction were not mapped. Hey1 can interfere with the ability of ARNT to activate a reporter, and this requires both the bHLH and Orange domains. Heterodimerization between subfamilies of bHLH-O proteins also has been reported. HES6, an E(spl) subfamily protein, can heterodimerize with mammalian HES1 as well as with Xenopus hairy1 and hairy2 (Bae et al., 2000; Koyano-Nakagawa et al., 2000). Coexpression of HES6 with HES1 prevents HES1 from inhibiting a MASH1/E47 driven reporter gene, suggesting that HES6 antagonizes HES1 function. However, others have reported that HES6 and HES1 can function cooperatively as heterodimers (Gao et al., 2001). Surprisingly, shortening the HES1 loop (part of the helix ± loop ± helix domain) to match the length of the HES6 loop allows HES1 to mimic HES6 in some functional assays (Bae et al., 2000). This change may alter the dimerization or other protein ± protein interaction properties of HES1. Hey2 has also been reported to heterodimerize with HES1 (Nakagawa et al., 2000), and the chicken Hey1 and Hey2 proteins interact with chicken hairy1 in a two-hybrid assay (Leimeister et al., 2000a), although the functional consequences of these interactions have not been established. While interactions between bHLH-O subfamilies may be inhibitory (e.g. by the formation of non-functional heterodimers), such interactions could also generate heterodimers with novel DNAbinding speci®cities and/or the ability to recruit new combinations of corepressors. It will be interesting to see if in vitro selection of binding sites for Hey/hairy heterodimers leads to the identi®cation of novel DNAbinding sites. Role of the Orange domain The Orange domain was identi®ed as a functional domain in the Drosophila hairy and E(spl) proteins (Dawson et al., 1995; Giebel and Campos-Ortega, 1997). In transgenic Drosophila embryos, the hairy protein can antagonize activation of the Sex-lethal gene by the activator bHLH protein scute (Parkhurst et al., 1990). In contrast, the E(spl) m8 protein does not prevent the activation of Sex-lethal by scute. Analysis of chimeric proteins between hairy and m8 demonstrated that the speci®city for scute inhibition in this system mapped to the hairy Orange domain (Dawson et al., 1995). While the bHLH domain also was required for inhibition, the m8 bHLH domain could substitute for the hairy bHLH domain. Since the Xenopus hairy1 protein also can inhibit the activation of Sex-lethal by scute in transgenic ¯y embryos (Dawson et al., 1995), Orange domain function may be associated with subfamily speci®city. In vertebrates, there has been little analysis of functional requirements for the Orange domain. However, the HES1 Orange domain is necessary for HES1 to inhibit the activation of the p21 promoter by MASH1 and E47 in cultured cells (Castella et al., 2000). The molecular function of the Orange domain remains unclear, although its conserved relationship with the HLH domain raises the possibility of a role in dimerization. As noted earlier, the Orange domain is always located C-terminal to the bHLH. Two other families of bHLH proteins have a conserved domain located C-terminal to the bHLH domain. In the bHLH-ZIP proteins, a leucine zipper is contiguous with the second helix of the HLH domain (Baxevanis and Vinson, 1993). Unlike the leucine zipper in bHLH-ZIP proteins, the Orange domain is separated from the bHLH domain by a short, variable length region of protein. The bHLH-PAS proteins have a similar organization, with the PAS domain located a short distance C-terminal to the Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8349 Figure 4 Simpli®ed schematic of Notch receptor pathway regulation of bHLH-O gene expression. A signaling cell expresses ligands of either the Delta or Serrate class. Ligand binding to Notch on the responding cell leads to regulated proteolysis of the Notch receptor and release of the Notch intracellular domain (NICD) from the plasma membrane. CSL protein complexes are generally thought to repress Notch targets in the absence of Notch signaling. Binding of the NICD alters the CSL complex to induce transcription of Notch target bHLH-O genes, such as HES1 bHLH domain (reviewed by Crews and Fan, 1999). However, the Orange domain is much smaller than the PAS domain. Both the leucine zipper of bHLHZIP proteins and the PAS domain of bHLH-PAS proteins mediate dimerization, raising the question of whether the Orange domain may also function as an extended dimerization domain. The chicken hairy1 Orange domain has been reported to enhance interaction between two hairy1 monomers in a two-hybrid assay, which would be consistent