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Journal of Cell Science 113, 3897-3905 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
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COMMENTARY
ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis
John D. Norton
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
E-mail: [email protected]
Published on WWW 31 October 2000
SUMMARY
The ubiquitously expressed family of ID helix-loop-helix
(HLH) proteins function as dominant negative regulators
of basic HLH (bHLH) transcriptional regulators that drive
cell lineage commitment and differentiation in metazoa.
Recent data from cell line and in vivo studies have
implicated the functions of ID proteins in other cellular
processes besides negative regulation of cell differentiation.
ID proteins play key roles in the regulation of lineage
commitment, cell fate decisions and in the timing of
differentiation during neurogenesis, lymphopoiesis and
neovascularisation (angiogenesis). They are essential for
embryogenesis and for cell cycle progression, and they
function as positive regulators of cell proliferation. ID
proteins also possess pro-apoptotic properties in a variety
of cell types and function as cooperating or dominant
oncoproteins in immortalisation of rodent and human cells
and in tumour induction in Id-transgenic mice. In several
human tumour types, the expression of ID proteins is
deregulated, and loss- and gain-of-function studies
implicate ID functions in the regulation of tumour growth,
vascularisation, invasiveness and metastasis. More recent
biochemical studies have also revealed an emerging
‘molecular promiscuity’ of mammalian ID proteins: they
directly interact with and modulate the activities of several
other families of transcriptional regulator, besides bHLH
proteins.
INTRODUCTION
function solely by dimerisation with other transcriptional
regulators, principally those of the bHLH type (Fig. 1). Such
ID-bHLH heterodimers are unable to bind to DNA, and hence
ID proteins act as dominant negative regulators of bHLH
proteins (Benezra et al., 1990; Garrell and Mondolell, 1990;
Ellis et al., 1990). Since most bHLH proteins positively
regulate sets of genes during cell fate determination and cell
differentiation, the term ‘ID’ conveniently alludes to the ability
of these proteins to inhibit both DNA binding and
differentiation.
Genes encoding ID proteins have been isolated from several
metazoan species, but have been most studied in Drosophila,
in which a single Id-like locus, extramacrochaetae (emc),
encodes an ID-like protein (Garrell and Mondolell, 1990; Ellis
et al., 1990), and in mammals (mouse and man), which possess
four Id family members (Id1-Id4) (Benezra et al., 1990; Sun et
al., 1991; Christy et al., 1991; Biggs et al., 1992; Riechmann
et al., 1994). These proteins range in size from 119 residues
(ID3) to 199 residues (EMC). Outside the highly conserved
HLH domain, the different ID proteins display extensive
sequence divergence. Although ID proteins traditionally have
been viewed as negative regulators of cell differentiation,
recent work has revealed much wider biological roles for this
family of regulatory proteins, which impinge on the fields of
developmental biology, cell cycle research and tumour biology
(see Table 1). Here, I highlight recent developments in our
The helix-loop-helix (HLH) family of transcription factors
comprises >200 members, which have been identified in
organisms from yeast to man (reviewed in Littlewood and
Evan, 1995; Massari and Murre, 2000). In metazoa, HLH
proteins function in the coordinate regulation of gene
expression, orchestrating cell cycle control, cell lineage
commitment and cell differentiation. An essential role has been
established for a number of HLH proteins in the development
of haemopoietic, myogenic, pancreatic and neurogenic
mammalian cell lineages. Four main groups of HLH protein
can be distinguished on the basis of the presence or absence of
additional functional domains (Fig. 1). The highly conserved
HLH region comprises two amphipathic α helices, each 15-20
residues long, which are separated by a shorter intervening
loop that has a more variable length and sequence. The HLH
domain primarily mediates homo- or hetero-dimerisation,
which is essential for DNA binding and transcriptional
regulation. Nearly all HLH proteins possess a region of highly
basic residues adjacent to the HLH domain (Fig. 1), which
facilitates binding to DNA containing the canonical ‘E box’
recognition sequence, CANNTG. Some HLH proteins also
bind the related ‘N box’ sequence, CACNAG (Massari and
Murre, 2000). However, a distinct subfamily of HLH proteins,
the ID proteins, lack such a DNA-binding region and instead
Key words: Helix-loop-helix protein, Cell growth, Cell
differentiation, Cell cycle, Tumorigenesis, Transcription factor
3898 J. D. Norton
helix-loop-helix
b
LZ
bHLH leucine
zipper
b
PAS
bHLH-PAS
b
Fig. 1. Schematic structure of different
HLH protein families. The basic DNAbinding region (b), leucine zipper (LZ) and
PAS domains that bound the HLH region
are shown (not to scale).
bHLH
Id HLH
understanding of the biological functions and mechanisms of
action of ID proteins. Comprehensive reviews of earlier work
can be found elsewhere (Norton et al., 1998; Israel et al., 1999).
