(Received for publication, March 21, 1997, and in revised form, June 5, 1997)
From the Dimerization of three Id proteins (Id1, Id2, and
Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two
groups of class B proteins, the myogenic regulatory factors (MRFs:
MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay. All three Ids bound with high affinity to E proteins, but a much broader range of interactions was observed between Ids and
the class B factors. Id1 and Id2 interacted strongly with MyoD and
Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacted
weakly with all four MRFs. Similar specificities were observed in
co-immunoprecipitation and mammalian 2-hybrid analyses. No interactions
were found between the Ids and any of the hematopoietic factors. Each
Id was able to disrupt the ability of E protein-MyoD complexes to
transactivate from a muscle creatine kinase reporter construct in
vivo. Finally, mutagenesis experiments showed that the
differences between Id1 and Id3 binding map to three amino acids in the
first helix and to a small cluster of upstream residues. The Id
proteins thus display a signature range of interactions with all of
their potential dimerization partners and may play a role in myogenesis
which is distinct from that in hematopoiesis.
An increasingly important role is ascribed to protein-protein
interactions in the regulation of cellular growth and differentiation pathways. Dimerization serves to convert inactive monomeric molecules into transcriptionally active dimeric complexes at specific times during cellular development. Deletional analysis has identified a
number of evolutionarily conserved regions that mediate these interactions. One such region, commonly associated with transcription factors involved in a range of proliferative and differentiation pathways, is the basic-helix-loop-helix
(bHLH)1 (1). This domain is
conserved from yeast to mammals and is composed of a positively charged
basic region followed by two amphipathic bHLH transcription factors can be broadly placed into two categories
(reviewed in Ref. 10). The class A factors, or E proteins, (E2-2, HEB,
and the E2A gene products E12 and E47) are expressed in a virtually
ubiquitous pattern and are able to dimerize efficiently with
tissue-restricted class B factors to activate gene expression (1,
11-13). Because each factor contributes a specific DNA recognition half-site, class A and class B heterodimeric complexes and class A
hetero- or homodimers theoretically provide distinct combinatorial E
box binding specificities (7). Class B members thus far characterized include the myogenic regulatory factors (MRFs) involved in skeletal muscle development (MyoD, myogenin, Myf-5, and MRF4/Myf-6) and the
hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) (14-21). Mammalian
homologues of the Drosophila bHLH achaete scute genes (mash1
and -2) have been implicated in neuronal development as has neurod
(beta2) which also has a role in insulin regulation (22-24). A range
of other class B bHLH proteins have been associated with early mesoderm
formation and later muscle development (Twist), adipocyte development
(Add1), and skeleton formation (Scl-1) (25-27). E box motifs have been
identified in the enhancer elements of a number of bHLH
factor-regulated genes such as myosin heavy chain, immunoglobulins, and
chymotrypsin (10).
The formation of active class A-class B complexes is modulated by the
Id (inhibitor of DNA binding) family members. The four Id proteins
identified thus far have an HLH domain that lacks the amino-terminal
associated basic region necessary for DNA binding (28-33). Id proteins
act to sequester class A factors, inhibiting the formation of active
class A-class B heterodimers and are therefore considered to act as
dominant negative regulators of differentiation pathways (28, 34, 35).
Ids are expressed in a largely overlapping but distinct fashion during
development, with the highest levels generally being achieved during
embryogenesis (33). Significant Id levels persist in a range of
actively proliferating tissues and in some tumor cell lines (28, 33).
Rapid Id down-regulation has been reported in myoblasts and
hematopoietic cells during terminal differentiation, consistent with
their negative regulatory role (28, 34). Indeed, forced Id expression
has been shown to inhibit the differentiation of each of these cell
types (36-40). Because considerable overlap exists in the expression
patterns of Id proteins, redundancy in their function has been inferred (30, 33).
Ids are known to bind avidly to class A factors such as E47, weakly to
the myogenic factors, and poorly, if at all, to the hematopoietic
factors (28, 34, 41). This has led to the assumption that
transcriptional control is exerted primarily at the level of Id-class A
interactions. However, these studies were performed with select members
of the class A and class B families. Thus far, no exhaustive studies
exist to compare the relative strengths of interactions among a broader
range of family members.
The yeast 2-hybrid system has become an increasingly popular method for
assessing protein-protein interactions and has been employed previously
to study a subset of bHLH interactions (35, 42-44). We sought to
develop a quantitative yeast 2-hybrid assay to investigate interactions
of three Id proteins with a range of class A and class B factor
targets. Our observations, confirmed and extended by
co-immunoprecipitation (IP) of in vitro translated proteins
and mammalian 2-hybrid analyses, indicate that discrete and
reproducible differences exist in relative binding preferences among
the Id proteins. As expected, Id proteins bound avidly to all the class
A factors tested, although a range of affinities were apparent. A
broader range of affinities for myogenic factors was observed, and no
interactions with the hematopoietic factors were seen despite their
expression in functional form. Transient transfection studies in C3H
myoblasts employing a muscle-specific creatinine kinase-
chloramphenicol acetyltransferase (CAT) reporter vector provided
independent confirmation of the hierarchical interactions of Id
proteins with class A factors. Site-directed mutagenesis enabled us to
map the regions responsible for establishing Id dimerization
preferences to the first helix of the HLH domain and to residues
immediately adjacent to this. Our findings have implications for the
mechanism by which Id proteins influence class A and class B
interactions and for the roles played by different Id proteins in
tissue-specific gene regulation.