with a role for the Orange domain in dimerization (Leimeister et al., 2000a). In addition, two-hybrid interactions between the chicken Hey1 or Hey2 proteins and chicken hairy1 are strongly enhanced by the presence of the Orange domain (Leimeister et al., 2000a). However, it remains possible that these enhanced interactions arise from an indirect eect of the Orange domain (e.g. protein stabilization). It should be mentioned that the PAS domain also acts as a ligand-binding domain in some vertebrate bHLH-PAS proteins (Crews and Fan, 1999), but at present there is no data to suggest that bHLH-O proteins require ligands for function in any system. Additional mechanisms of bHLH-O protein function Under certain experimental conditions, the WRPW motif has been shown to be dispensable for repression by speci®c hairy or E(spl) proteins. A Drosophila hairy protein missing the WRPW motif can antagonize ectopic activation of Sex-lethal by the scute bHLH protein, although this same hairy protein cannot block endogenous Sex-lethal expression (Dawson et al., 1995). Similarly, a truncated zebra®sh her4 protein that does not contain the WRPW motif can repress neurogenesis in zebra®sh embryos (Takke et al., 1999). DNA-binding is also not always required: Drosophila E(spl) proteins with a mutation in the DNA-binding domain can still function to regulate bristle formation (Giebel and Campos-Ortega, 1997). A similar observation has been made for the rat HES1 protein (Castella et al., 2000). Even more strikingly, the HES6 protein remains functional in ectopic expression assays in Xenopus embryos with either a mutation in the DNA binding region or a truncation that removes the WRPW motif (Koyano-Nakagawa et al., 2000). The simplest explanation for these results is that these Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8350 bHLH-O proteins act as competitive inhibitors of dimerization in these experiments, and thus do not require the ability to recruit groucho/TLE proteins to DNA. However, this does not readily explain the ability of the truncated ¯y hairy protein to inhibit scute function, since hairy does not dimerize with either scute or its dimerization partner (Alifragis et al., 1997). Another possibility is that the bHLH-O proteins repress transcription through additional mechanisms that remain intact in these mutant proteins. These might include interactions with other corepressors or DNA-binding repressors. It will be interesting to see if additional proteins that interact with the bHLH-O proteins can be isolated. One candidate for mediating additional protein ± protein interactions is the HC domain present in all members of the hairy subfamily (Figure 2). While no role has been determined for this domain, its conservation from invertebrate hairy proteins through human hairy-related proteins implies a strong functional selection. Regulation of vertebrate bHLH-O gene expression by Notch signaling It has become clear that the Notch signaling pathway plays a major role in regulating the transcription of many vertebrate bHLH-O genes. Notch signaling controls cell fate decisions and other developmental processes in both vertebrates and invertebrates (Artavanis-Tsakonas et al., 1999). As a consequence of ligand binding, the intracellular domain of the Notch transmembrane receptor is released by proteolysis, and translocates to the nucleus (Mumm and Kopan, 2000). The intracellular domain of Notch then associates with a CSL (CBF1, Su(H), Lag-1) transcription factor, converting the CSL protein from a repressor into an activator (Figure 4). Israel and coworkers showed that a constitutively active form of Notch could activate the expression of a transiently transfected reporter based on the HES1 promoter, and this activation required CSL binding sites in the promoter (Jarriault et al., 1995). This suggested that HES1 transcription is activated directly in response to Notch signaling, thus positioning HES1 as a potential eector of Notch signaling. Later studies have shown that HES1 expression is dependent on Notch function in the segmenting mesoderm (Jouve et al., 2000). However, it remains unclear whether all expression of HES1 depends on Notch signaling. Rossant and coworkers reported that HES1 expression in the developing mouse nervous system is not signi®cantly altered by targeted disruptions of Notch1 or RBP-JK, a mouse CSL gene (de la Pompa et al., 1997). However, a constitutively active Notch protein stimulates HES1 gene expression in retinal progenitors (Ohtsuka et al., 1999). Thus, HES1 may act as an eector for Notch within the nervous system in some cells, as well