Oncogene (2001) 20, 8342 ± 8357 ã 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 di€erentiation and embryonic patterning

Robert L Davis1 and David L Turner*,2

1Department 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 super- frogs, and zebra®sh. As we discuss below, the family of DNA-binding transcription factors that vertebrate hairy and E(spl) related proteins can be regulate numerous biological processes in both inverte- grouped into distinct subfamilies based on their brates and vertebrates. One family of bHLH transcrip- primary structures. However, all proteins in these tional repressors is related to the Drosophila hairy and subfamilies contain a conserved amino acid sequence Enhancer-of-split proteins. These repressors contain a known as the Orange domain located just C-terminal tandem arrangement of the bHLH domain and an to the bHLH domain. The tandem arrangement of the adjacent sequence known as the Orange domain, so we bHLH and Orange domains is the major structural refer to these proteins as bHLH-Orange or bHLH-O feature shared among these proteins, so for conve- proteins. Phylogenetic analysis reveals the existence of nience we refer to all hairy and E(spl) related proteins four bHLH-O subfamilies, with distinct, evolutionarily collectively as bHLH-Orange (bHLH-O) proteins. conserved features. A principal function of bHLH-O In both vertebrates and invertebrates, bHLH-O proteins is to bind to speci®c DNA sequences and recruit proteins function as DNA-binding transcriptional transcriptional corepressors to inhibit target gene repressors, and regulate a wide variety of biological expression. However, it is likely that bHLH-O proteins processes. These include negative control of di€erentia- repress transcription by additional mechanisms as well. tion (Fisher and Caudy, 1998a; Kageyama et al., 2000), Many vertebrate bHLH-O proteins are e€ectors of the anteroposterior segmentation in both invertebrates and Notch signaling pathway, and bHLH-O proteins are vertebrates (probably by distinct mechanisms; Palmeir- involved in regulating neurogenesis, vasculogenesis, im et al., 1997; Jen et al., 1999; Damen et al., 2000 and mesoderm segmentation, myogenesis, and T lymphocyte references therein), and sex determination in ¯ies development. In this review, we discuss mechanisms of (Parkhurst et al., 1990; Younger-Shepherd et al., action and biological roles for the vertebrate bHLH-O 1992). In many but not all of these processes, bHLH- proteins, as well as some of the unresolved questions O proteins function as e€ectors of the Notch signaling about the functions and regulation of these proteins pathway (Artavanis-Tsakonas et al., 1999). As might during development and in human disease. Oncogene be expect from their diverse roles, both structural and (2001) 20, 8342 ± 8357 functional analyses suggest that di€erences exist between the bHLH-O subfamilies, and even among Keywords: basic-helix-loop-helix; transcription; Notch; members of the same subfamily. Here we review the corepressor; hairy bHLH-O protein subfamilies and domain structures, known and proposed mechanisms of bHLH-O- mediated repression, and our current knowledge about Introduction the roles and regulation of bHLH-O proteins in vertebrates. We also consider some unresolved ques- Over the past decade, numerous vertebrate proteins tions about these proteins and their functions in structurally related to the Drosophila hairy and vertebrates. 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 Vertebrate bHLH-O proteins can be divided into four related proteins designated HES2 ± 5 (Akazawa et al., distinct subfamilies 1992; Sasai et al., 1992; Feder et al., 1993). Subse- quently, additional related proteins have been identi®ed Most sequence comparisons and derived phylogenetic in humans and other mammals, as well as chickens, 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 *Correspondence: DL Turner, Neuroscience Laboratory Building, 1103 East Huron, Ann Arbor, Michigan, MI 48104-1687, USA; a phylogenetic tree derived from pairwise comparisons E-mail: [email protected] 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,

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Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8344 comparison of full-length protein sequences, suggests hairy-like and E(spl)-like proteins have been named that there are four major subfamilies of these proteins. HES (hairy and enhancer of split) or her (hairy and We refer to the four subfamilies by the names of the enhancer of split related) in mammals and zebra®sh prototypic protein for each: hairy, E(spl), Hey, and respectively, and numbered by the order of isolation. Stra13. Except for Stra13, each of these subfamilies has Hey proteins have also been named HRT or Hesr, or in members from Drosophila to humans. Although all of the case of Hey2, Gridlock or CHF1, while Stra13 these proteins are transcriptional repressors, the proteins have also been named SHARP, DEC, CLAST, conserved di€erences in the primary structures imply or BHLHB2 (see Figure 1 legend). For brevity, we that members of di€erent subfamilies have distinct generally use only a single name for a bHLH-O protein functions and/or post-translational regulation. with multiple names in a given species. Most bHLH-O proteins have been isolated based on The recent completion of sequencing of the sequence similarity within the bHLH domain. However, Drosophila and human genomes permits a comparison as noted above, a second domain, denoted either as the of bHLH-O family complexity between a highly Orange domain (Dawson et al., 1995) or as helix III/IV evolved invertebrate and humans. While many tran- (Knust et al., 1992) is conserved in every member of each scription factor families have expanded in number in subfamily. Figure 2a shows a schematic of the domains humans relative to ¯ies (e.g. homeobox and activator present in each bHLH-O subfamily, while Figure 2b bHLH proteins), the bHLH-O family has not shows sequence alignments of representative domains. signi®cantly increased in size. Drosophila has 13 known The bHLH domains share typical features of the bHLH bHLH-O proteins (Moore et al., 2000), including three superfamily, although residues at certain positions are hairy proteins (hairy, deadpan and side), eight E(spl) speci®c to bHLH-O proteins (see below). The interven- bHLH-O proteins (seven m-type, and her), and two ing sequence between the bHLH and Orange domains Hey-like proteins (Hesr-1 and sticky/ch1). Humans ranges from seven to 41 amino acids. Although not appear to have 12 bHLH-O proteins. These include shown in Figure 2, this intervening sequence is highly two hairy-like proteins (HES1 and HES4). Zebra®sh similar among members of the hairy, Hey, and Stra13 and chickens also have two hairy-like proteins, subfamilies, respectively, while in the E(spl) subfamily it suggesting that this is the common number for shows more variation. The Orange domain is about 30 vertebrates. Humans have ®ve E(spl) bHLH-O proteins amino acids in length, with a well-conserved N-terminal (HES2, 3, 5, 6, 7), three Hey proteins (1, 2, L), and 2 boundary in most family