Parental yeast vectors pGBT9 and pGAD
were kindly supplied by Dr. S. Elledge (Baylor College of Medicine).
Parental expression vectors for mammalian 2-hybrid analysis, pSG424 and
pNLVP16, were obtained from Drs. C. Dang (Johns Hopkins University
School of Medicine) and M. Green (University of Massachusetts) (45,
46). Murine Id1 was obtained from Dr. H. Weintraub (Fred Hutchinson Cancer Research Center), and murine Id2 and Id3 cDNAs were obtained from Dr. D. Nathans (Johns Hopkins University) (28, 30, 34). Fragments
encoding the HLH regions plus 15-20 flanking amino acid residues were
generated by PCR primers incorporating EcoRI (forward primer) and BamHI (reverse primer) sites and cloned
directionally and in-frame into pGAD, pGBT9, and pSG424. The fragments
amplified encoded amino acids 73-138 of Id1, 72-140 of Id2, and
28-91 of Id3. All products were sequenced to confirm the fidelity of
the PCR amplification reaction. Full-length cDNAs were cloned into pRcCMV (Invitrogen, San Diego, CA) for use in in vitro
transcription/translation reactions and transient transfection assays.
Murine MyoD, murine myogenin, and human Myf-5 were supplied by Dr. D. Shapiro (St. Jude Children's Research Hospital) and rat MRF4/Myf-6 by
Dr. S. Konieczny (Purdue University) (8, 15, 16, 20). Fragments encoding the bHLH domains were again amplified by PCR, incorporating EcoRI and BamHI sites for MyoD or
BamHI and PstI sites for myogenin, Myf-5, and
MRF4/Myf-6 and cloned in-frame into pGBT9 and pGAD424. The regions
amplified included codons 83-184 of MyoD, 55-155 of myogenin, 56-143
of Myf-5, and 65-168 of MRF4/Myf-6. Due to the presence of an internal
PstI site in the second helix of Myf-5 and MRF4/Myf-6, these
were cloned as BamHI/blunt fragments. The bHLH domains from
MyoD and Myf-5 were also subcloned in-frame into pNLVP16 as blunt-ended
fragments. Full-length MyoD and MRF4/Myf-6 cDNAs were cloned into
pRcCMV. The cDNAs encoding each of the class A factors were
isolated from a yeast 2-hybrid screen of a murine embryo library in the
pGAD10 vector (CLONTECH, Palo Alto, CA) using an
Id2 bait. These contained residues 118-264 of murine E47 (A1),
residues 379-666 of murine E2-2 (ME-2), and residues 574-729 of
murine HEB (Alf-1) (47-49). The E47 fragment was also cloned into both
pNLVP16 and pGBT9. A carboxyl-terminal bHLH containing fragment of
human E12 (residues 508-654) in the pAS1 vector, a gift from Dr. E. Olsen (University of Texas), was excised and sub-cloned into pGAD424
(44). None of the class A clones contained the putative leucine zipper
regions that are associated with transcriptional activation domains
(50). An almost full-length E12 clone in bluescript (E12R, a gift from
Dr. H. Weintraub), including the putative leucine zipper domain, was
used for in vitro translation (6). Full-length E12 and E47
in the mammalian expression vector pGK were a gift from Dr. G. Kato
(Johns Hopkins University School of Medicine). The cDNAs for human
hematopoietic factors Scl/Tal-1, Tal-2, and Lyl-1 were provided by Drs.
I. Kirsch (National Cancer Institute), R. Baer (University of Texas),
and M. Cleary (Stanford University), respectively (14, 19, 21). bHLH
domains were again amplified by PCR with primers containing
EcoRI and BamHI sites and were cloned in-frame
into both pGAD424 and PGBT9. The amplified fragments encoded amino
acids residues 66-138 of Scl/Tal-1, 2-69 of Tal-2, and 127-200 of
Lyl-1. Full-length Scl/Tal-1 was subcloned into pBluescript SK+
(Stratagene, La Jolla, CA). The multimerized gal4:CAT reporter
construct pGal5E472CAT was supplied by Dr. M. Green (51). The muscle
creatinine kinase (MCK) CAT construct was obtained from Dr. S. Hauschka
(University of Washington).