as the segmenting mesoderm, while possibly functioning in a Notch independent manner in other cells. Oncogene Expression of the HES5 gene, which encodes an E(spl)-like protein, depends on an intact Notch signaling pathway. HES5 expression is reduced or abolished in mice mutant for Notch1, RBP-JK, or presenilin1 and 2 (presenilins are required for Notch proteolytic activation) (de la Pompa et al., 1997; Barrantes et al., 1999; Donoviel et al., 1999; Handler et al., 2000). These observations suggest that HES5 is likely to be an eector for Notch signaling in the developing nervous system and elsewhere. In Xenopus, genes for several E(spl)-related proteins can be activated by Notch signaling, including ESR1 and ESR7, which are closely related to HES5, as well as ESR4, ESR5, and ESR6e (Wettstein et al., 1997; Deblandre et al., 1999; Jen et al., 1999; KoyanoNakagawa et al., 2000). In at least some cases, this expression can be blocked by a dominant negative ligand for Notch or a dominant negative CSL protein (Chitnis et al., 1995; Jen et al., 1997; Wettstein et al., 1997; Jen et al., 1999). Similarly, the zebra®sh E(spl)like genes her1 and her4 are activated by Notch signals (Takke and Campos-Ortega, 1999; Takke et al., 1999). Notch signaling has also been observed to regulate expression of the Hey genes in mice. Expression of the Hey genes in mouse embryos depends on function of the Notch ligand Dll-1 (Kokubo et al., 1999). Consistent with this, the promoter regions of the three mammalian Hey genes contain binding sites for CSL proteins, and these promoters are responsive in cultured cells to activated Notch signaling (Maier and Gessler, 2000; Nakagawa et al., 2000). Transgenic expression of activated Notch in mouse hair follicles leads to ectopic expression of the HeyL gene (Lin et al., 2000), while mice mutant for Notch1 or the Notch ligand Dll-1 lose expression of HeyL in the presomitic mesoderm (Leimeister et al., 2000b). Although a number of bHLH-O genes have been shown to be immediate targets of Notch signaling, some bHLH-O genes do not appear to function in the Notch pathway. The mouse and Xenopus HES6 genes do not respond to activated Notch1 (Bae et al., 2000; Koyano-Nakagawa et al., 2000), nor do the mouse HES3 (Hirata et al., 2000) or Xenopus ESR2 genes (DL Turner, unpublished). There are suggestions that HES6 is regulated by neural bHLH activators in both Xenopus and mouse (Koyano-Nakagawa et al., 2000). The Stra13 and SHARP1 genes have been shown to be activated by a variety of Notch-independent signals in cultured cells (Boudjelal et al., 1997; Rossner et al., 1997; Sun and Taneja, 2000; Ivanova et al., 2001); whether these genes also can be regulated by Notch signaling is not known. It will be interesting to further characterize the upstream factors or signals that regulate these genes. In Drosophila, expression of most E(spl) genes depends on Notch signaling, while expression of genes for hairy and related proteins is not known to require the Notch pathway (Bier et al., 1992; Langeland and Carroll, 1993; Jennings et al., 1994; Bailey and Posakony, 1995). This apparent distinction between these two subfamilies of bHLH-O genes has not been Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner evolutionarily conserved, since members of both subfamilies are regulated by Notch in vertebrates (e.g. HES1 and HES5). However, additional transcriptional inputs are also required to generate the normal expression patterns for the E(spl) genes during Drosophila development (Nellesen et al., 1999), and this is also true for vertebrate bHLH-O genes regulated by Notch. The Xenopus ESR6e gene responds to activated Notch within the developing epidermis, but not within the developing nervous system, while the ESR1 and ESR7 genes can be activated by Notch in both neural and epidermal cells (Deblandre et al., 1999). In contrast, the ESR4 and ESR5 genes only respond to Notch in the segmenting mesoderm (Jen et al., 1999). The hairy subfamily gene Xenopus hairy2 and its chicken ortholog c-hairy1 have dynamic expression patterns in the segmenting mesoderm (discussed below) that have been interpreted to mean that they are not direct targets of Notch signaling. However, recent data obtained with Xenopus hairy2 promoter constructs in transgenic Xenopus embryos shows that hairy2 expression in the segmenting mesoderm (as well as the neuroectoderm) depends on a paired CSL binding motif in the promoter, implying a requirement for Notch signaling (Davis et al., 2001). Two additional cis-regulatory elements are required to reconstitute the hairy2 pattern in the mesoderm, demonstrating