members. The C-terminal Stra13 proteins (Stra13/DEC1, DEC2). In Table 1, we boundary shows more variation in some of the vertebrate show the current human protein set, as well as the E(spl)-like proteins. Thus far, an Orange domain has not probable orthologs in other animal models, when such been identi®ed in any non-bHLH protein. Except for the assignments can be made. The surprisingly similar size Stra13 proteins, members of each subfamily also have a of the bHLH-O families in ¯ies and humans suggests conserved C-terminal tetrapeptide motif, either WRPW that the increase in the number of vertebrate for hairy and E(spl) subfamilies, or YXXW for the Hey transcriptional activator proteins has not required a subfamily. The Hairy subfamily proteins also have a parallel increase in the number of bHLH-O repressors. short sequence between the Orange domain and the C- terminus that is conserved in all invertebrate and vertebrate members (HC domain, Figure 2b). Additional Molecular basis of bHLH-O protein function sequences conserved among subfamily members, though not necessarily conserved between invertebrates and DNA binding vertebrates, can be discerned by alignments of the individual subfamilies (not shown). Taken together, all In contrast to the Id/emc HLH repressor proteins of these sequence features allow clear assignment of each described in the accompanying articles, the bHLH-O bHLH-O protein to one of the subfamilies. proteins bind to DNA. As for other bHLH proteins, The nomenclature for the bHLH-O family is compli- bHLH-O DNA-binding is mediated by a region of cated by the common problem of independent isolation basic amino acids immediately N-terminal to the HLH in multiple laboratories, as well as by the subfamily dimerization domain. The basic regions of proteins in structure. In both frogs and chickens, names for hairy- the hairy and E(spl) subfamilies di€er from other like and E(spl)-like proteins have maintained the bHLH proteins by the presence of a proline residue at distinction between these subfamilies. However, the 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 di€erent 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 and glycine residues in the hairy/E(spl)/Hey proteins region of the Stra13 proteins has a proline at a are on the face of the basic region pointing into the di€erent position (Figure 2b). The functional signi®- major groove. Intriguingly, a threonine residue in cance of these signature residues is not entirely clear. MyoD present at the same position as the proline in Based upon the crystal structure of the myogenic the hairy and E(spl) bHLH-O proteins is essential for bHLH protein MyoD (Ma et al., 1994), the proline 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 Sub- Human Mouse Rat Chicken Frog Zebrafish family

HES1/HRY HES1 HES1 hairy2 hairy1 her6 hairy HES2 HES2 HES2 E(spl) HES3 HES3 HES3 her3 E(spl) HES4 hairy1 hairy2a/b hairy1 hairy HES5 HES5 HES5 ESR1 E(spl) HES6 HES6 HES6 E(spl) HES7 HES7 ESR5 her1 E(spl) Hey1 Hey1 Hey1 Hesr1/Hey1 Hey Hey2 Hey2 Hey2 gridlock Hey HeyL HeyL Hey Stra13/DEC1 Stra13 SHARP2 Stra13 DEC2 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 di€erent local structure in the bound bHLH-O protein that con- tributes 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 Figure 3 Mechanisms of transcriptional repression by bHLH-O HES2 proteins preferentially bind to an E-box instead proteins. (a) The most common mechanism for bHLH-O protein of the N-box, although they can bind to the N-box repression is recruitment of corepressors (shown here as groucho/ (Ishibashi et al., 1993; Jennings et al., 1999). In TLE proteins) to target gene promoters by DNA binding of contrast, HES1 preferentially binds to the N-box (Sasai bHLH-O hetero- or homodimers at speci®c binding sites. (b) et al., 1992), although it also can bind to an E-box Vertebrate bHLH-O proteins (e.g. HES5) inhibit reporter activation by activator bHLH heterodimers, such as MASH1- (Hirata et al., 2000), while Drosophila hairy homo- E47, and interact directly with bHLH activator proteins in vitro. dimers prefer a sequence similar to the N-box, This is consistent with either the formation of inactive CACGCG (Ohsako et al., 1994; Van Doren et al., heterodimers between bHLH-O proteins and activator bHLH 1994). The Hey proteins have been observed to bind to proteins, or inhibition through other protein ± protein interac- tions. (c) It has been proposed that bHLH-O proteins can an E-box, but not the N-box (Nakagawa et al., 2000). compete with activator bHLH proteins for the same DNA- At present, a binding site for the Stra13 proteins has binding sites (see text). In this hypothetical example, bHLH-O not been identi®ed, although it has been reported that homodimers compete with MASH1-E47 heterodimers for DNA- they do not bind to either E-boxes or N-boxes binding to an E-box; the bHLH-O proteins also may recruit (Boudjelal et al., 1997; Garriga-Canut et al., 2001). In corepressors to inhibit promoter function 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 for the Drosophila E(spl) bHLH-O proteins is also an al., 1994; Van Doren et al., 1994; Jennings et al., 1999). optimal binding site for heterodimers of the Drosophila The ability to bind an E-box raises the possibility that daughterless and lethal of scute bHLH activators, and bHLH-O proteins could compete with bHLH activator these proteins can compete for this site in in vitro DNA proteins for binding to E-box target sequences (Figure binding assays (Jennings et al., 1999). Competition for 3c). Consistent with this, an optimal E-box binding site binding sites has not yet been demonstrated for

Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8347 vertebrate bHLH-O proteins, but the ability of HES2 yet known if this motif can recruit groucho/TLE and other vertebrate proteins to bind to E-boxes proteins. Expression of Hey proteins can repress suggests that this may occur. transcription of a reporter based on the mouse Hey2 promoter (Nakagawa et al., 2000). Unlike groucho/ TLE-mediated repression, inhibition of the Hey2 Interactions between the bHLH-O proteins and promoter by Hey proteins does not require HDAC transcriptional corepressors function. However, it also does not require the YXXW A key discovery for understanding the mechanism of motif, leaving open the question of whether this motif bHLH-O protein function was made by Ish-Horowicz may recruit TLE proteins or other HDAC associated and colleagues. Using a two-hybrid screen, they corepressors in another context. The Stra13 proteins do determined that the Drosophila hairy protein binds to not contain any sequences related to the WRPW or the transcriptional corepressor groucho, and they YXXW motifs. However, the Stra13 and SHARP1 showed that this interaction requires the conserved C- proteins can repress transcription by an HDAC- terminal tetrapeptide WRPW motif (Paroush et al., dependent mechanism and the sequences required for 1994). Since the WRPW motif or a related sequence is this repression map to near their C-termini (Sun and present at the C-terminus of most bHLH-O proteins, Taneja, 2000; Garriga-Canut et al., 2001). Whether this