ID PROTEINS IN DEVELOPMENTAL BIOLOGY
Drosophila
In Drosophila, the emc locus is required for many
developmental processes; sensory organ development and
wing morphogenesis have been most extensively studied
(reviewed in Jan and Jan, 1993). Although null mutations in
emc are embryonic lethal, studies on various partial lossand gain-of-function emc mutants have revealed a role for
the EMC gene product in regulating cell commitment,
differentiation and cell proliferation (Jan and Jan, 1993). The
EMC protein heterodimerises with and antagonises the
functions of several bHLH proteins, including those encoded
by the achaete-scute complex genes involved in neurogenesis
and sex determination (Garrell and Mondolell, 1990; Ellis et
al., 1990; Jan and Jan, 1993). These genes are positively
autoregulated by the Achaete and Scute proteins and are
negatively regulated by EMC (Martinez et al., 1993). Genetic
studies strongly support the notion that a critical balance
between the relative levels of EMC protein and its bHLH
targets is important in regulating cell fate determination in
Drosophila (Jan and Jan, 1993).
More recent genetic studies have revealed how EMC is
integrated with upstream signalling pathways that regulate cell
fate during Drosophila wing morphogenesis. Proteins that
function in the Ras signalling pathway (Torpedo, Vein, Veinlet
and Gap) appear to cooperate with EMC during cell
proliferation but antagonise it during differentiation (Baonza
and Garcia-Bellido, 1999). Strong genetic interaction has also
been found between emc and genes that encode different
components of the Notch signalling pathway (Baonza et al.,
2000). Notch signalling defines an evolutionarily conserved
cell-cell interaction mechanism that, throughout development,
controls the ability of primitive cells to respond to
developmental signals (Artavanis-Tsakonas et al., 1999). Data
on genetic complementation between different mutants
affecting wing morphogenesis are consistent with regulation of
the emc gene or protein by Notch and its collaboration with
other proteins downstream of Notch to control wing vein
differentiation (Baonza et al., 2000). The biochemical
mechanisms underlying genetic interaction between emc and
Notch remain to be elucidated. Since both Ras and Notch
signalling play pivotal roles in higher organisms, these studies
may well provide a paradigm for understanding how ID
proteins are integrated with upstream signalling pathways in
mammals.
Mammals
Embryonic lethality of double-knockout mice
As in flies, Id function in mice is essential for development.
However, because of the functional redundancy among the four
Table 1. Biological functions of ID proteins
Function
References*
Act as ‘global’ regulators of lineage commitment
and cell fate determination
Ellis et al., 1990; Martinsen and Bronner-Fraser, 1998; Yokota et al., 1999; Lyden et al., 1999;
Blom et al., 1999; Jaleco et al., 1999; Rivera et al., 2000; Janatpour et al., 2000
Required for embryogenesis/organogenesis
Garrell and Modolell, 1990; Ellis et al., 1990; Yokota et al., 1999; Lyden et al., 1999
Promote cell growth; arrest cell differentiation
Jen et al., 1992; Iavarone et al., 1994; Sun, 1994; Desprez et al., 1995; Atherton et al., 1996;
Kondo and Raff, 2000; Morrow et al., 1999
Required for cell cycle progression
Barone et al., 1994; Hara et al., 1994; Peverali et al., 1994
Induce apoptosis
Norton and Atherton, 1998; Florio et al., 1998; Kim et al., 1999
Immortalise primary cells
Norton and Atherton, 1998; Alani et al., 1999
Function as oncoproteins in vivo
Wice and Gordon, 1998; Kim et al., 1999; Morrow et al., 1999
Required for angiogenesis in vivo and promote
tumour invasiveness
Desprez et al., 1998; Lyden et al., 1999; Lin et al., 2000
*Only selected references are given; for additional references refer to text and to Norton et al., 1998.
ID helix-loop-helix proteins 3899
members of the mammalian Id family and their widespread,
overlapping expression patterns, only crosses between mice
that lack different Id genes are embryonic lethal. All
combinations of double knockouts of Id1-Id3 are non-viable
and die before birth (R. Benezra, personal communication).
However, only the Id1−/− Id3−/− phenotype has so far been
reported (Lyden et al., 1999). Early embryos from these mice
display aberrant neurogenesis with premature withdrawal of
neuroblasts from the cell cycle and inappropriate expression of
neural-specific markers. In addition, embryos from Id1−/−
Id3−/− mice exhibit vascular malformations and an absence
of branching and sprouting of blood vessels into the
neuroectoderm. These defects in angiogenesis manifest from
aberrant vascular endothelial cell development (Lyden et al.,
1999). The apparent restriction of these lesions to the
embryonic brain probably reflects the endothelial expression
patterns of Id genes. All three Id genes are expressed in
endothelia outside of the brain, whereas only Id1 and Id3 are
expressed in embryonic endothelial cells in the brain. However,
in adult Id1+/− Id3−/− mice, which are viable owing to the
presence of a single Id1 allele, the endothelial cell vasculature
of tumour xenografts is also defective, and this leads to failure
of tumour growth and metastasis (Lyden et al., 1999) (see
below). It seems likely that analysis of other combinations of
crosses between mice that lack different Id genes will reveal
ID functions in other cell lineages that are essential for
development.