a more complex regulation of hairy2 in this tissue. Biological roles of the bHLH-O proteins in vertebrates In this section, we discuss the function and regulation of bHLH-O proteins in neurogenesis, neural cell fate, vascular development, mesoderm segmentation, and myogenesis. While roles for bHLH-O proteins in these processes are well established, expression patterns and functional analyses implicate bHLH-O proteins in other processes as well. For example, Xenopus ESR6e is involved in patterning of embryonic epidermis (Deblandre et al., 1999), and the expression of Xenopus hairy2 suggests a role in patterning the early neural plate (Turner and Weintraub, 1994). Stra13 has been linked to both mesoderm and neural cell fate regulation in cultured P19 embryonal carcinoma cells (Boudjelal et al., 1997), zebra®sh her5 is involved in mesendoderm cell fate (Bally-Cuif et al., 2000), and mammalian HES1 regulates T lymphocyte dierentiation (see next section). It seems likely that the list of processes regulated by bHLH-O proteins will continue to expand as the functional roles of these proteins are better characterized. Regulation of vertebrate neurogenesis by bHLH-O proteins In vertebrates, the bHLH-O proteins and the Notch pathway play essential roles in restricting the dierentiation of neurons from neural precursor cells. In the developing mammalian nervous system, the expression of HES1 and HES5 is generally restricted to regions containing undierentiated neural precursors (Akazawa et al., 1992; Sasai et al., 1992; Ishibashi et al., 1995). Constitutive expression of HES1 in neural precursors, using a retroviral vector, prevents neuronal dierentiation in both the brain (Ishibashi et al., 1994) and the retina (Tomita et al., 1996). Conversely, targeted disruption of the HES1 gene in mice leads to premature dierentiation of neurons in the telencephelon (Ishibashi et al., 1995), olfactory placode (Cau et al., 2000), inner ear (Zheng et al., 2000), and the retina (Tomita et al., 1996). Similarly, transfection of primary rat hippocampal neural precursors with HES1 inhibits dierentiation (Castella et al., 1999). Consistent with these results, ectopic expression of E(spl)-related proteins can inhibit dierentiation of primary neurons in zebra®sh (Takke et al., 1999) and Xenopus (Koyano-Nakagawa et al., 2000; Schneider et al., 2001). In Drosophila, the E(spl) and hairy proteins appear to restrict neurogenesis both by directly repressing expression of the proneural bHLH genes, and by antagonizing the ability of proneural bHLH proteins to activate target genes (Ohsako et al., 1994; Van Doren et al., 1994). A similar regulatory relationship between the bHLH-O repressors and proneural activators has been conserved in vertebrates. In both the telencephalon and olfactory placode of HES1 null mice, mRNA for the proneural gene MASH1 is upregulated (Ishibashi et al., 1995; Cau et al., 2000). This is likely to re¯ect the lack of direct repression of MASH1 by HES1, based on studies in cell culture (Chen et al., 1997). In addition, HES1 can inhibit MASH1 DNA binding in vitro, and antagonize MASH1 activation of reporters and target genes in cultured mouse cells (Sasai et al., 1992; Castella et al., 1999, 2000; Hirata et al., 2000). It also appears that neuroendocrine dierentiation in mammalian lungs is regulated by the repression of MASH1 by HES1 (Ito et al., 2000). Intriguingly, mice with a targeted disruption of HES5 do not upregulate MASH1 in the olfactory placode, but HES1/HES5 double null mice upregulate mRNA levels for both MASH1 and a second bHLH gene, neurogenin1 (ngn1) (Cau et al., 2000). In the olfactory bulb, ngn1 is expressed in a population of precursors with restricted potential, and this expression depends on MASH1 (Cau et al., 1997). These observations suggest that HES1 restricts MASH1 expression in early olfactory progenitors, while HES1 and HES5 act redundantly to restrict the expression of ngn1 in later progenitors. Thus, HES1 and HES5 can regulate transitions in gene expression within a mitotic neural precursor population, in addition to restricting the terminal dierentiation of neurons. It seems likely that HES1 and HES5 function in precursors at least in part by inhibiting the activation of the ngn1 gene by MASH1. Similarly, forced expression of zebra®sh her4 can repress neurogenin expression in developing zebra®sh embryos (Takke et al., 1999). Loss of function for Notch pathway components within the developing nervous system also leads to 8351 Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8352 excessive and premature dierentiation of neurons (Chitnis et