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Oncogene (2001) 20, 8342 ± 8357
2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
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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
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 e€ectors 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 di€erentiation (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 e€ectors 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 di€erences 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
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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)
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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 di€erences in the primary structures imply
that members of di€erent 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 di€er 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 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
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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
di€erent 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
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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 di€erent 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
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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 sucient 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'.
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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 di€erent 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 e€ect 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
e€ector 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
e€ector 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 e€ector 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 di€erentiation (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 di€erentiation of neurons from neural precursor cells. In the
developing mammalian nervous system, the expression
of HES1 and HES5 is generally restricted to regions
containing undi€erentiated 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 di€erentiation 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 di€erentiation 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 di€erentiation (Castella et al., 1999).
Consistent with these results, ectopic expression of
E(spl)-related proteins can inhibit di€erentiation 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
di€erentiation 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 di€erentiation 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 di€erentiation 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 di€erentiation of neural precursors
from wild-type mice and from mice with either HES1
or HES5 disrupted. In contrast, activated Notch did
not eciently inhibit neuronal di€erentiation of neural
precursors missing both HES1 and HES5. This
indicates that HES1 and HES5 are required redundantly for e€ective Notch mediated repression of
di€erentiation. Since activated Notch still had some
inhibitory function in the HES1/HES5 double null
neural precursors, there are likely to be additional
e€ectors for Notch in the nervous system.
A few bHLH-O proteins are expressed in di€erentiating or di€erentiated 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 di€erentiating 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 di€erentiation, 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 di€erentiation (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 di€erentiation, 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 di€erentiating Muller glial cells. While Tomita
et al. (1996) reported that forced expression of HES1 in
retinal cells inhibited all di€erentiation, 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
di€erence 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 di€erentiation.
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 e€ect 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
di€erentiation. 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 di€erentiation.
Analysis of neural precursors from the MASH1/
neurogenin2 double mutant mice indicates that, in the
absence of neural bHLH function, precursors that
would normally di€erentiate as neurons can adopt a
glial fate. In addition, forced expression of the neural
bHLH protein ngn1 in neural precursors not only
promotes neuronal di€erentiation, but inhibits glial
di€erentiation (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
di€erentiation of somitic muscle. However, both
HES1 and HES6 expression patterns in mouse
embryos suggest that these proteins may function
during muscle di€erentiation, 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 di€erentiation in vitro (Kuroda et al., 1999). Mouse C2C12
myoblasts, which can di€erentiate 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 di€erentiation 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 di€erentiation. These observations, together
with other studies of Notch inhibition of muscle
di€erentiation (Kopan et al., 1994; Nye et al., 1994;
Hirsinger et al., 2001) suggest that HES1 functions as
an e€ector 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 di€erentiation, 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
di€erentiation 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 di€erentiation.
Retroviral expression of constitutively active Notch1
in mouse thymocytes prevents formation of more
di€erentiated single positive CD4 or CD8 cells from
double positive CD4/CD8 precursors (Izon et al.,
2001). In a di€erent 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 e€ect 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 di€erentiation 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
di€erentiation of precursor cells in a variety of tissues.
However, we know of only a few genes involved in
Oncogene
di€erentiation 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 di€erentiation. 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 sucient 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 di€erent
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 di€erent
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 di€erent 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 di€erentiation 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. Finally, the involvement of bHLH-O proteins in
the regulation of T-cell formation raises the question of
whether these proteins have additional roles in immune
system functions.
8355
Acknowledgments
We thank our colleagues, including Anne Vojtek, Susan
Parkhurst, and Marc Kirschner, for helpful discussions and
comments. DL Turner thanks the National Institutes of
Health and the University of Michigan Frontiers in
Neuroscience for their support.
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