This was performed according to the
protocol developed in the laboratory of Dr. S. Elledge, who also
supplied the Saccharomyces cerevisiae strain Y153 (MATa,
leu2-3, trp1-901, his3- Yeast transformants
were grown to stationary phase in complete EGG medium containing 2%
ethanol, 2% galactose, and 3% glycerol and lacking tryptophan and
leucine. 107 cells were pelleted and resuspended in 50 µl
of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM
MgCl2, 50 mM Yeast were grown in EGG medium until
they reached stationary phase. They were then pelleted and subject to
two cycles of freeze-thawing followed by boiling in standard
SDS-polyacrylamide gel electrophoresis (PAGE) lysis buffer for 10 min
to ensure complete lysis (53). 100 µg of protein was resolved by 12%
PAGE and transferred to Immobilon-P membranes (Sigma) by semi-dry
electroblotting. Membranes were preincubated in wash solution (1 × phosphate-buffered saline + 0.1% Tween 20). This was followed by a
1-h incubation in block solution (5% non-fat dry milk powder,
phosphate-buffered saline + 0.1% Tween 20) containing either an
anti-yeast gal4 DNA binding domain or activation domain antibody (at
1:500 and 1:2000 dilutions, respectively) (Upstate Biotechnology Inc.,
Lake Placid, NY). Membranes were washed four times before the addition
of a horseradish peroxidase-linked goat anti-mouse antibody (1:1000
dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in blocking
solution and incubated for a further 1 h at room temperature.
After a further four washes, proteins were visualized using an enhanced
chemiluminescence detection kit according to the manufacturer's
instructions (Amersham International, Buckinghamshire, UK).
Full-length E12, Id1, Id2, Id3, Scl/Tal-1,
MyoD, and MRF4/Myf-6 cDNAs in either pBluescript SK+ or pRcCMV were
transcribed and translated in vitro using a coupled
reticulocyte lysate kit (TNT, Promega, Madison, WI) in the presence of
[35S]methionine (1 mCi/mmol). Labeled proteins were mixed
and incubated at 37 °C for 20 min before the addition of 100 µl of
IP buffer (250 mM NaCl, 0.25% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol). Anti-Id, E12, or MyoD polyclonal
antibodies were added and reactions incubated on ice for 30 min prior
to the addition of 30 µl of a 1:1 mix of protein A-Sepharose
(Bio-Rad) in IP buffer. After a further 60 min incubation, samples were washed four times in IP buffer before resolution by 12% SDS-PAGE. The
intensity of each partner was quantified by PhosphorImage analysis
(Molecular Dynamics) using Image-Quant software and normalized relative
to methionine content of each protein. The value was then expressed as
a ratio of the amount of input proteins. All antibodies were tested for
cross-reactivity and shown to be specific for their respective protein
under the conditions described here.
5 µg
of each vector DNA was introduced into HeLa cells by calcium phosphate
precipitation, and the cells were maintained in Dulbecco's modified
Eagle medium (DMEM) supplemented with 10% calf serum (Life
Technologies, Inc.) (54). All transfections included a CMV
A determination of the ability of Ids to repress E protein-MyoD
interactions was performed in C3H myoblasts. The amounts of MyoD and E
protein vector DNAs used were determined empirically so as to generate
the maximum amount of transcriptional activation in combination over
either factor alone, prior to determining the influence of
co-transfected Id. This ensured that CAT activity was a reflection of a
specific MyoD-E2A interaction, minimizing the influence of additional
endogenous factors. 2 days post-transfection, DMEM + 10% calf serum
was replaced with DMEM + 2% horse serum, and incubation was continued
for a further 2 days. Under these conditions, endogenous Id levels are
repressed, allowing for myogenic differentiation to proceed (55). All
transfections were adjusted to give uniform DNA concentrations with the
empty pRcCMV vector.
Helix
swaps between Id1 and Id3 were generated by PCR amplification of
individual helices followed by a "cut and paste" cloning strategy.
Sixteen residues upstream of the amino terminus and 14 residues
downstream of the carboxyl terminus of each HLH domain were maintained
to promote correct folding. A fragment of Id1 encompassing helix 1 was
amplified with a forward primer containing an EcoRI site.
The reverse primer mapped to the helix 1/loop junction and contained a
BamHI site. A SmaI site was introduced at the helix 1/loop boundary (residues 102 and 103 of Id1) where the codons
CCC ACC were altered to CCC GGG.
EcoRI/BamHI-digested fragments were cloned into
pBluescript SK+ to create plasmid A. Helix 2 from Id3 was amplified
from a 5 A quantitative 2-hybrid approach was
used to study interactions between Id proteins and a series of class A
and class B targets expressed as gal4 binding domain and gal4
transactivation domain fusions, respectively (Fig.
1). All Id-class A interactions were determined to be quite strong by this assay (Fig. 1A).
Interactions varied over a 5-fold range from the weakest (Id2-HEB) to
the strongest (Id2-E47). The consistently strong Id-class A
interactions observed stand in contrast to those seen between the Ids
and the class B myogenic factors (Fig. 1B). These latter
interactions were considerably weaker than those of Id-class A
interactions and showed a far greater degree of variability. Id2
displayed the strongest binding which, in the case of MyoD and Myf-5,
was 3-4-fold more avid than that of the corresponding Id1
interactions. In turn, Id1 bound MyoD and Myf-5 with a 5-10-fold
greater affinity than Id3 which demonstrated barely detectable
interaction with any of the myogenic factors. Of note, however, was
that the strength of the Id2-Myf-5 and Id2-MyoD interactions rivaled
the weakest Id-class A factor interactions. MyoD and Myf-5 showed the
strongest interactions with all three Ids tested. Although Id3 bound
weakly, if at all, to the myogenic factors, its binding to class A
factors was comparable to that of the other Ids (Fig. 1A).