al., 1995; de la Pompa et al., 1997; Handler et al., 2000), consistent with observations that Notch signaling regulates HES1 and HES5 expression. To directly test the requirement for HES1 and HES5 in Notch signaling, Ohstuka et al. (1999) prepared neural precursors from mice with targeted disruptions of HES1, HES5 or both, and then infected these cells with a retroviral vector expressing a constitutively active Notch1 protein. Activated Notch almost completely inhibited the dierentiation of neural precursors from wild-type mice and from mice with either HES1 or HES5 disrupted. In contrast, activated Notch did not eciently inhibit neuronal dierentiation of neural precursors missing both HES1 and HES5. This indicates that HES1 and HES5 are required redundantly for eective Notch mediated repression of dierentiation. Since activated Notch still had some inhibitory function in the HES1/HES5 double null neural precursors, there are likely to be additional eectors for Notch in the nervous system. A few bHLH-O proteins are expressed in dierentiating or dierentiated neurons. These include one form of HES3 (Hirata et al., 2000), which is expressed in the Purkinje cells of the cerebellum, and HES6, which is found in dierentiating and mature neurons in many parts of the nervous system (Bae et al., 2000; Koyano-Nakagawa et al., 2000; Pissarra et al., 2000; Vasiliauskas and Stern, 2000). Surprisingly, ectopic expression of HES6 in Xenopus embryos promotes ectopic neuronal dierentiation, as well as upregulation of a number of neural bHLH genes including neuroD and ngn2 (Koyano-Nakagawa et al., 2000). Similarly, forced expression of HES6 in retinal progenitors promotes neuronal dierentiation (Bae et al., 2000). It has been proposed that HES6 functions by inhibiting other bHLH-O factors such as HES1 and Xenopus hairy1/2 (Bae et al., 2000; Koyano-Nakagawa et al., 2000). Regulation of neural cell fate by bHLH-O proteins In addition to regulating neuronal dierentiation, the HES1, HES5, and Hey2 proteins have been found to alter the choice of neuronal versus glial cell fate in the developing retina. Retinas from HES5 null mice have fewer Muller glial cells, while forced expression of HES5 in retinal cells using a retroviral vector leads to most infected cells becoming Muller glia (Hojo et al., 2000). Consistent with this, postnatal HES5 expression in the retina becomes localized to the layer that contains dierentiating Muller glial cells. While Tomita et al. (1996) reported that forced expression of HES1 in retinal cells inhibited all dierentiation, Furukawa et al. (2000) found that HES1 could divert retinal cells to a Muller glial fate, as was observed for HES5. The reason for this discrepancy is not clear, although a dierence in the expression levels of the introduced HES1 genes seems a plausible explanation. Furukawa et al. (2000) also found that a putative dominant negative HES1 (a HES1 protein defective in DNA Oncogene binding) interfered with Muller glial dierentiation. The Hey1, Hey2, and HeyL genes are also expressed in the developing retina, and as observed for HES5 and HES1, forced expression of Hey2 diverts the majority of infected cells to a Muller glial fate (Satow et al., 2001). However, forced expression of Hey1 or HeyL has no eect on cell fate. Although the HES and Hey proteins may directly promote gliogenesis, it seems more likely that they allow precursor cells to adopt a glial instead of neuronal fate by repressing neuronal dierentiation. As discussed above, the HES1 and HES5 proteins can inhibit the function of neural bHLH proteins such as MASH1. Interestingly, mice double mutant for either MASH1 and MATH3 (Tomita et al., 2000), or MASH1 and neurogenin2 (Nieto et al., 2001) have excessive glial dierentiation. Analysis of neural precursors from the MASH1/ neurogenin2 double mutant mice indicates that, in the absence of neural bHLH function, precursors that would normally dierentiate as neurons can adopt a glial fate. In addition, forced expression of the neural bHLH protein ngn1 in neural precursors not only promotes neuronal dierentiation, but inhibits glial dierentiation (Sun et al., 2001). Thus, bHLH-O proteins may direct neural precursor cells toward a glial fate by inhibiting the expression and/or function of the neural bHLH proteins. Hey proteins and blood vessel formation The mouse Hey1 and Hey2 genes are expressed in the developing heart and in the cardiac vessels, including the aorta, with HeyL expressed later in the cardiac vessels (Leimeister et al., 1999; Nakagawa et al., 1999). The zebra®sh gridlock mutant contains a point mutation in the zebra®sh