immediately suggested a common mechanism of TLE proteins or other corepressors participate in this action for these proteins in both Drosophila and repression has not been determined. In addition, Stra13 vertebrates (Figure 3a). Subsequently it was shown can repress transcription by a second, HDAC- that attaching the WRPW motif to the C-terminus of independent, mechanism (Boudjelal et al., 1997; Sun the yeast gal-4 DNA-binding domain is sucient to and Taneja, 2000). recruit groucho to a promoter with gal-4 binding sites The bHLH-O proteins may interact with corepres- and thus repress transcription (Fisher et al., 1996). sors other than groucho/TLE proteins. The CtBP Groucho has several vertebrate homologs, known as corepressor protein has been shown to bind to the C- TLE proteins, and the TLE proteins interact with terminus of Drosophila hairy (Poortinga et al., 1998). mammalian HES1 via its WRPW motif (Grbavec and This interaction maps to a PLSLV motif in Drosophila Stifani, 1996; Grbavec et al., 1998). hairy, and CtBP can also bind to a similar motif in Groucho/TLE proteins are complex transcriptional E(spl)mdelta. The PLSLV motif is not present in the corepressors that do not bind directly to DNA, but vertebrate hairy-like proteins, although it remains instead are recruited to target genes by a variety of possible that CtBP proteins interact with vertebrate DNA bound repressors (Fisher and Caudy, 1998b). bHLH-O proteins via a divergent motif. CtBP has been Groucho/TLE proteins appear to function at least in reported to function as a short-range corepressor in part by recruiting histone deacetylases (HDACs) to ¯ies (Nibu et al., 1998), suggesting that CtBP could repress target genes (reviewed by Chen and Courey, participate in hairy-mediated repression. However, 2000). Recently it has been reported that TLE proteins CtBP can interfere with groucho-mediated repression can mediate an interaction between HES1 and the (Zhang and Levine, 1999; Phippen et al., 2000). Since winged-helix repressor BF-1, and this interaction CtBP can bind to hairy simultaneously with groucho, potentiates HES-1 repression (Yao et al., 2001). This this suggests that CtBP may negatively regulate the raises the possibility that groucho/TLE proteins could function of a hairy/groucho complex, a model mediate cooperative interactions between bHLH-O consistent with the inhibitory genetic interaction proteins and multiple types of repressors. Such between CtBP and hairy in early Drosophila embryos cooperative repression would parallel the cooperative (Poortinga et al., 1998; Phippen et al., 2000). It is also activation permitted by coactivator-mediated interac- possible that CtBP inhibits hairy function in the early tions between transcriptional activators, and might be embryo, but cooperates with hairy to repress transcrip- expected to permit the evolution of more complex tion in another context. The likelihood of additional control of transcriptional repression. corepressor interactions with bHLH-O proteins sug- While Drosophila hairy and E(spl) proteins end gests that the assembly of a DNA bound, bHLH-O precisely with the WRPW tetrapeptide, some vertebrate `repressor complex' may turn out to be as sophisticated bHLH-O proteins have a short extension to this as the interactions between transcriptional activator sequence (see Figure 2b). For example, the mammalian proteins and coactivators required to form a DNA HES1 protein ends with WRPWRN. The signi®cance, bound `activator complex'. if any, of the two additional amino acids is unclear, and these extra amino acids still permit HES1 to HLH dimerization interact with the TLE1 protein (Grbavec and Stifani, 1996). Since vertebrates have multiple TLE proteins, Like other bHLH proteins, the bHLH-O proteins one intriguing possibility is that variations on the form heterodimers or homodimers via their HLH WRPW motif might restrict which TLE proteins bind domain. Members of the hairy subfamily including to a speci®c bHLH-O protein. HES1 have been reported to homodimerize in vitro Proteins in the Hey subfamily do not contain the (Sasai et al., 1992; Van Doren et al., 1994) and in WRPW tetrapeptide, but they do contain a related two-hybrid assays (Alifragis et al., 1997), and the motif, YXXW, located near their C-termini. It is not SHARP1 protein can homodimerize in vivo (Garriga-

Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8348 Canut et al., 2001). Other vertebrate HES proteins al., 2001). Surprisingly, shortening the HES1 loop and the ¯y E(spl) bHLH-O proteins can homodimer- (part of the helix ± loop ± helix domain) to match the ize and in some cases also heterodimerize, either with length of the HES6 loop allows HES1 to mimic HES6 related bHLH-O proteins, or with activator bHLH in some functional assays (Bae et al., 2000). This proteins (Akazawa et al., 1992; Ishibashi et al., 1993; change may alter the dimerization or other protein ± Alifragis et al., 1997; Hirata et al., 2000). HES1 can protein interaction properties of HES1. Hey2 has also interfere with DNA binding by heterodimers of the been reported to heterodimerize with HES1 (Nakaga- myogenic activator MyoD and E47 and it can inhibit wa et al., 2000), and the chicken Hey1 and Hey2 reporter activation by E47 heterodimerized with either proteins interact with chicken hairy1 in a two-hybrid MyoD or MASH1 (Sasai et al., 1992; Hirata et al., assay (Leimeister et al., 2000a), although the func- 2000). HES5 can interact with the MASH1 or E47 tional consequences of these interactions have not bHLH proteins in vitro, and it can inhibit E47 from been established. While interactions between bHLH-O binding to E-boxes in vitro, as well as attenuate subfamilies may be inhibitory (e.g. by the formation reporter activation by E47 in cultured cells (Akazawa of non-functional heterodimers), such interactions et al., 1992). This suggests that, in addition to acting could also generate heterodimers with novel DNA- as DNA-binding repressors, the bHLH-O proteins binding speci®cities and/or the ability to recruit new may titrate activator bHLH proteins by dimerization, combinations of corepressors. It will be interesting to analogous to the Id/emc proteins (Figure 3b). see if in vitro selection of binding sites for Hey/hairy However, most experimental tests of activator titration heterodimers leads to the identi®cation of novel DNA- by bHLH-O proteins have used large amounts of binding sites. bacterially synthesized protein for in vitro binding assays, or overexpressed bHLH-O proteins for Role of the Orange domain reporter assays. It is not certain that dimerization between bHLH-O proteins and activator bHLH The Orange domain was identi®ed as a functional proteins occurs in vivo at physiological expression domain in the Drosophila hairy and E(spl) proteins levels. In addition, the domains required for interac- (Dawson et al., 1995; Giebel and Campos-Ortega, tions between bHLH-O proteins and activator pro- 1997). In transgenic Drosophila embryos, the hairy teins have rarely been tested, leaving open the protein can antagonize activation of the Sex-lethal gene possibility that some of these interactions may not by the