Hence the differences in binding seen here cannot be attributed to
differential expression. This was also confirmed by Western blot
analysis of yeast lysates showing comparable levels of expression of
all proteins under study (Fig. 2). When
tested for their ability to bind the class A factors E47 or E12, all
the myogenic factors (including MRF4/Myf-6) interacted strongly (Fig.
1B), again indicating that the differences in myogenic factor-Id binding were not due to relative differences in MRF protein
expression. None of the hematopoietic factors displayed discernible
interactions with the Id proteins, whereas all were found to interact
with the class A target E2-2 (Fig. 1C).
MRFs were also investigated for homodimerization ability. MyoD,
myogenin, and Myf-5 displayed weak homodimerization (less than 50 pg of
Co-IP of selected full-length
in vitro translated proteins was employed to confirm the
differences in dimerization properties identified by yeast 2-hybrid
analysis (Fig. 3). Increasing amounts of
Id1, Id2, or Id3 proteins were incubated with E12, and resultant heterodimers were precipitated with an anti-E12 polyclonal antibody (Fig. 3A). Consistent with the yeast 2-hybrid data (Fig. 1),
all three Ids interacted strongly with E12 with any differences being on the order of less than 2-fold. In contrast, interactions between Id
proteins and MyoD were considerably weaker and showed more pronounced
differences than those observed with Id-E protein interactions. For
example, Id1 and Id2 showed comparable interactions with MyoD, whereas
Id3-MyoD interactions were significantly weaker. These results were in
good agreement with the results obtained by the 2-hybrid system. All
Ids also bound very weakly to MRF4/Myf-6 (Fig. 3C) and not
at all to Scl/Tal-1 (Fig. 3D). From these experiments, we
conclude that the strengths of Id-E protein and Id-MRF interactions qualitatively and quantitatively reflected those observed in yeast. Furthermore, our results indicate that the dimerization properties of
full-length proteins are not significantly different from those of the
isolated HLH domains expressed in yeast.
The observation that the Ids
displayed the widest range of interactions with MyoD and Myf-5 led us
to investigate whether such differences were maintained in mammalian
cells (Fig. 4). Id1, Id2, and Id3 were
expressed as gal4 DNA binding domain fusions, whereas MyoD and Myf-5
were expressed as VP16 transactivation domain fusions (56). Each was
tested for its ability to activate a reporter vector in HeLa cells
(Fig. 4A). E47, which displays strong interactions with each
Id in yeast (Fig. 1A), was also expressed as a VP16 fusion
as a positive control for Id dimerization activities. Appreciable CAT
conversion was seen when either Id1 or Id2 was co-expressed with either
MyoD or Myf-5 (Fig. 4B), whereas Id3 interaction with either
of the myogenic factors was only marginally above background. These
observations were generally consistent with both the yeast 2-hybrid
data and with the co-IP experiments (Figs. 1 and 3). As the Ids
differed in their ability to dimerize with the MRFs, we investigated
their ability to bind E47 (Fig. 4B). Each was found to
generate comparable high levels of CAT activity as might have been
predicted from our yeast 2-hybrid results (Fig. 1A). Thus
the reduced ability of Id3 to interact with MyoD or Myf-5 cannot simply
be explained by its differential expression. Our results in both
mammalian cells and in yeast appear to reflect the true dimerization
preferences of the Id proteins under study.
By
having established that the Ids interacted similarly with each E
protein (Fig. 1A), we sought to determine the influence of
each Id on the transcriptional activity of full-length E2A·MyoD heterodimers (Fig. 5). We had previously
established that this combination of factors gave the highest levels of
transactivation from the MCK-CAT
reporter.2 The relative
consistency of Id-E2A interactions in yeast suggested that each Id
should disrupt E2A·MyoD complexes with comparable ability. Low levels
of transactivation were observed when either 5 µg of MyoD or 1 µg
of E47 or E12 was transfected alone. Co-transfection of these factors,
however, generated significant CAT conversion, indicating that
transactivation was mediated by an E2A·MyoD heterodimer and not by
endogenous factors. The enhanced ability of MyoD to form heterodimers
with E47 compared with E12 in yeast (Fig. 1B) was reflected
in an increased ability of MyoD·E47 heterodimers to activate the
MCK-CAT reporter. The introduction of increasing amounts of each Id led
to the suppression of CAT activity in a dose-dependent
fashion, with a maximal 2-3-fold reduction in transactivation observed
at the highest input Id concentration. In metabolic labeling experiments, the expression of each Id was determined to be comparable (data not shown).
To map
the amino acid residues responsible for the differential binding
capabilities of Id proteins with respect to the MRFs, helix swaps were
generated, and site-directed mutagenesis was performed (Fig.