The embryonic lethality of Id double-knockout mice
contrasts markedly with the phenotype observed in mice that
lack a single Id gene. Only the Id2-null mouse displays obvious
phenotypic abnormalities of retarded growth and neonatal
morbidity (Yokota et al., 1999). Male Id2−/− mice exhibit a
profound defect in spermatogenesis (F. Sablitzky, personal
communication), which is consistent with the stage-specific
expression pattern of Id2 during spermatogenesis (Sablitzky et
al., 1998).
Id proteins in lymphopoiesis
Surviving Id2−/− mice also display a cell-intrinsic defect in
production of natural killer (NK) cells, which are involved in
immune function (Yokota et al., 1999). A role for Id function
in NK-cell fate determination is also supported by the
observation that overexpression of Id3 in primary foetal
thymocyte cultures promotes NK cell generation from CD34+
progenitor cells at the expense of T cell production
(Heemskerk et al., 1997). Id2-null mice also lack secondary
lymphoid follicles of Peyer’s patches in the intestine, although
this defect evidently arises from an absence of lymphotoxinproducing, CD4+ CD3− IL-7Rα+ cells that are essential for
Peyer’s patch development (Yokota et al., 1999).
Progression
through S phase
E2F
pRB
pRB
E2F
S phase genes
ID
Mitogenic signalling
P
pRB
cyclin D
CDK4/6
TCF
cyclin E/A
CDK2
p21
P
ID
FOS
SRF
TCF
ID
EGR1
bHLH
Immediate
early genes
EGR1
bHLH
Id genes
bHLH
bHLH
p21 gene
Fig. 2. Tentative scheme showing how the functions of ID proteins are integrated with mitogenic gene signalling cascades and cell cycle
progression. During early G1 phase of the cell cycle, mitogenic signalling pathways activate a cascade of gene expression changes and activate
the assembly of functional cyclin-dependent kinase (CDK) complexes (see Sherr and Roberts, 1999, for recent review). This leads to sequential
phosphorylation of the tumour suppressor protein pRB (and related ‘pocket proteins’, p107 and p130), resulting in its dissociation from
members of the E2F family of transcriptional regulators. Free E2F-DP1 (E2F) transcriptional complexes then activate the expression of genes
required for progression through S phase of the cell cycle. The functions of ID proteins are integrated at multiple points in G1 cell cycle control
(see text). Only ID2 and ID4 interact with pRB. All ID proteins except for ID1 are substrates for CDK2-dependent phosphorylation. The ID1interacting protein MIDA1 (not shown in the figure) is thought to be coupled indirectly to the cell proliferative response by modulating the
expression of growth factor genes (see text).
3900 J. D. Norton
No abnormalities or defective cell functions have so far been
reported for Id1-deficient mice, and data for Id4-deficient mice
are not yet available. However, the Id3-null mouse, although
apparently normal, displays subtle abnormalities that again
affect lymphopoietic cells involved in immune function. Data
from studies employing enforced expression of Id3 in
lymphopoietic cells also implicate this Id family member in
multiple stages of lymphopoiesis. Mature B cells from Id3−/−
mice exhibit a defective in vitro proliferative response induced
specifically by signalling through the B cell surface
immunoglobulin (Ig) receptor (Pan et al., 1999). This probably
accounts at least in part for the defective in vivo immune
response observed in these mice. B cells from Id3−/− mice are
also enhanced in their Ig-class-switching capacity during in
vitro differentiation (Pan et al., 1999). The inference from this
that ID3 modulates class-switch recombination of Ig genes is
further supported by the observation that bHLH transcription
factors such as the E2A-encoded E12 and E47 proteins actively
promote class switching, whereas enforced expression of Id3
impairs class switching (Quong et al., 1999). ID3 might also
play a role in B cell fate determination at an early stage during
lymphopoiesis, since enforced expression of Id3 in primitive
lymphomyeloid progenitors from foetal liver selectively
inhibits B cell, but not NK or myeloid cell, differentiation
(Jaleco et al., 1999).
During the final stages of thymocyte differentiation, the
ID3 protein also plays a role in positive selection of T cells
in the cell-mediated immune response. Id3−/− mice exhibit
defective positive selection of both MHC-class-I- and
MHC-class-II-restricted thymocytes (Rivera et al., 2000).