Hey2 gene, which is also expressed in the developing heart and aorta (Zhong et al., 2000). Gridlock mutants have defective circulation due to disrupted assembly of the aorta, and this defect can be rescued by injection of mRNA encoding the zebra®sh Hey2 protein. The point mutation in gridlock removes the stop codon for Hey2, adding a C-terminal extension to the protein. It is likely that this extension interferes with a required function of the C-terminal YXXW motif, since injection of an mRNA for a Hey2 protein without this motif fails to rescue the gridlock mutation (Zhong et al., 2000). The function of Hey2 in the aorta is unknown, but in mammalian cell culture, expression of Hey2 inhibits the VEGF promoter, which is activated by the bHLH-PAS protein ARNT (Chin et al., 2000). This raises the possibility that Hey2 regulates signals associated with vascular growth. Intriguingly, overexpression of Hey1 downregulates VEGF receptor 2 in cultured endothelial cells (Henderson et al., 2001), suggesting that the Hey proteins may regulate both VEGF signals and the response to those signals. HeyL may also have a role in vascular development. Leimeister (2000b) noted that expression of HeyL overlaps with Notch3 in vascular smooth muscle. Mutations in Notch3 give rise to CADASIL (cerebral autosomal dominant arteriopathy Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner with subcortical infarcts and leukoencephalopathy), a condition in which strokes arise from a vascular smooth muscle defect (Joutel et al., 1996). It will be interesting to see if HeyL expression is regulated by Notch3, and whether the HeyL protein is required for proper formation of arterial smooth muscle cells. bHLH-O gene expression during mesoderm segmentation In Drosophila, hairy functions in a complex genetic hierarchy that controls anteroposterior segmentation, while the expression patterns of hairy-like genes in other invertebrates are consistent with a function in segmentation (Lawrence, 1992; Sommer and Tautz, 1993; Damen et al., 2000). In vertebrates, hairy, E(spl), and Hey genes are expressed in dynamic patterns in the unsegmented paraxial, or pre-somitic mesoderm (PSM), again consistent with roles in segmentation (Hrabe de Angelis et al., 1997; Jen et al., 1997, 1999; Palmeirim et al., 1997, 1998; Stern and Vasiliauskas, 1998; Leimeister et al., 1999, 2000b; Takke and Campos-Ortega, 1999; Holley et al., 2000; Jouve et al., 2000; Bessho et al., 2001). For instance, cyclic expression of the chicken hairy1 and hairy2 genes begins with RNA localized to the posterior PSM (Palmeirim et al., 1997; Jouve et al., 2000). RNA then appears in more anterior PSM cells and sweeps forward, while RNA disappears in the posterior cells. A new cycle begins every 90 min, the same period as somite formation in the chick embryo. Cycle duration is 180 min and ends with hairy RNA expressed in a stripe in the caudal half (hairy1) or rostral half (hairy2) of a prospective somite two removed from the last completed somite. Thus, PSM cells experience multiple cycles of hairy expression and extinction before they are incorporated into a newly formed somite. The rhythmic expression of the two hairy genes suggests a clock-like mechanism controlling somite formation, consistent with many older experiments and theories of vertebrate segmentation (Cooke, 1975; Meinhardt, 1986; Cooke, 1998; Pourquie, 2000; Stern and Vasiliauskas, 2000). Similar rhythmic expression has been observed for her1 in zebra®sh, and HES1 in the mouse (Holley et al., 2000; Jouve et al., 2000). Lunatic fringe, a modulator of Notch signaling, also has been shown to have a rhythmic expression pattern similar to the hairy genes in chick and mouse embryos (Forsberg et al., 1998; Aulehla and Johnson, 1999; Moloney et al., 2000). This observation supports a connection between spatio-temporally regulated Notch signaling, and the segmentation `clock' (Evrard et al., 1998; Jiang et al., 1998; McGrew et al., 1998; Zhang and Gridley, 1998; Barrantes et al., 1999; Pourquie, 1999). Disruption of Notch signaling by transient expression of dominant interfering forms of Notch pathway components has been shown to disrupt both bHLH-O gene expression and normal segmentation in the frog (Jen et al., 1997, 1999). These ®ndings are supported by zebra®sh mutants, and targeted knockouts in the mouse (Conlon et al., 1995; Oka et al., 1995; Holley et al., 2000; Jiang et al., 2000; Jouve et al., 2000). Since disruption of Notch signaling disrupts the dynamic pattern of bHLH-O gene expression in the PSM, it is likely that the regulated pattern of bHLH-O genes in the PSM is crucial to vertebrate segmentation. 