6). All mutants were tested in yeast for
their ability to bind E12, and all were found to bind with similar
avidity, varying over an approximate 2-fold range (Fig. 6, last
column). These results were anticipated based on our observations
that wild-type Id1 and Id3 interacted to a comparable extent with E12 (Fig. 1A). Initially, chimeras containing the first helix of
Id1 and the loop-helix 2 region of Id3 or the first helix of Id3 and the loop-helix 2 region of Id1 were constructed and tested for interaction with all four MRFs (Fig. 6, lines 3 and
4). These experiments clearly demonstrated that the region
determining the specificity of interactions resided in the first helix
and/or in the amino-terminal region immediately adjacent to this. Based on this observation, the three amino acid residues that distinguish the
first helices of Id1 and Id3 were altered sequentially to convert Id1
to Id3 and vice versa. Altering individual tyrosine, glycine, and lysine residues of Id1 to the corresponding aspartic acid,
histidine, and arginine residues of Id3, respectively, reduced binding
in each case (Fig. 6, lines 5, 7, and 9).
However, the relative importance of each residue for dimerization was
dependent upon the MRF under study. For example Id1/3 (G92H) showed a
3-fold reduction in the ability to bind MyoD but a 14-fold reduction in
the ability to bind Myf-5 (compare Fig. 6, lines 3 and
7). In contrast Id1/3 (K98R) showed a 12-fold reduction in
MyoD binding but only a 4-fold reduction in myogenin binding (compare
Fig. 6, lines 3 and 9). Similarly, no single Id3
to Id1 mutation restored maximal binding (Fig. 6, lines 6, 8, and 10). This demonstrates that each residue, even
the conserved lysine of Id1 and arginine of Id3, contributes to binding
specificity although the relative importance of each residue is
dependent upon the MRF target. To investigate possible additive
effects, two further sets of Id3 mutations were investigated for their
ability to restore Id1-like binding. A double mutant (Fig. 6,
line 11), created by altering the aspartic acid and
histidine residues of Id3/1 to the complementary tyrosine and glycine
residues of Id1, resulted in almost full binding to MyoD, increased
binding to Myf-5, and weak binding to MRF4/Myf-6. This double mutant
did not, however, restore myogenin binding. Even when all three
residues were altered to those of Id1 (Fig. 6, line 12), no
increase was seen in Myf-5 binding when compared with the double
mutant. Some binding was seen to myogenin, whereas MRF4/Myf-6 binding
was undetectable. These data suggested that helix 1 residues are
sufficient for MyoD recognition but that additional residues upstream
of helix 1 appear to be required for full dimerization with the other
three myogenic factors. To investigate the upstream requirement
further, a series of deletions were made in the Id1/3 swap background.
The initial deletion of six amino acids at the extreme NH2
terminus of the Id1/3 sequence (Fig. 6, line 13) had a
minimal effect on binding (<2-fold) with the exception of Myf-5 whose
binding was reduced approximately 3-fold (Fig. 6, lines 3 and 13). The deletion of a further six residues (Fig. 6,
line 14) resulted in the abrogation of myogenin and
MRF4/Myf-6 binding although little additional effect was seen with
respect to Myf-5 and MyoD. Finally, an internal deletion of the six
residues immediately adjacent to the amino terminus of the first helix
(Fig. 6, line 15) severely inhibited dimerization with all
MRFs, although this was least obvious with MyoD. Interestingly, all of
the individual deletion mutants (Fig. 6, lines 13-15) bound E12 as well as either of the wild-type Ids (with <2-fold differences). From these studies, it appears that complete MyoD and Myf-5
interactions with Id1 require similar amino acid residues. The residues
facilitating MyoD and Myf-5 binding are not sufficient for myogenin and
MRF4/Myf-6 binding, and these latter factors require additional,
upstream amino acids. Binding by E12 would appear not to require any of these upstream residues.
A select number of the Id "helix swap" proteins depicted in Fig. 4
were examined using the mammalian two-hybrid system. In these
experiments, the HLH domains of Id1/3, Id3/1, and Id1/3 D80-85 were
cloned into the pSG424 vector and transfected into HeLa cells together
with either pNLVPMyoD or pNLVPMyf5. As shown in Fig.
7, these results recapitulated those
observed in yeast. Neither Id3, Id3/1, nor Id1/3 D80-85 gave a
detectable interaction with either MyoD or Myf5. In contrast, Id1
interacted strongly. That each of the Id proteins was expressed was
confirmed in control experiments showing that all five Id fusion
proteins depicted in Fig. 7 interacted strongly with a pNLVP16 fusion
of the E12 bHLH domain (not shown).
bHLH proteins are involved in diverse aspects of cellular
physiology, from establishing the topography of the early embryo (e.g. Twist) to promoting cellular transformation,
proliferation, and apoptosis (e.g. c-Myc) and ultimately to
the regulation of terminal differentiation programs such as myogenesis
(the MRFs) (25, 57, 58). As an increasing number of such proteins are characterized, it is apparent that spatial and temporal expression patterns combine with a hierarchical network of specific
protein-protein interactions to provide precise control of many
cellular processes.
A variety of biochemical, genetic, and in vivo approaches
have been taken in the investigation of dimerization potential, sequence-specific DNA binding, and transcriptional activity of bHLH
factors (35, 41, 43, 56, 59-62). Although our investigation generally
considers only one aspect of these activities, dimerization, we feel
that a comprehensive understanding of the relative affinities displayed
by a group of key factors may provide insight into mechanisms of
transcriptional activation. As the function of the myogenic factors
both in establishing the myoblast lineage and initiating terminal
differentiation is reasonably well described, myogenesis may provide a
useful model for other tissue-specific differentiation pathways
(reviewed in Ref. 58).