Furthermore, enforced expression of Id3 in committed
thymocyte progenitors inhibits commitment to and
production of the αβ subset but not the γδ subset of T cells
(Blom et al., 1999). Finally, enforced expression of Id3
promotes maturation of an immature T cell line (Bain et al.,
1999); this finding is consistent with the observation that,
in the absence of the bHLH protein E47, mature-T-cell
production is increased (Bain et al., 1999). ID3 probably
plays a role partially overlapping but complimentary to that
of ID1 and ID2 during lymphopoiesis. Enforced expression
of Id1 or Id2 in committed thymocytes in transgenic mice
leads to a block in further maturation (Kim et al., 1999;
Morrow et al., 1999). Similarly, enforced expression of an Id1
transgene in early B cells blocks B lymphocyte maturation at
the pro-B-cell stage (Sun, 1994).
ID proteins in other cell lineages
Studies on other developmental systems, particularly the
nervous system (Andres-Barquin et al., 2000), also highlight
the complex role played by ID proteins in cell fate
determination. For example, enforced expression of Id2 in
primitive ectodermal cells in an avian system leads to
overgrowth and premature neurogenesis of the dorsal neural
tube, which is consistent with ID2 directing ectodermal
precursor cells to neural crest and neurogenic fates rather than
to epidermal lineages (Martinsen and Bronner-Fraser, 1998).
Similarly, during myelopoiesis, Id2 is markedly upregulated
as cells progress to terminal differentiation of monocytemacrophage, granulocyte and erythroid lineages (Ishiguro et
al., 1996; Cooper and Newburger, 1998). Enforced expression
of Id genes reportedly promotes the differentiation of
erythroleukaemia cells, albeit in cell lines (Deed et al., 1998;
Holmes et al., 1999). Interestingly, the ID2 and ID4 proteins
differ in their abilities to suppress the myogenic differentiation
programme in a cell line model, which indicates that different
Id family members have distinct functions in the regulation of
individual developmental pathways (Melnikova et al., 1999).
Finally, Kondo and Raff (Kondo and Raff, 2000) have recently
implicated ID4, which has been less well studied than its
relatives, in the ‘molecular clock’ mechanism controlling the
timing of oligodendrocyte differentiation.
The above studies clearly illustrate the complex role played
by ID proteins in mammalian cell fate determination.
Numerous reports show that ID proteins function as negative
regulators of differentiation, both in vitro and in vivo, and ID
proteins have been shown to inhibit bHLH-dependent
expression of several differentiation-linked genes (Norton et
al., 1998; Israel et al., 1999). These observations are in accord
with the highly complex pattern of temporal and tissuespecific expression of Id genes in vivo. Essentially all
cell lineages express one or, more usually, multiple Id
family members. In general, expression is highest in
undifferentiated, self-renewing populations and is
downregulated as cells exit the cycle and terminally
differentiate (Norton et al., 1998 and references therein).
However, it is now clear that, depending on the particular cell
lineage and developmental stage, individual ID proteins can
act as either negative or positive regulators of differentiation
in addition to performing a regulatory role in cell lineage
commitment decisions.
ID PROTEINS IN CELL CYCLE CONTROL
As might be expected for regulatory molecules that have a
major role in negative regulation of cell differentiation, ID
proteins also act as positive regulators of cell growth, and
indeed their functions are required for cell cycle progression
in cell line models. Genetic studies in Drosophila and mice
(discussed above) now provide direct evidence for a role for
ID proteins in cell proliferation and cell cycle control in vivo.
Quiescent cells express low/undetectable levels of Id genes.
However, following mitogenic stimulation of, for example,
fibroblasts, Id expression is rapidly induced (within 1-2 hours)
as part of a cascade of ‘delayed’ early response genes. After
an initial decline, Id expression is sustained throughout the G1
phase of the cell cycle and is further upregulated as cells enter
S phase (Hara et al., 1994; Norton et al., 1998 and references
therein). ID proteins function at multiple stages in cell cycle
control by modulating the transcription of several known target
genes, in some instances by directly interacting with nonbHLH proteins (see Table 2).
Fig. 2 summarises the known pathways through which ID
proteins are integrated into mammalian cell cycle control.
Following mitogenic signalling, ID proteins downregulate the
expression of immediate early genes (e.g. c-fos and egr-1) by
antagonising the ETS domain proteins SAP-1 and ELK-1,
which are components of the ternary complex factor (TCF)
responsible for upregulating immediate-early-gene expression
(Yates et al., 1999). Interestingly, the expression of Id1 (and
probably also of Id3) is itself positively regulated by EGR-1
(Tournay and Benezra, 1996; Deed et al., 1994) as part of a
ID helix-loop-helix proteins 3901
Table 2. Non-bHLH proteins whose functions are modulated by direct interaction with ID proteins
Target protein
Notes
References
Retinoblastoma (pRB) tumour suppressor protein
and related ‘pocket’ proteins, p107 and p130
Only ID2 and ID4 display this interaction, which antagonises pRB
function. Involved in cell cycle and growth control
Iavarone et al., 1994; Lasorella
et al., 1996; R. Deed, F.
