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 dierentiation 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 dierentia- repress transcription by additional mechanisms as well. tion (Fisher and Caudy, 1998a; Kageyama et al., 2000), Many vertebrate bHLH-O proteins are eectors 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 eectors 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 dierences 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, (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 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 dierences in the primary structures imply or BHLHB2 (see Figure 1 legend). For brevity, we that members of dierent 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