activator bHLH protein scute (Parkhurst et al., be mediated by HLH dimerization. An interesting 1990). In contrast, the E(spl) m8 protein does not variation on inhibition by dimerization appears to prevent the activation of Sex-lethal by scute. Analysis result from alternate promoter usage in the mamma- of chimeric proteins between hairy and m8 demon- lian HES3 gene. Hes3 protein is expressed in two strated that the speci®city for scute inhibition in this forms, one with a bHLH domain and one that lacks system mapped to the hairy Orange domain (Dawson part of the DNA binding basic region, but retains the et al., 1995). While the bHLH domain also was HLH dimerization domain (Hirata et al., 2000). The required for inhibition, the m8 bHLH domain could two HES3 proteins are expressed at di€erent times substitute for the hairy bHLH domain. Since the during development, suggesting distinct functional Xenopus hairy1 protein also can inhibit the activation roles (Hirata et al., 2000). The truncated HES3 of Sex-lethal by scute in transgenic ¯y embryos protein seems likely to titrate other bHLH proteins (Dawson et al., 1995), Orange domain function may by dimerization, but whether its targets are other be associated with subfamily speci®city. In vertebrates, bHLH-O proteins or bHLH activator proteins is not there has been little analysis of functional requirements known. for the Orange domain. However, the HES1 Orange It was recently observed that the Hey1 and Hey2 domain is necessary for HES1 to inhibit the activation proteins interact with the ARNT bHLH-PAS protein of the p21 promoter by MASH1 and E47 in cultured using a two-hybrid screen (Chin et al., 2000). This cells (Castella et al., 2000). suggests that these proteins may heterodimerize, The molecular function of the Orange domain although the speci®c domains mediating the interac- remains unclear, although its conserved relationship tion were not mapped. Hey1 can interfere with the with the HLH domain raises the possibility of a role ability of ARNT to activate a reporter, and this in dimerization. As noted earlier, the Orange domain requires both the bHLH and Orange domains. is always located C-terminal to the bHLH. Two Heterodimerization between subfamilies of bHLH-O other families of bHLH proteins have a conserved proteins also has been reported. HES6, an E(spl) domain located C-terminal to the bHLH domain. In subfamily protein, can heterodimerize with mamma- the bHLH-ZIP proteins, a leucine zipper is con- lian HES1 as well as with Xenopus hairy1 and hairy2 tiguous with the second helix of the HLH domain (Bae et al., 2000; Koyano-Nakagawa et al., 2000). (Baxevanis and Vinson, 1993). Unlike the leucine Coexpression of HES6 with HES1 prevents HES1 zipper in bHLH-ZIP proteins, the Orange domain is from inhibiting a MASH1/E47 driven reporter gene, separated from the bHLH domain by a short, suggesting that HES6 antagonizes HES1 function. variable length region of protein. The bHLH-PAS However, others have reported that HES6 and HES1 proteins have a similar organization, with the PAS can function cooperatively as heterodimers (Gao et domain located a short distance C-terminal to the

Oncogene 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). Additional mechanisms of bHLH-O protein function However, the Orange domain is much smaller than the PAS domain. Both the leucine zipper of bHLH- Under certain experimental conditions, the WRPW ZIP proteins and the PAS domain of bHLH-PAS motif has been shown to be dispensable for repression proteins mediate dimerization, raising the question of by speci®c hairy or E(spl) proteins. A Drosophila hairy whether the Orange domain may also function as an protein missing the WRPW motif can antagonize extended dimerization domain. The chicken hairy1 ectopic activation of Sex-lethal by the scute bHLH Orange domain has been reported to enhance protein, although this same hairy protein cannot block interaction between two hairy1 monomers in a endogenous Sex-lethal expression (Dawson et al., two-hybrid assay, which would be consistent with a 1995). Similarly, a truncated zebra®sh her4 protein role for the Orange domain in dimerization that does not contain the WRPW motif can repress (Leimeister et al., 2000a). In addition, two-hybrid neurogenesis in zebra®sh embryos (Takke et al., 1999). interactions between the chicken Hey1 or Hey2 DNA-binding is also not always required: Drosophila proteins and chicken hairy1 are strongly enhanced E(spl) proteins with a mutation in the DNA-binding by the presence of the Orange domain (Leimeister et domain can still function to regulate bristle formation al., 2000a). However, it remains possible that these (Giebel and Campos-Ortega, 1997). A similar observa- enhanced interactions arise from an indirect e€ect of tion has been made for the rat HES1 protein (Castella the Orange domain (e.g. protein stabilization). It et al., 2000). Even more strikingly, the HES6 protein should be mentioned that the PAS domain also acts remains functional in ectopic expression assays in as a ligand-binding domain in some vertebrate Xenopus embryos with either a mutation in the DNA bHLH-PAS proteins (Crews and Fan, 1999), but at binding region or a truncation that removes the present there is no data to suggest that bHLH-O WRPW motif (Koyano-Nakagawa et al., 2000). The proteins require ligands for function in any system. 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 Expression of the HES5 gene, which encodes an dimerization in these experiments, and thus do not E(spl)-like protein, depends on an intact Notch require the ability to recruit groucho/TLE proteins to signaling pathway. HES5 expression is reduced or DNA. However, this does not readily explain the abolished in mice mutant for Notch1, RBP-JK,or ability of the truncated ¯y hairy protein to inhibit scute presenilin1 and 2 (presenilins are required for Notch function, since hairy does not dimerize with either proteolytic activation) (de la Pompa et al., 1997; scute or its dimerization partner (Alifragis et al., 1997). Barrantes et al., 1999; Donoviel et al., 1999; Handler Another possibility is that the bHLH-O proteins et al., 2000). These observations suggest that HES5 is repress transcription through additional mechanisms likely to be an e€ector for Notch signaling in the that remain intact in these mutant proteins. These developing nervous system and elsewhere. In Xenopus, might include interactions with other corepressors or genes for several E(spl)-related proteins can be DNA-binding repressors. It will be interesting to see if activated by Notch signaling, including ESR1 and additional proteins that interact with the bHLH-O ESR7, which are closely related to HES5, as well as proteins can be isolated. One candidate for mediating ESR4, ESR5, and ESR6e (Wettstein et al., 1997; additional protein ± protein interactions is the HC Deblandre et al., 1999; Jen et al., 1999; Koyano- domain present in all members of the hairy subfamily Nakagawa et al., 2000). In at least some cases, this (Figure 2). While no role has been determined for this expression can be blocked by a dominant negative domain, its conservation from invertebrate hairy ligand for Notch or a dominant negative CSL protein proteins through human hairy-related proteins implies (Chitnis et al., 1995; Jen et al., 1997; Wettstein et al., a strong functional selection. 