Sablitzky and J. Norton,
unpublished observations
ETS-domain transcription factors
Demonstrated for the ternary complex factors (TCF), SAP-1 and
ELK-1. Interaction with ID1-ID3 inhibits DNA binding and inhibits
immediate early gene induction coupled to MAP kinase signalling
Yates et al., 1999
PAX-2/PAX-5/PAX-8 paired-domain homeobox
transcription factors
ID1-ID3 disrupt DNA binding by PAX proteins. Shown to inhibit PAXmediated trans-activation of the B-cell-specific MB-1 promoter
A. Sharrocks and J. Norton,
unpublished observations
MIDA1, a Z-DNA-binding protein displaying an
additional sequence-specific DNA-binding
function
Implicated in positive regulation of cell growth. Association with ID1
potentiates sequence-specific DNA binding of MIDA1
Shoji et al., 1995; Inoue et al.,
1999; Inoue et al., 2000
ADD1/SREBP-1c (adipocyte determination and
differentiation factor 1/sterol regulatory element
binding protein 1c)
bHLH-leucine-zipper transcription factor controlling expression of
adipocyte genes. Interaction with ID2/ID3 inhibits ADD1-regulated
promoter function implicated in lipogenesis
Moldes et al., 1999
negative feedback loop in mitogenic signalling cascades (Fig.
2). Two distinct pathways couple ID function to positive
regulation of the cell cycle machinery during mid-late G1
phase. The first involves a direct interaction between the
retinoblastoma proteins (pRB, and related ‘pocket’ proteins
p107 and p130), and ID2 and ID4 (but not ID1 or ID3)
(Iavarone et al., 1994; Lasorella et al., 1996; R. Deed, F.
Sablitzky and J. Norton, unpublished observations). This is
thought to potentiate S phase progression by attenuating pRB
suppression of E2F-DP1 transcription factors, which drive
expression of genes required for S phase progression (Fig. 2).
The second pathway, described for ID1 but probably also used
by other ID family members, involves inhibition of E2A
bHLH-regulated expression of the gene encoding the cyclindependent kinase (CDK) inhibitor p21Cip1/Waf1 (Prabhu et al.,
1997). This relieves p21 suppression of cyclin-A/E–CDK2
activity, which in turn leads to phosphorylation of pRB. The
phosphorylation of pRB causes its dissociation from E2F-DP1
complexes (Fig. 2), which can then activate genes required for
progression into S phase.
Interestingly, ID2, ID3 and ID4 (but not ID1) are
themselves substrates for CDK2-dependent phosphorylation
during late-G1/early-S phase (Hara et al., 1997; Deed et al.,
1997). This alters the bHLH dimerisation specificity of the
ID2 and ID3 proteins and is essential for cell cycle
progression, since mutants of ID2 and ID3 that lack CDK2
phosphorylation sites elicit S phase arrest and cell death
(Deed et al., 1997). The p21-CDK2 pathway provides an
elegant feedback mechanism for temporal control of ID
phosphorylation during late G1 (Fig. 2). The functions of the
phosphorylated forms of ID proteins during S and G2/M
phases of the cell cycle are not known.
ID1 also associates with a novel transcription-factor-like
protein, MIDA1 (mouse ID-associated protein 1 – see Table 2),
which is ubiquitously expressed (T. Inoue and M. Obinata,
personal communication) and is required for cell growth, at
least in erythroleukaemia cells (Shoji et al., 1995). The
association with ID1 potentiates the sequence-specific DNA
binding of the MIDA1 protein to a 7-bp sequence, GTCAAGC,
present in the 5′ flanking region of several growthfactor/cytokine genes. MIDA1 might therefore couple ID
function to the cell cycle control apparatus through indirect
mechanisms (Inoue et al., 1999; Inoue et al., 2000).
ID PROTEINS IN TUMOUR BIOLOGY
Oncogenic properties of ID proteins
Early studies showed that enforced expression of Id genes in
immortalised fibroblast cell lines causes disruption of
cytoskeletal organisation and loss of adhesion (Deed et al.,
1993). Subsequently, Id genes have been shown to function as
either cooperating oncogenes or as dominant oncogenes in
various contexts. For example, Id genes can immortalise
primary rodent fibroblasts when co-transfected with the Bcl-2
gene (Norton and Atherton, 1998), and Id1 can immortalise
primary human keratinocytes (Alani et al., 1999). The extended
lifespan of Id1-immortalised keratinocytes is accompanied by
activation of telomerase activity and inhibition of pRB function
(Alani et al., 1999). Enforced expression of Id1 also promotes
mammary epithelial cell invasion of basement membranes and
is associated with induction of a 120-kDa gelatinase activity
(Desprez et al., 1998). Enforced expression of Id1 targeted to
intestinal epithelial cells in transgenic mice is associated with
development of intestinal adenomas (Wice and Gordon, 1998).
Similarly Id1 and Id2 transgenes expressed in thymocytes
induce an aggressive thymic lymphoma in vivo (Kim et al.,
1999; Morrow et al., 1999).