Numerous groups have reported the ability of class A, class B, and Id
proteins to homo- and heterodimerize. Sun and Baltimore (60) reported
the dissociation constants (Kd) for MyoD homodimers,
E47 homodimers, and E47·MyoD heterodimers to be 6.8 × 10 All three Id proteins tested here interacted strongly with all class A
factors, consistent with their role in the sequestration of these
ubiquitous proteins. Although Id-class A interactions displayed the
strongest and most consistent interactions among the series of factors
tested, each Id nevertheless displayed a discrete "fingerprint" of
preferred class A partner. Specific patterns of Id-MRF interactions
were also observed, although these interactions were considerably
weaker and showed greater variability than those of Id-class A
interactions. The finding that Id-MRF interactions were conserved, in
contrast to Id-hematopoietic factor interactions, suggests that the Ids
may have a role in myogenesis that is distinct from that in
hematopoiesis. Finally, the overlap in Id-class B and Id-class A
interaction strengths provides additional reason to believe that the
former may be physiologically relevant. When the Ids were expressed as
full-length proteins in vitro, or as HLH domains in human
cells, the differences in relative affinities observed in yeast
persisted (Figs. 3 and 4), indicating that the discrete range of
interactions accurately reflects the in vivo situation and
was not merely a consequence of the expression of mammalian genes in
yeast.
Homodimerization of bHLH proteins seems to be important in certain
contexts. For example, E47 homodimers are sufficient to activate
immunoglobulin gene expression (65). MyoD is also capable of binding
DNA as a homodimer, although it has not been established if this is
biologically significant as the concentrations required for
homodimerization in vitro may not be achievable in
vivo (8). On the other hand, it is conceivable that apparently
weak interactions are stabilized by accessory factors or that low
concentrations of MyoD homodimers are sufficient to initiate the
myogenic cascade, and this may be sensitive to direct Id-mediated
suppression. Therefore, cell fate determination may in part depend on
the relative level of distinct Id proteins to create a permissive
environment. Interestingly, our observations indicate that the Ids bind
with the highest affinity those myogenic factors involved in myoblast
lineage determination (MyoD and Myf-5), whereas those MRFs active
post-mitotically (myogenin and MRF4/Myf-6), a time when Id levels are
generally low or undetectable, bind Ids considerably less well.
The differential affinities of the Id proteins for both the class A and
class B MRFs could be explained if these latter proteins homodimerized.
Under such conditions, there might exist a differential availability of
E proteins or MRFs for Ids due to their sequestration in homodimeric
form. In other experiments, we have examined the ability of the Ids,
the E proteins, and the class B MRFs and hematopoietic factors to
homodimerize using the same two-hybrid system shown in Fig.
1.3 We detected
homodimerization only with E47, although at levels that were
10-30-fold lower than its interaction with any of the three Ids. Thus,
limitation of E protein accessibility to the Ids due to homodimeric
sequestration appears unable to explain our results.
Having demonstrated that Id proteins bind each class A molecule and a
subset of class B molecules, we tested the ability of each Id to
suppress transactivation from a muscle-specific E box by an E2A·MyoD
complex in vivo (Fig. 5). Id1, Id2, and Id3 were found to
disrupt E2A·MyoD complexes with comparable ability, consistent with
the yeast data. Interestingly, an apparent excess of Id was unable to
completely abrogate binding. Other studies on the effect of Id1 or Id3
on the transactivation potential of MyoD or E proteins have also
reported a less than complete suppression of activity, with even
greater excesses of Id than we have utilized (28, 35). Investigation of
the activity of any transcription factor in isolation is complicated by
the influence of endogenous factors. Having established conditions in
which each factor alone results in minimal transactivation, we can be
certain that the repressive effect of the Ids is exerted only on the
factors under study.
The observation that the Ids differed in their ability to recognize
myogenic partners led us to investigate the precise residues responsible for this specificity (Fig. 6). Dimeric bHLH complexes bound
to DNA are predicted to form a parallel 4-helix bundle stabilized by a
hydrophobic core as well as by electrostatic interactions (2, 8, 62).