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). Regulation of vertebrate bHLH-O gene expression by Notch signaling has also been observed to regulate Notch signaling expression of the Hey genes in mice. Expression of the Hey genes in mouse embryos depends on function of It has become clear that the Notch signaling pathway the Notch ligand Dll-1 (Kokubo et al., 1999). plays a major role in regulating the transcription of Consistent with this, the promoter regions of the three many vertebrate bHLH-O genes. Notch signaling mammalian Hey genes contain binding sites for CSL controls cell fate decisions and other developmental proteins, and these promoters are responsive in processes in both vertebrates and invertebrates cultured cells to activated Notch signaling (Maier and (Artavanis-Tsakonas et al., 1999). As a consequence Gessler, 2000; Nakagawa et al., 2000). Transgenic of ligand binding, the intracellular domain of the expression of activated Notch in mouse hair follicles Notch transmembrane receptor is released by proteo- leads to ectopic expression of the HeyL gene (Lin et lysis, and translocates to the nucleus (Mumm and al., 2000), while mice mutant for Notch1 or the Notch Kopan, 2000). The intracellular domain of Notch then ligand Dll-1 lose expression of HeyL in the presomitic associates with a CSL (CBF1, Su(H), Lag-1) tran- mesoderm (Leimeister et al., 2000b). scription factor, converting the CSL protein from a Although a number of bHLH-O genes have been repressor into an activator (Figure 4). Israel and shown to be immediate targets of Notch signaling, coworkers showed that a constitutively active form of some bHLH-O genes do not appear to function in the Notch could activate the expression of a transiently Notch pathway. The mouse and Xenopus HES6 genes transfected reporter based on the HES1 promoter, and do not respond to activated Notch1 (Bae et al., 2000; this activation required CSL binding sites in the Koyano-Nakagawa et al., 2000), nor do the mouse promoter (Jarriault et al., 1995). This suggested that HES3 (Hirata et al., 2000) or Xenopus ESR2 genes HES1 transcription is activated directly in response to (DL Turner, unpublished). There are suggestions that Notch signaling, thus positioning HES1 as a potential HES6 is regulated by neural bHLH activators in both e€ector of Notch signaling. Later studies have shown Xenopus and mouse (Koyano-Nakagawa et al., 2000). that HES1 expression is dependent on Notch function The Stra13 and SHARP1 genes have been shown to be in the segmenting mesoderm (Jouve et al., 2000). activated by a variety of Notch-independent signals in However, it remains unclear whether all expression of cultured cells (Boudjelal et al., 1997; Rossner et al., HES1 depends on Notch signaling. Rossant and 1997; Sun and Taneja, 2000; Ivanova et al., 2001); coworkers reported that HES1 expression in the whether these genes also can be regulated by Notch developing mouse nervous system is not signi®cantly signaling is not known. It will be interesting to further altered by targeted disruptions of Notch1 or RBP-JK, characterize the upstream factors or signals that a mouse CSL gene (de la Pompa et al., 1997). regulate these genes. However, a constitutively active Notch protein In Drosophila, expression of most E(spl) genes stimulates HES1 gene expression in retinal progenitors depends on Notch signaling, while expression of genes (Ohtsuka et al., 1999). Thus, HES1 may act as an for hairy and related proteins is not known to require e€ector for Notch within the nervous system in some the Notch pathway (Bier et al., 1992; Langeland and cells, as well as the segmenting mesoderm, while Carroll, 1993; Jennings et al., 1994; Bailey and possibly functioning in a Notch independent manner Posakony, 1995). This apparent distinction between in other cells. these two subfamilies of bHLH-O genes has not been

Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8351 evolutionarily conserved, since members of both of HES1 and HES5 is generally restricted to regions subfamilies are regulated by Notch in vertebrates (e.g. containing undi€erentiated neural precursors (Akazawa HES1 and HES5). However, additional transcriptional et al., 1992; Sasai et al., 1992; Ishibashi et al., 1995). inputs are also required to generate the normal Constitutive expression of HES1 in neural precursors, expression patterns for the E(spl) genes during using a retroviral vector, prevents neuronal di€erentia- Drosophila development (Nellesen et al., 1999), and tion in both the brain (Ishibashi et al., 1994) and the this is also true for vertebrate bHLH-O genes regulated retina (Tomita et al., 1996). Conversely, targeted by Notch. The Xenopus ESR6e gene responds to disruption of the HES1 gene in mice leads to activated Notch within the developing epidermis, but premature di€erentiation of neurons in the telen- not within the developing nervous system, while the cephelon (Ishibashi et al., 1995), olfactory placode ESR1 and ESR7 genes can be activated by Notch in (Cau et al., 2000), inner ear (Zheng et al., 2000), and both neural and epidermal cells (Deblandre et al., the retina (Tomita et al., 1996). Similarly, transfection 1999). In contrast, the ESR4 and ESR5 genes only of primary rat hippocampal neural precursors with respond to Notch in the segmenting mesoderm (Jen et HES1 inhibits di€erentiation (Castella et al., 1999). al., 1999). The hairy subfamily gene Xenopus hairy2 Consistent with these results, ectopic expression of and its chicken ortholog c-hairy1 have dynamic E(spl)-related proteins can inhibit di€erentiation of expression patterns in the segmenting mesoderm primary neurons in zebra®sh (Takke et al., 1999) and (discussed below) that have been interpreted to mean Xenopus (Koyano-Nakagawa et al., 2000; Schneider et that they are not direct targets of Notch signaling. al., 2001). However, recent data obtained with Xenopus hairy2 In Drosophila, the E(spl) and hairy proteins appear promoter constructs in transgenic Xenopus embryos to restrict neurogenesis both by directly repressing shows that hairy2 expression in the segmenting expression of the proneural bHLH genes, and by mesoderm (as well as the neuroectoderm) depends on antagonizing the ability of proneural bHLH proteins to a paired CSL binding motif in the promoter, implying activate target genes (Ohsako et al., 1994; Van Doren a requirement for Notch signaling (Davis et al., 2001). et al., 1994). A similar regulatory relationship between Two additional cis-regulatory elements are required to the bHLH-O repressors and proneural activators has reconstitute the hairy2 pattern in the mesoderm, been conserved in vertebrates. In both the telencepha- demonstrating a more complex regulation of hairy2 lon and olfactory placode of HES1 null mice, mRNA in this tissue. 