These oncogenic properties of Id genes and their welldocumented ability to promote cell proliferation are in accord
with the tumour suppressor properties of some bHLH proteins
whose activities are antagonised by ID proteins. For example,
overexpression of bHLH proteins such as E47 in cell lines
typically leads to suppression of growth by inducing cell cycle
arrest in G1 (Peverali et al., 1994 and references therein). This
probably occurs at least in part through transcriptional
activation of the gene encoding the p21 CDK2 inhibitor
(Prabhu et al., 1997) (see Fig. 2). Restoration of E12/E47
bHLH protein function to tumour cell lines also leads to growth
arrest and in some instances to apoptosis (Park et al., 1999;
Engel and Murre, 1999). In addition, mice lacking the E2A
gene that encodes the E12 and E47 bHLH proteins develop T
cell tumours at a high frequency (Yan et al., 1997). However,
crossing of E2A-deficient mice with Id1−/− mice fails to
suppress tumour incidence (Yan et al., 1997). Also, crossing of
Id3−/− mice with mice lacking the related bHLH protein, HEB,
leads to T cell tumorigenesis not seen in individual singleknockout animals (Barndt and Zhuang, 1999). Although the
3902 J. D. Norton
etiological mechanisms involved are unclear, these
observations nonetheless highlight the important role of IDbHLH interactions in tumorigenesis in vivo.
A feature shared by some positive regulators of mammalian
cell cycle progression such as MYC, cyclins D and E and
E2F1, is their ability to drive apoptosis when ectopically
overexpressed. Like ID proteins, these cell cycle regulatory
proteins also display oncogenic properties. Several groups have
independently reported on the pro-apoptotic properties of ID
proteins in cell line and primary cell models (Norton and
Atherton, 1998; Florio et al., 1998; Nakajima et al., 1998;
Tanaka et al., 1998; Andres-Barquin et al., 1999). Enforced
expression of Id genes in vivo can also induce apoptosis – in
transgenic mice expressing an Id1 transgene in thymocytes, the
differentiation arrest and T cell lymphomagenesis mentioned
above is also accompanied by widespread apoptosis (Kim et
al., 1999). Interestingly, ID2 might drive apoptosis through a
pathway that is distinct from that utilised by other ID family
members. ID2-induced apoptosis is independent of HLHmediated dimerisation and does not therefore depend on
interaction with bHLH or other known ID target proteins (see
Table 2). Instead, the ability of ID2 to promote apoptosis
resides in the N-terminal region of the protein and is associated
with enhanced expression of the pro-apoptotic BAX protein
(Florio et al., 1998).
ID proteins in primary tumours
Immortalised cell lines from a number of different tumour
types display markedly elevated levels of one or more Id
mRNAs (reviewed in Israel et al., 1999). More recent work has
extended these studies, analysing protein expression in primary
tumours. Table 3 lists the major human tumour types in which
ID protein levels have been reported to be deregulated. Note
that constitutive Id expression is not simply a generalised
feature of tumorigenesis per se – in most leukaemias and
lymphomas, for example, the Id expression profile mirrors that
of normal haemopoietic progenitor cells (Ishiguro et al., 1996).
Given the known ‘oncogenic’ properties of ID proteins and the
observation that loss of Id expression in tumour cell lines leads
to suppression of cell growth (Hara et al., 1994; Kleef et al.,
1998; Lin et al., 2000), it seems likely that ID proteins perform
a causal role in tumorigenesis mechanisms. Presumably, the
tumour-cell-associated deregulation of Id gene expression
arises through perturbations in upstream signalling pathways.
A number of oncogenes and tumour suppressor genes encode
components of signalling pathways; qualitative and or
quantitative changes in these constitute the primary genetic
lesion in cancer cells.
Two studies highlight distinct roles for ID proteins in
tumorigenesis mechanisms associated with invasiveness and
metastasis. In primary breast cancer (Table 3), the expression
of Id1 is more frequently associated with infiltrating, more
aggressive tumours than with indolent ductal tumours, which
suggests a link between Id expression and disease progression
(Lin et al., 2000). Overexpression of the Id1 gene, as
mentioned above, causes mammary epithelial cells to invade
the basement membrane (Desprez et al., 1998). Moreover,
constitutive expression of Id1 in a non-aggressive breast cancer
cell line confers a more aggressive phenotype, as assessed by
growth and invasiveness (Lin et al., 2000). ID1 also regulates
steroid-hormone-responsive growth of breast cancer cells.
Table 3. Primary human tumour types in which ID
expression is deregulated
Tumour type
References
Pancreatic cancer
Kleef et al., 1998; Maruyama et al., 1999
Astrocytic tumours
Andres-Barquin et al., 1997
High-grade neural tumour
endothelial cells
Lyden et al., 1999
Invasive breast carcinoma
Lin et al., 2000
Seminomas
Sablitzky et al., 1998
Colorectal adenocarcinoma
J. Wilson and J. Norton, unpublished
observations
Multiple myeloma
L. Peterson and J. Norton, unpublished
observations
These observations implicate loss of appropriate regulation of
Id1 expression in the process of tumour progression and
suggest that Id1 provides a useful marker for aggressive breast
tumours (Lin et al., 2000).