Shirakata et al. (62) reported that the alteration of five
non-hydrophobic residues distinguishing chicken MyoD from the
Drosophila MyoD homologue nautilus (which does not bind E12)
led to a progressive reduction in MyoD·E12 dimer formation, suggesting that these residues confer an additive effect on binding (62). Likewise, in our study, no single residue was able to confer
Id1-like characteristics. Rather a combination of non-conserved residues was involved in MRF binding. However, these residues did not
appear to be important for Id-class A interaction (Fig. 6). Two
non-conserved residues in the first helix were sufficient for almost
complete MyoD binding. These residues, Tyr-88 and Gly-92 of Id1, are
uncharged, whereas the corresponding residues of Id3 (Asp-42 and
His-46) confer a negative and positive charge, respectively. Thus, the
differences in Id binding (with respect to MyoD) may be consistent with
the "charged pair" model which suggests that attractive or
repulsive forces between contacting residues in the helices of aligned
molecules act to stabilize or destabilize dimer formation (62). The
residue at the third position also influences heterodimerization even
though this represents a conservative change (Lys-98 in Id1 to Arg-52
in Id3). In this case, the larger arginine side chain may act to
destabilize Id3·MRF complexes. Despite the obvious importance of
these residues, the complete conversion of Id3 helix 1 to that of Id1
helix 1 restored only 50% Myf-5 binding and only minimal interaction
with myogenin and MRF4/Myf-6, suggesting that additional residues exert
an effect. Indeed, a series of amino-terminal deletions (Fig. 6,
lines 13 and 14) demonstrated that a region
outside the HLH domain was important for Id1-MRF interactions,
particularly with respect to myogenin and MRF4/Myf-6. Interestingly,
these deletions did not appear to influence E12 binding. Upstream
sequences may not be involved in establishing dimeric complexes as
such, but rather in stabilizing pre-formed interactions determined by
residues in the HLH region. Such subtle cooperative effects might be
expected to contribute more to weak Id-myogenin or Id-MRF4/Myf-6
interactions than to stronger associations. Goldfarb et al.
(43) used random mutagenesis in conjunction with a yeast 2-hybrid
system to generate both hydrophobic and hydrophilic substitutions which
enhanced the ability of Scl/Tal-1 to recognize E2-2 (43). Such
alterations were again found to have a synergistic effect on dimer
stability. More extensive mutagenesis is required to fully understand
the diversity of dimer preferences displayed by HLH factors. The
quantitative yeast 2-hybrid system described in this report provides a
useful tool for this purpose.
Regions other than the HLH domain, such as the putative leucine zipper
present in the class A proteins, have the potential to modulate
interactions (1, 11, 12). Because our co-IP findings with full-length
proteins are consistent with both our yeast and mammalian 2-hybrid
results, we infer that other potential dimerization domains do not
contribute significantly to the interactions observed here. However, we
have not evaluated the possible influence of post-translational
modifications on dimerization (47, 59, 66, 67).
Although HLH activity may be modulated in vivo, the key
differences in binding characteristics presented in this study may play
a fundamental role in the establishment of differentiation programs.
There still exists, however, a great deal of redundancy. One apparent
paradox is that there is little evidence, as yet, for the combinatorial
variation in E box binding allowed by heterodimerization between
different class B groups and any class A factor. For example, all class
A·MRF dimers so far studied efficiently bind an element in the muscle
creatine kinase enhancer, and binding site selection indicates that
Scl/Tal-1, Tal-2 and Lyl-1 heterodimers with E2-2 all recognize the
same putative hematopoietic-specific element (68, 69). Spatial and
temporal differences in the expression of factors with apparently
conserved function may also explain some of the apparent overlap.
Alternatively, redundancy may reflect the need for cooperative
occupancy of proximal promoter elements by accessory factors. A
distinct group of tissue-restricted non-bHLH transcription factors have
been identified, such as the myocyte enhancer family (MEF-2) in muscle
and the zinc finger GATA factors in erythropoiesis (70, 71). These may
act to enhance the transcriptional activity of bHLH dimers, thereby
modulating lineage-specific gene expression. Indeed, the apparent
redundancy in function may mask the requirement for a subtle gradient
of both ubiquitous and tissue-specific factors to facilitate the
controlled establishment of the differentiated phenotype.
We thank members of our laboratory for
helpful discussions and advice and Geneva Jordan for skilled
secretarial assistance.
Section of Hematology/Oncology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helices separated by a
spacer loop. Dimers are stabilized by a series of hydrophobic and
electrostatic interactions between the helices of compatible molecules
(2-5). The juxtaposition of two basic regions resulting from
dimerization forms a DNA binding interface able to insert into the
major groove in a sequence-specific manner (2, 6). Although bHLH
proteins have no discernible DNA binding activity as monomers, dimers
recognize a consensus DNA sequence (CANNTG), termed the E box (4,
7-9).
Plasmid Construction
200, ura5-32, ade2-101, gal4
,
gal80
GGAL-HIS3, LYS2::GAL-HIS3). Potential partner genes
in either pGAD424 or pGBT9 were co-transformed into yeast by a lithium
chloride/polyethylene glycol precipitation method (52).
Semi-quantitative determination of interaction strengths was performed
by in situ assessment of
-galactosidase activity.
-Galactosidase Assay
-mercaptoethanol) containing
0.01% SDS. Two microliters of CHCl2 were added followed by
two cycles of freeze-thawing in liquid nitrogen. Lysates were transferred to 96-well plates, and 50 µl of a fluorogenic substrate (8 mM
3-carboxyumbelliferyl-
-D-galactopyranoside, Molecular
Probes Inc., Eugene, OR) was added. After 30 min, 100 µl of stop
buffer (300 mM glycine, 15 mM EDTA, pH 11.5)
was added, and the reactions were allowed to stabilize for 1 h.
Fluorescence was determined in a Perkin-Elmer microtiter plate reader
(Foster City, CA; excitation 390 nm, emission 460 nm), and the amount
of
-galactosidase synthesized was calculated relative to a dilution
series of
-galactosidase enzyme standards (Sigma) assayed
simultaneously with the yeast lysates. All assays were performed in
triplicate with standard errors of less than 10% in each case. None of
the individual vectors used in this series displayed a background
higher than 10 pg of
-galactosidase enzyme/107 cells
which represents the lower limit of detection with this assay.