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 Biological roles of the bHLH-O proteins in vertebrates HES1, based on studies in cell culture (Chen et al., 1997). In addition, HES1 can inhibit MASH1 DNA In this section, we discuss the function and regulation binding in vitro, and antagonize MASH1 activation of of bHLH-O proteins in neurogenesis, neural cell fate, reporters and target genes in cultured mouse cells vascular development, mesoderm segmentation, and (Sasai et al., 1992; Castella et al., 1999, 2000; Hirata et myogenesis. While roles for bHLH-O proteins in these al., 2000). It also appears that neuroendocrine processes are well established, expression patterns and di€erentiation in mammalian lungs is regulated by functional analyses implicate bHLH-O proteins in the repression of MASH1 by HES1 (Ito et al., 2000). other processes as well. For example, Xenopus ESR6e Intriguingly, mice with a targeted disruption of is involved in patterning of embryonic epidermis HES5 do not upregulate MASH1 in the olfactory (Deblandre et al., 1999), and the expression of Xenopus placode, but HES1/HES5 double null mice upregulate hairy2 suggests a role in patterning the early neural mRNA levels for both MASH1 and a second bHLH plate (Turner and Weintraub, 1994). Stra13 has been gene, neurogenin1 (ngn1) (Cau et al., 2000). In the linked to both mesoderm and neural cell fate olfactory bulb, ngn1 is expressed in a population of regulation in cultured P19 embryonal carcinoma cells precursors with restricted potential, and this expression (Boudjelal et al., 1997), zebra®sh her5 is involved in depends on MASH1 (Cau et al., 1997). These mesendoderm cell fate (Bally-Cuif et al., 2000), and observations suggest that HES1 restricts MASH1 mammalian HES1 regulates T lymphocyte di€erentia- expression in early olfactory progenitors, while HES1 tion (see next section). It seems likely that the list of and HES5 act redundantly to restrict the expression of processes regulated by bHLH-O proteins will continue ngn1 in later progenitors. Thus, HES1 and HES5 can to expand as the functional roles of these proteins are regulate transitions in gene expression within a mitotic better characterized. neural precursor population, in addition to restricting the terminal di€erentiation of neurons. It seems likely that HES1 and HES5 function in precursors at least in Regulation of vertebrate neurogenesis by bHLH-O part by inhibiting the activation of the ngn1 gene by proteins MASH1. Similarly, forced expression of zebra®sh her4 In vertebrates, the bHLH-O proteins and the Notch can repress neurogenin expression in developing zebra- pathway play essential roles in restricting the di€er- ®sh embryos (Takke et al., 1999). entiation of neurons from neural precursor cells. In the Loss of function for Notch pathway components developing mammalian nervous system, the expression within the developing nervous system also leads to

Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8352 excessive and premature di€erentiation of neurons binding) interfered with Muller glial di€erentiation. (Chitnis et al., 1995; de la Pompa et al., 1997; Handler The Hey1, Hey2, and HeyL genes are also expressed in et al., 2000), consistent with observations that Notch the developing retina, and as observed for HES5 and signaling regulates HES1 and HES5 expression. To HES1, forced expression of Hey2 diverts the majority directly test the requirement for HES1 and HES5 in of infected cells to a Muller glial fate (Satow et al., Notch signaling, Ohstuka et al. (1999) prepared neural 2001). However, forced expression of Hey1 or HeyL precursors from mice with targeted disruptions of has no e€ect on cell fate. Although the HES and Hey HES1, HES5 or both, and then infected these cells proteins may directly promote gliogenesis, it seems with a retroviral vector expressing a constitutively more likely that they allow precursor cells to adopt a active Notch1 protein. Activated Notch almost com- glial instead of neuronal fate by repressing neuronal pletely inhibited the di€erentiation of neural precursors di€erentiation. As discussed above, the HES1 and from wild-type mice and from mice with either HES1 HES5 proteins can inhibit the function of neural or HES5 disrupted. In contrast, activated Notch did bHLH proteins such as MASH1. Interestingly, mice not eciently inhibit neuronal di€erentiation of neural double mutant for either MASH1 and MATH3 precursors missing both HES1 and HES5. This (Tomita et al., 2000), or MASH1 and neurogenin2 indicates that HES1 and HES5 are required redun- (Nieto et al., 2001) have excessive glial di€erentiation. dantly for e€ective Notch mediated repression of Analysis of neural precursors from the MASH1/ di€erentiation. Since activated Notch still had some neurogenin2 double mutant mice indicates that, in the inhibitory function in the HES1/HES5 double null absence of neural bHLH function, precursors that neural precursors, there are likely to be additional would normally di€erentiate as neurons can adopt a e€ectors for Notch in the nervous system. glial fate. In addition, forced expression of the neural A few bHLH-O proteins are expressed in di€er- bHLH protein ngn1 in neural precursors not only entiating or di€erentiated neurons. These include one promotes neuronal di€erentiation, but inhibits glial form of HES3 (Hirata et al., 2000), which is expressed di€erentiation (Sun et al., 2001). Thus, bHLH-O in the Purkinje cells of the cerebellum, and HES6, proteins may direct neural precursor cells toward a which is found in di€erentiating and mature neurons in glial fate by inhibiting the expression and/or function many parts of the nervous system (Bae et al., 2000; of the neural bHLH proteins. Koyano-Nakagawa et al., 2000; Pissarra et al., 2000; Vasiliauskas and Stern, 2000). Surprisingly, ectopic Hey proteins and blood vessel formation expression of HES6 in Xenopus embryos promotes ectopic neuronal di€erentiation, as well as upregulation The mouse Hey1 and Hey2 genes are expressed in the of a number of neural bHLH genes including neuroD developing heart and in the cardiac vessels, including and ngn2 (Koyano-Nakagawa et al., 2000). Similarly, the aorta, with HeyL expressed later in the cardiac forced expression of HES6 in retinal progenitors vessels (Leimeister et al., 1999; Nakagawa et al., 1999). promotes neuronal di€erentiation (Bae et al., 2000). The zebra®sh gridlock mutant contains a point It has been proposed that HES6 functions by inhibiting mutation in the zebra®sh Hey2 gene, which is also other bHLH-O factors such as HES1 and Xenopus expressed in the developing heart and aorta (Zhong et hairy1/2 (Bae et al., 2000; Koyano-Nakagawa et al., al., 2000). Gridlock mutants have defective circulation 2000). 