As mentioned previously, the Id1+/− Id3−/− knockout mouse,
although viable, has a severely impaired ability to support the
growth and metastasis of tumour xenografts, owing to a defect
in tumour cell vascularisation (Lyden et al., 1999). Significant
regression is observed for some tumour types even in mice
lacking just one Id allele (Id1+/− Id3+/+), which implies that
there is a gene-dosage effect on ability to support tumour
xenografts. The ability to support tumour growth in Id1-Id3deficient mice is also paralleled by impairment in the ability to
support metastasis. This defect in angiogenesis can be readily
explained by the loss of αvβ3 integrin and its associated
MMP2 metalloproteinase on tumour-infiltrating vascular
endothelial cells (Lyden et al., 1999). More recent studies have
revealed that tumours of diverse histological origin display a
similar defect in growth/metastasis in Id-deficient mice and
concomitant haemorrhage, necrosis and tumour regression (R.
Benezra, personal communication). Significantly, in at least
some high-grade human tumours, for example of neural origin,
Id1 and Id3 are expressed in cells of the infiltrating vascular
endothelium (Table 3). This contrasts with normal adult
vascular endothelial cells, in which ID protein levels are very
low (Lyden et al., 1999).
Since growth and angiogenesis of tumour cells appear to be
strictly dependent on Id function and can be modulated by
small changes in intracellular levels of Id protein, it has been
suggested that small-molecule-based inhibitors of Id proteins
might provide useful drugs in the treatment of human cancers
(Lyden et al., 1999; Lin et al., 2000). Whether therapeutically
useful drugs can be developed with sufficient specificity for ID
proteins remains to be determined.
MECHANISMS OF ACTION OF ID PROTEINS
ID-bHLH protein interactions
Compelling biochemical and genetic data from studies in both
Drosophila and mammals support the view that the primary
mechanisms through which ID proteins function is through
antagonism of bHLH transcriptional regulators. Moreover,
several studies illustrate how subtle changes in the equilibrium
of heterodimeric interactions between bHLH and ID proteins
ID helix-loop-helix proteins 3903
cause dramatic changes in cell fate determination (see Barndt
and Zhuang, 1999; Jan and Jan, 1993). The mammalian ID
proteins preferentially target the ubiquitously expressed ‘E
proteins’, which belong to the Class A bHLH proteins (E12,
E47, E2-2, HEB) (Benezra et al., 1990; Sun et al., 1991;
reviewed in Norton et al., 1998). These typically function as
heterodimeric partners for the larger family of tissue-specific
Class B bHLH proteins (Norton et al., 1998; Littlewood and
Evan, 1995; Massari and Murre, 2000). Although all ID
proteins interact avidly with each of the four E proteins,
biochemical data indicate that individual ID proteins have
distinct preferences for their E protein targets (Langlands et al.,
1997; Deed et al., 1998). More recent data have also revealed
differences in the interaction between E proteins and different
IDs at a genetic level. The postnatal lethality associated with
HEB-deficient mice can be partially rescued by crossing them
with Id3−/− but not with Id1−/− mouse strains (Barndt and
Zhuang, 1999).
Although ID proteins, because of their lack of a basic, DNAbinding region, are not known to bind DNA, either in vitro
or in vivo, under certain circumstances they can act as
transcriptional activators. All four mammalian ID proteins,
when fused to a heterologous Gal4-DNA binding domain, can
activate Gal4-dependent transcription (Bounpheng et al.,
1999b). Since this is apparently mediated through
sequestration of bHLH protein(s) (which contribute the
transactivation function), and ID-bHLH heterodimers do not
bind DNA, this mechanism is unlikely to be physiological.
However, ID proteins might provide such an adapter function
as constituents of DNA-bound, multicomponent transcriptional
complexes.
Interactions with non-bHLH proteins
In addition to the pRB, ETS and MIDA1 proteins that interact
with ID proteins in the context of cell cycle and growth control
(see above and Table 2), two additional non-bHLH proteins
also interact with and are functionally antagonised by ID
proteins (Table 2). Members of the paired homeobox family of
transcription factors (PAX-2, PAX-5; PAX-8) associate in vitro
and in vivo with ID1-ID3 (A. Sharrocks and J. Norton,
unpublished observations). As shown for the B-cell-specific,
mb-1 promoter, ID proteins disrupt PAX-5 DNA binding and
transcriptional activation of PAX-responsive genes. The
adipocyte determination and differentiation factor-1 (ADD1),
a member of the bHLH-leucine-zipper family of transcription
factors (Fig. 1) that controls expression of genes during
lipogenesis also interacts with ID2 and ID3 (Moldes et al.,
1999). Again, the association with ID protein abrogates
binding to and transcriptional activation of ADD1-regulated
promoters. These and the other known examples listed in Table
2 illustrate the emerging molecular promiscuity of the ID
family. Evidently, ID proteins can form intermolecular
associations with diverse structural motifs on different partner
proteins. However, as with the interaction between bHLH and
ID proteins, in every reported example listed in Table 2, protein
interaction is mediated by the ID HLH domain. Also, with the
exception of MIDA1 (discussed above; see Table 2), the
association with ID protein antagonises the function of the
DNA-binding protein, typically by inhibiting DNA binding –
as occurs with bHLH proteins. It is important to emphasise,
however, that all of the reported functional interactions
between ID and non-bHLH proteins (Table 2) have so far only
been documented in biochemical and cell line ‘in vivo’ studies.