8353 bHLH-O proteins and myogenesis The expression of most bHLH-O genes involved in paraxial mesoderm segmentation ends prior to the dierentiation of somitic muscle. However, both HES1 and HES6 expression patterns in mouse embryos suggest that these proteins may function during muscle dierentiation, subsequent to their apparent roles during somite formation. In the initial report describing the rat HES1 gene, the authors showed that high-level co-expressed HES1 could inhibit the ability of MyoD to convert 10T1/2 mouse embryo ®broblasts to muscle cells (Sasai et al., 1992). Subsequently, Honjo and coworkers used a co-culture assay to study Notch signaling and muscle dierentiation in vitro (Kuroda et al., 1999). Mouse C2C12 myoblasts, which can dierentiate in culture under appropriate conditions, were mixed with a myeloma cell line expressing the Notch ligand mouse Dll-1 (Delta-like 1) on its surface. This co-culture inhibited C2C12 dierentiation and HES1 mRNA increased signi®cantly in the C2C12 cells within 1 h of coculture. Subsequent to HES1 up-regulation, expression of the myogenic bHLH protein MyoD decreased, but forced expression of MyoD could rescue muscle dierentiation. These observations, together with other studies of Notch inhibition of muscle dierentiation (Kopan et al., 1994; Nye et al., 1994; Hirsinger et al., 2001) suggest that HES1 functions as an eector of Notch signaling to inhibit MyoD expression in myoblasts and thereby restrict muscle formation. However, additional experiments suggest that there may be other pathways, independent of HES1, that are also required for inhibition of myogenesis by Notch signaling (Shawber et al., 1996). In contrast, recent observations indicate that expression of HES6 promotes myogenic dierentiation, possibly by repressing expression of the bHLH repressor MyoR (Gao et al., 2001). bHLH-O proteins and oncogenesis The ability of bHLH-O proteins to negatively regulate dierentiation during development points to a potential role in oncogenesis. Inappropriate, sustained expression of bHLH-O proteins could act as one of the molecular insults leading to malignant disease. In addition, the suspected roles of the Hey proteins in controlling VEGF and the VEGF receptor expression suggest intriguing links to angiogenesis, a known in¯uence on tumor growth and/or metastasis. However, the best circumstantial evidence to date for the involvement of a bHLH-O protein in oncogenesis focuses on HES1 as a target of misregulated Notch signaling in T-cells. Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8354 Targeted knockout of Notch1 has demonstrated a requirement for Notch signaling at early stages of Tcell thymic development; later stages of T-cell development do not seem to depend on Notch1 (Radtke et al., 1999; Wolfer et al., 2001). However, a knockout of HES1 results in an almost complete loss of thymus. Immune system reconstitution experiments show that the defect is in thymocyte development rather than in the thymus stroma (Tomita et al., 1999). The speci®c defect is in proliferative expansion of CD4/CD8 double negative T-cell precursors, consistent with a role for HES1 in maintaining the proliferative capacity of cells during progressive steps of dierentiation. Retroviral expression of constitutively active Notch1 in mouse thymocytes prevents formation of more dierentiated single positive CD4 or CD8 cells from double positive CD4/CD8 precursors (Izon et al., 2001). In a dierent setting, transgenic mice expressing active Notch1 under a thymocyte speci®c promoter show an altered ratio of mature T-cell development, with single positive CD8 T-cells increased at the expense of single positive CD4 T-cells (Robey et al., 1996). This eect is presumably in part a function of the ability of HES1 to downregulate the CD4 promoter (Kim and Siu, 1998), and probably other CD4 T-cell speci®c genes. In a subset of human T-cell lymphoblastic leukemia (T-ALL), a t(7;9) chromosome translocation joins the beta T-cell receptor to the human Notch1 gene, TAN-1, creating a truncated form of Notch1 and hence constitutive Notch signaling in leukemic Tcells (Ellisen et al., 1991). In a mouse model of this process, a T-cell line from a spontaneous T-cell lymphoma has a retroviral insertion into the Notch1 locus that generates a similar truncated and constitutively active form of mouse Notch1 (Lee et al., 1999). This line has elevated levels of HES1. More importantly, in a bone marrow reconstitution assay using cells infected with retroviruses expressing the activated TAN-1 protein, only clonal leukemias of immature T-cell