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 Regulation of neural cell fate by bHLH-O proteins removes the stop codon for Hey2, adding a C-terminal In addition to regulating neuronal di€erentiation, the extension to the protein. It is likely that this extension HES1, HES5, and Hey2 proteins have been found to interferes with a required function of the C-terminal alter the choice of neuronal versus glial cell fate in the YXXW motif, since injection of an mRNA for a Hey2 developing retina. Retinas from HES5 null mice have protein without this motif fails to rescue the gridlock fewer Muller glial cells, while forced expression of mutation (Zhong et al., 2000). The function of Hey2 in HES5 in retinal cells using a retroviral vector leads to the aorta is unknown, but in mammalian cell culture, most infected cells becoming Muller glia (Hojo et al., expression of Hey2 inhibits the VEGF promoter, which 2000). Consistent with this, postnatal HES5 expression is activated by the bHLH-PAS protein ARNT (Chin et in the retina becomes localized to the layer that al., 2000). This raises the possibility that Hey2 contains di€erentiating Muller glial cells. While Tomita regulates signals associated with vascular growth. et al. (1996) reported that forced expression of HES1 in Intriguingly, overexpression of Hey1 downregulates retinal cells inhibited all di€erentiation, Furukawa et VEGF receptor 2 in cultured endothelial cells al. (2000) found that HES1 could divert retinal cells to (Henderson et al., 2001), suggesting that the Hey a Muller glial fate, as was observed for HES5. The proteins may regulate both VEGF signals and the reason for this discrepancy is not clear, although a response to those signals. HeyL may also have a role in di€erence in the expression levels of the introduced vascular development. Leimeister (2000b) noted that HES1 genes seems a plausible explanation. Furukawa expression of HeyL overlaps with Notch3 in vascular et al. (2000) also found that a putative dominant smooth muscle. Mutations in Notch3 give rise to negative HES1 (a HES1 protein defective in DNA CADASIL (cerebral autosomal dominant arteriopathy

Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8353 with subcortical infarcts and leukoencephalopathy), a 2000). Since disruption of Notch signaling disrupts the condition in which strokes arise from a vascular dynamic pattern of bHLH-O gene expression in the smooth muscle defect (Joutel et al., 1996). It will be PSM, it is likely that the regulated pattern of bHLH-O interesting to see if HeyL expression is regulated by genes in the PSM is crucial to vertebrate segmentation. Notch3, and whether the HeyL protein is required for proper formation of arterial smooth muscle cells. bHLH-O proteins and myogenesis The expression of most bHLH-O genes involved in bHLH-O gene expression during mesoderm segmentation paraxial mesoderm segmentation ends prior to the In Drosophila, hairy functions in a complex genetic di€erentiation of somitic muscle. However, both hierarchy that controls anteroposterior segmentation, HES1 and HES6 expression patterns in mouse while the expression patterns of hairy-like genes in embryos suggest that these proteins may function other invertebrates are consistent with a function in during muscle di€erentiation, subsequent to their segmentation (Lawrence, 1992; Sommer and Tautz, apparent roles during somite formation. In the initial 1993; Damen et al., 2000). In vertebrates, hairy, E(spl), report describing the rat HES1 gene, the authors and Hey genes are expressed in dynamic patterns in the showed that high-level co-expressed HES1 could unsegmented paraxial, or pre-somitic mesoderm inhibit the ability of MyoD to convert 10T1/2 mouse (PSM), again consistent with roles in segmentation embryo ®broblasts to muscle cells (Sasai et al., 1992). (Hrabe de Angelis et al., 1997; Jen et al., 1997, 1999; Subsequently, Honjo and coworkers used a co-culture Palmeirim et al., 1997, 1998; Stern and Vasiliauskas, assay to study Notch signaling and muscle di€er- 1998; Leimeister et al., 1999, 2000b; Takke and entiation in vitro (Kuroda et al., 1999). Mouse C2C12 Campos-Ortega, 1999; Holley et al., 2000; Jouve et myoblasts, which can di€erentiate in culture under al., 2000; Bessho et al., 2001). For instance, cyclic appropriate conditions, were mixed with a myeloma expression of the chicken hairy1 and hairy2 genes cell line expressing the Notch ligand mouse Dll-1 begins with RNA localized to the posterior PSM (Delta-like 1) on its surface. This co-culture inhibited (Palmeirim et al., 1997; Jouve et al., 2000). RNA then C2C12 di€erentiation and HES1 mRNA increased appears in more anterior PSM cells and sweeps signi®cantly in the C2C12 cells within 1 h of co- forward, while RNA disappears in the posterior cells. culture. Subsequent to HES1 up-regulation, expres- A new cycle begins every 90 min, the same period as sion of the myogenic bHLH protein MyoD de- somite formation in the chick embryo. Cycle duration creased, but forced expression of MyoD could rescue is 180 min and ends with hairy RNA expressed in a muscle di€erentiation. These observations, together stripe in the caudal half (hairy1) or rostral half (hairy2) with other studies of Notch inhibition of muscle of a prospective somite two removed from the last di€erentiation (Kopan et al., 1994; Nye et al., 1994; completed somite. Thus, PSM cells experience multiple Hirsinger et al., 2001) suggest that HES1 functions as cycles of hairy expression and extinction before they an e€ector of Notch signaling to inhibit MyoD are incorporated into a newly formed somite. expression in myoblasts and thereby restrict muscle The rhythmic expression of the two hairy genes formation. However, additional experiments suggest suggests a clock-like mechanism controlling somite that there may be other pathways, independent of formation, consistent with many older experiments and HES1, that are also required for inhibition of theories of vertebrate segmentation (Cooke, 1975; myogenesis by Notch signaling (Shawber et al., Meinhardt, 1986; Cooke, 1998; Pourquie, 2000; Stern 1996). In contrast, recent observations indicate that and Vasiliauskas, 2000). Similar rhythmic expression expression of HES6 promotes myogenic di€erentia- has been observed for her1 in zebra®sh, and HES1 in tion, possibly by repressing expression of the bHLH the mouse (Holley et al., 2000; Jouve et al., 2000). repressor MyoR (Gao et al., 2001). 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 bHLH-O proteins and oncogenesis (Forsberg et al., 1998; Aulehla and Johnson, 1999; Moloney et al., 2000). This observation supports a The ability of bHLH-O proteins to negatively regulate connection between spatio-temporally regulated Notch di€erentiation during development points to a potential signaling, and the segmentation `clock' (Evrard et al., role in oncogenesis. Inappropriate, sustained expres- 1998; Jiang et al., 1998; McGrew et al., 1998; Zhang sion of bHLH-O proteins could act as one of the and Gridley, 1998; Barrantes et al., 1999; Pourquie, molecular insults leading to malignant disease. In 1999). Disruption of Notch signaling by transient addition, the suspected roles of the Hey proteins in expression of dominant interfering forms of Notch controlling VEGF and the VEGF receptor expression pathway components has been shown to disrupt both suggest intriguing links to angiogenesis, a known bHLH-O gene expression and normal segmentation in in¯uence on tumor growth and/or metastasis. How- the frog (Jen et al., 1997, 1999). These ®ndings are ever, the best circumstantial evidence to date for the supported by zebra®sh mutants, and targeted knock- involvement of a bHLH-O protein in oncogenesis outs in the mouse (Conlon et al., 1995; Oka