The relevance of these various interactions in mediating
biological functions of ID proteins in a truly physiological
context in vivo has yet to be established.
Regulation of ID function
In mammalian cell lineages, Id expression responds to a
multitude of cell surface, ligand-receptor interactions – as
might be expected for early response genes that are pivotally
involved in cell fate determination (Norton et al., 1998).
Although some data on the mechanisms of regulation of Id
expression at the promoter level are available (see Norton et
al., 1998), very little is known about the upstream signalling
pathways that couple Id expression to extracellular signals. The
ID proteins themselves are regulated at the post-translational
level. In addition to cell-cycle-linked phosphorylation (see
above), intracellular levels of ID proteins are regulated through
the ubiquitin-proteasome degradation pathway (Bounpheng et
al., 1999a). In common with most other proteins encoded by
early response genes, ID proteins rapidly turn over in the cell,
having a reported half-life of 20-60 min, depending on the cell
type (Deed et al., 1996; Bounpheng et al., 1999a). ID4 is
apparently much less sensitive to inhibitors of the 26S
proteasome pathway, and, although its degradation is
dependent on ubiquitin-activating enzyme activity, this ID
family member might also be degraded through an alternative
pathway (Bounpheng et al., 1999a).
Heterodimerisation with bHLH proteins extends the half-life
of ID3 (and also enhances the degradation of its heterodimeric
bHLH protein partner); ID proteins might therefore be less
susceptible to degradation by the 26S proteasome pathway in
the heterodimer state (Deed et al., 1996; Bounpheng et al.,
1999a). In common with several other positive regulators of
cell cycle progression, ID proteins are also regulated by
subcellular localisation. ID proteins do not possess nuclear
localisation signals, whereas their bHLH protein partners
possess an efficient nuclear localisation signal. Co-transfection
of cells with constructs expressing ID and bHLH proteins leads
to sequestration of the ID protein into the nucleus, which
suggests that heterodimerisation regulates the subcellular
distribution of IDs (Deed et al., 1996). The physiological
relevance of such a mechanism has yet to be established.
CONCLUSIONS
In the ten year period since ID proteins were first described in
flies and mammals, interest in this family of HLH proteins has
increased exponentially. Inhibition of cell differentiation
through antagonism of bHLH proteins, as originally described
ten years ago, still remains the single most important facet
of ‘Ideology’. However, the diversity of other biological
processes in which the functions of ID proteins have been
implicated and the apparent promiscuity of molecular
interactions that might mediate ID functions now presents a
rather bewildering picture. One of the problems associated with
the field is the questionable generality of many of the
observations made. For example, most of the known molecular
mechanisms through which ID functions are integrated in cell
cycle control have been modelled in fibroblasts. Given that
3904 J. D. Norton
cells from different lineages and developmental stages express
distinct repertoires of ID proteins and different ID proteins
have different properties, it seems highly likely that the
mechanisms through which ID proteins regulate the cell cycle
machinery will turn out to be cell type specific. Also, the role
of ID proteins in lymphopoiesis has been particularly well
studied, primarily because of the long-standing interest of
developmental immunologists in the bHLH E proteins, whose
functions are pivotal for development and function of the
lymphopoietic system. However, it is far from clear whether
the mode of action of ID proteins in lymphopoiesis, at both the
molecular and biological levels, can be extrapolated to other
mammalian cell lineages in which ID proteins undoubtedly
play a role.
At a mechanistic level, it will clearly be important to
establish the biological significance of the interactions between
ID proteins and their various non-bHLH protein partners in a
physiological context. Some of these ID-interacting proteins,
such as pRB, ETS and PAX transcription factors, are well
known for their roles in regulating various cellular processes,
particularly cell fate determination in diverse cell lineages, and
indeed have a functional status to match that of bHLH proteins.
Also, the upstream signalling pathways that couple individual
ligand-receptor interactions to Id gene regulation in
mammalian cell fate determination deserve more serious
investigation, particularly given the provocative data from
Drosophila implicating signalling through Notch and Ras
pathways.
Work in the author’s laboratory is supported by the UK Cancer
Research Campaign and the Leukaemia Research Fund.
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