origin developed in tumor bearing mice, even though retroviral expression of TAN-1 occurred in most hematopoietic cells (Pear et al., 1996). These leukemias were similar to those in human T-ALL involving TAN-1. Taken together, these studies support a role for increased HES1 expression, mediated by excess Notch signaling, in dampening cell dierentiation and promoting cell proliferation associated with T-ALL. The widespread role of Notch signaling in the development of other tissues leaves open the question of how extensive the role of bHLH-O proteins may be in the formation of other tumor types. Unresolved questions about bHLH-O function and regulation Vertebrate bHLH-O proteins act to restrict the dierentiation of precursor cells in a variety of tissues. However, we know of only a few genes involved in Oncogene dierentiation that are direct targets of bHLH-O repression. For example, in neural precursors and myoblasts, bHLH-O proteins antagonize the ability of the bHLH activators to initiate dierentiation. Are targets for bHLH-O repression primarily regulatory genes, such as those that encode the bHLH activators, or are genes for structural proteins also directly repressed by bHLH-O proteins? Do bHLH-O proteins block activation of all genes activated by the bHLH activators, or only an essential subset? In addition, almost nothing is known about the targets of bHLH-O function during patterning of the mesoderm and other tissues. With the advent of cDNA microarray analysis and other technologies for large-scale analysis of gene expression, we may soon learn answers to these questions. Signi®cant gaps remain in our understanding of the regulation of the bHLH-O genes. Although the Notch pathway regulates many of these genes in vertebrates, it is unlikely that Notch signaling alone is sucient to explain the expression patterns of these genes. What other factors interact with Notch signals to allow the expression of speci®c bHLH-O genes? Do dierent vertebrate Notch receptors or ligands regulate distinct subsets of bHLH-O proteins? What regulates bHLH-O genes that function independently of Notch? There is also the question of whether negative feedback and/or cross-regulation between vertebrate bHLH-O genes contributes to the control of their expression in vivo (see Takebayashi et al., 1994). Detailed answers to these questions will likely require systematic analysis of the regulatory elements from bHLH-O genes in transgenic animal models. A related issue is whether vertebrate bHLH-O proteins are regulated by posttranslational modi®cations. There is some evidence for control of HES1 function by phosphorylation (Strom et al., 1997), but to date this issue remains largely unexplored. While the most common mechanism for repression by bHLH-O proteins appears to be the recruitment of the groucho/TLE transcriptional corepressors to target genes, the lack of a requirement for the WRPW motif and/or DNA-binding in some systems suggests additional modes of action. Titration of activator bHLH proteins by dimerization or other direct interactions with bHLH-O proteins has long been proposed, but not yet clearly demonstrated in vivo. The recruitment of additional corepressors, instead of or in addition to the groucho/TLE proteins, is another possibility, especially in the case of the Hey and Stra13 proteins. The molecular roles of the Orange and HC domains remain unclear to date, but given the possibilities of activator titration and/or additional corepressor recruitment, studies of protein ± protein interactions dependent on these domains seem likely to provide additional insights into bHLH-O function. The identi®cation of protein ± protein interactions speci®c to dierent bHLH-O subfamilies also could help to elucidate why these subfamilies have been evolutionarily conserved. It also would be of interest to know if heterodimers between dierent bHLH-O subfamilies exist in vivo, Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner and if so, whether they have functional roles distinct from bHLH-O homodimers. The role of bHLH-O proteins in oncogenesis and other diseases has not been extensively explored. Are bHLH-O proteins expressed in speci®c types of tumors beyond T-ALL? Activator bHLH proteins are present in tumors of neuroendocrine and neural origin (Chen et al., 1997; Rostomily et al., 1997), suggesting that bHLH-O antagonism of dierentiation could play a role in the formation of these tumors. The possibility that Hey proteins could contribute to the control of tumor vascularization also warrants further investiga- tion. 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