et al., focuses on HES1 as a target of misregulated Notch 1995; Holley et al., 2000; Jiang et al., 2000; Jouve et al., 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 di€erentiation that are direct targets of bHLH-O requirement for Notch signaling at early stages of T- repression. For example, in neural precursors and cell thymic development; later stages of T-cell devel- myoblasts, bHLH-O proteins antagonize the ability of opment do not seem to depend on Notch1 (Radtke et the bHLH activators to initiate di€erentiation. Are al., 1999; Wolfer et al., 2001). However, a knockout of targets for bHLH-O repression primarily regulatory HES1 results in an almost complete loss of thymus. genes, such as those that encode the bHLH activators, Immune system reconstitution experiments show that or are genes for structural proteins also directly the defect is in thymocyte development rather than in repressed by bHLH-O proteins? Do bHLH-O proteins the thymus stroma (Tomita et al., 1999). The speci®c block activation of all genes activated by the bHLH defect is in proliferative expansion of CD4/CD8 double activators, or only an essential subset? In addition, negative T-cell precursors, consistent with a role for almost nothing is known about the targets of bHLH-O HES1 in maintaining the proliferative capacity of cells function during patterning of the mesoderm and other during progressive steps of di€erentiation. tissues. With the advent of cDNA microarray analysis Retroviral expression of constitutively active Notch1 and other technologies for large-scale analysis of gene in mouse thymocytes prevents formation of more expression, we may soon learn answers to these di€erentiated single positive CD4 or CD8 cells from questions. double positive CD4/CD8 precursors (Izon et al., Signi®cant gaps remain in our understanding of the 2001). In a di€erent setting, transgenic mice expressing regulation of the bHLH-O genes. Although the Notch active Notch1 under a thymocyte speci®c promoter pathway regulates many of these genes in vertebrates, show an altered ratio of mature T-cell development, it is unlikely that Notch signaling alone is sucient to with single positive CD8 T-cells increased at the explain the expression patterns of these genes. What expense of single positive CD4 T-cells (Robey et al., other factors interact with Notch signals to allow the 1996). This e€ect is presumably in part a function of expression of speci®c bHLH-O genes? Do di€erent the ability of HES1 to downregulate the CD4 promoter vertebrate Notch receptors or ligands regulate distinct (Kim and Siu, 1998), and probably other CD4 T-cell subsets of bHLH-O proteins? What regulates bHLH-O speci®c genes. genes that function independently of Notch? There is In a subset of human T-cell lymphoblastic also the question of whether negative feedback and/or leukemia (T-ALL), a t(7;9) chromosome translocation cross-regulation between vertebrate bHLH-O genes joins the beta T-cell receptor to the human Notch1 contributes to the control of their expression in vivo gene, TAN-1, creating a truncated form of Notch1 (see Takebayashi et al., 1994). Detailed answers to and hence constitutive Notch signaling in leukemic T- these questions will likely require systematic analysis of cells (Ellisen et al., 1991). In a mouse model of this the regulatory elements from bHLH-O genes in process, a T-cell line from a spontaneous T-cell transgenic animal models. A related issue is whether lymphoma has a retroviral insertion into the Notch1 vertebrate bHLH-O proteins are regulated by post- locus that generates a similar truncated and con- translational modi®cations. There is some evidence for stitutively active form of mouse Notch1 (Lee et al., control of HES1 function by phosphorylation (Strom 1999). This line has elevated levels of HES1. More et al., 1997), but to date this issue remains largely importantly, in a bone marrow reconstitution assay unexplored. using cells infected with retroviruses expressing the While the most common mechanism for repression activated TAN-1 protein, only clonal leukemias of by bHLH-O proteins appears to be the recruitment of immature T-cell origin developed in tumor bearing the groucho/TLE transcriptional corepressors to target mice, even though retroviral expression of TAN-1 genes, the lack of a requirement for the WRPW motif occurred in most hematopoietic cells (Pear et al., and/or DNA-binding in some systems suggests addi- 1996). These leukemias were similar to those in tional modes of action. Titration of activator bHLH human T-ALL involving TAN-1. Taken together, proteins by dimerization or other direct interactions these studies support a role for increased HES1 with bHLH-O proteins has long been proposed, but expression, mediated by excess Notch signaling, in not yet clearly demonstrated in vivo. The recruitment of dampening cell di€erentiation and promoting cell additional corepressors, instead of or in addition to the proliferation associated with T-ALL. The widespread groucho/TLE proteins, is another possibility, especially role of Notch signaling in the development of other in the case of the Hey and Stra13 proteins. The tissues leaves open the question of how extensive the molecular roles of the Orange and HC domains remain role of bHLH-O proteins may be in the formation of unclear to date, but given the possibilities of activator other tumor types. titration and/or additional corepressor recruitment, studies of protein ± protein interactions dependent on these domains seem likely to provide additional Unresolved questions about bHLH-O function and insights into bHLH-O function. The identi®cation of regulation protein ± protein interactions speci®c to di€erent bHLH-O subfamilies also could help to elucidate why Vertebrate bHLH-O proteins act to restrict the these subfamilies have been evolutionarily conserved. It di€erentiation of precursor cells in a variety of tissues. also would be of interest to know if heterodimers However, we know of only a few genes involved in between di€erent bHLH-O subfamilies exist in vivo,

Oncogene Vertebrate hairy and Enhancer of split related proteins RL Davis and DL Turner 8355 and if so, whether they have functional roles distinct tion. Finally, the involvement of bHLH-O proteins in from bHLH-O homodimers. the regulation of T-cell formation raises the question of The role of bHLH-O proteins in oncogenesis and whether these proteins have additional roles in immune other diseases has not been extensively explored. Are system functions. 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 Acknowledgments et al., 1997; Rostomily et al., 1997), suggesting that We thank our colleagues, including Anne Vojtek, Susan bHLH-O antagonism of di€erentiation could play a Parkhurst, and Marc Kirschner, for helpful discussions and role in the formation of these tumors. The possibility comments. DL Turner thanks the National Institutes of that Hey proteins could contribute to the control of Health and the University of Michigan Frontiers in tumor vascularization also warrants further investiga- Neuroscience for their support.

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Oncogene