-galactosidase reporter (CLONTECH) to
standardize transformation efficiencies. CAT assays were performed on
cell lysates harvested after 48 h and relative conversion
calculated by PhosphorImage analysis.
primer derived from the loop/helix 2 region, also containing
a SmaI site and a 3
primer containing a BamHI
site. BamHI/SmaI-digested fragments were then
ligated into BamHI/SmaI-digested plasmid A, thus
generating an Id1/3 chimera (plasmid B). A complementary approach was
taken to constructing the Id3/1 chimera. Finally,
EcoRI/BamHI fragments were excised from plasmid B
and cloned into the yeast vector pGBT9 or into an m13 vector for
further point mutagenesis. Oligonucleotide-mediated site-directed
mutagenesis was performed with the Muta-gene kit (Bio-Rad) according to
the manufacturer's recommendations using the chimeric Id1/3 or Id3/1
single-stranded phage DNAs. All clones were completely sequenced to
ensure the presence of the desired mutation prior to subcloning into
pGBT9. Each construct was tested for interaction with pGAD E12 and
shown to interact with consistent affinity.
Yeast 2-Hybrid Analysis
Fig. 1.
Quantitative yeast 2-hybrid analysis. Id
proteins expressed as gal4 DNA binding domain fusions were tested with
class A and class B targets expressed as gal4 transactivation domain fusions. A, interaction of Id proteins with class A factors.
B, interaction of Id proteins with MRFs. E12 and E47 (also
expressed as binding domain fusions) are shown as strong positive
controls. C, interaction of Id proteins with the
hematopoietic factors. E2-2 (also expressed as a binding domain fusion)
is shown as a positive control. The values shown represent the average
of three independent determinations (with standard errors of <10% in
each case). Expression of each protein was independently verified by Western blotting (Fig. 2).
[View Larger Version of this Image (29K GIF file)]
Fig. 2.
Western blot analysis of yeast 2-hybrid
fusion proteins. A, Id proteins expressed in pGBT9 were
detected with an anti-gal4 DNA binding domain antibody.
B-D, expression of the hematopoietic factors, E proteins,
and MRFs, expressed in pGAD424, were detected with an anti-gal4
activation domain antibody. Untransformed Y153 lysates were used as
controls. A size marker (in kilodaltons) is shown. In other experiments
(not shown), we have determined that the levels of proteins shown here
do not change when they are co-expressed with other bHLH member
proteins.
[View Larger Version of this Image (35K GIF file)]
-galactosidase detected), whereas MRF4/Myf-6 homodimerization was
not observed. Neither the Ids nor the hematopoietic factors displayed
discernible homodimerization ability in this assay (data not
shown).
Fig. 3.
Co-immunoprecipitation of in
vitro translated full-length proteins. E12 (A),
MyoD (B), MRF4/Myf-6 (C), and Scl/Tal-1 (D) programmed reticulocyte lysates were incubated with Id1,
Id2, and Id3 programmed lysates. Protein complexes were captured with anti-E12, MyoD, or Id polyclonal antibodies and reactions resolved by
12% SDS-PAGE. Quantitative determination of "captured" protein relative to Id was performed by PhosphorImage analysis, and values were
normalized for methionine content. Determinations were performed at
least twice, and representative graphs are shown.
[View Larger Version of this Image (26K GIF file)]
Fig. 4.
Mammalian 2-hybrid analysis. A, an
interaction between an MRF (expressed in pNLVP16) and an Id protein
(expressed in pSG424) will result in the activation of a CAT reported
gene bearing tandemly repeated gal4 binding sites in its promoter.
B, HLH domains of Id1, Id2, and Id3, expressed as gal4 DNA
binding domain (BD) fusions, were tested for their ability
to interact with MyoD, Myf-5, or E47 expressed as VP16 transactivation
domain (TAD) fusions. CAT conversion over background was
determined by PhosphorImage analysis. Values shown represent the
average of three independent experiments with standard errors of less
than 10%.
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Disruption of E2A·MyoD complexes by
Ids. Ids were tested in their ability to disrupt E47·MyoD
complexes (dark shading) or E12·MyoD complexes
(light shading). Values are normalized relative to
MyoD·E47 conversion. The average of at least three experiments is
shown.
[View Larger Version of this Image (33K GIF file)]
Fig. 6.
Id protein helix swaps and site-directed
mutagenesis. A series of Id hybrid molecules were tested in their
ability to bind all four MRFs and E12 using a quantitative yeast
2-hybrid assay, performed in triplicate. The amount of
-galactosidase enzyme generated per 107 cells (ng) for
each set of interactions is shown in the right hand columns.
Standard errors were less than 5% in each case.
[View Larger Version of this Image (35K GIF file)]
Fig. 7.
Mammalian 2-hybrid interactions of MyoD and
Myf5 with select Id1 and Id3 mutants. The pNLVP vectors used were
the same as those described in Fig. 6. The Id1/3, Id3/1, and Id1D80-85 mutant HLH domains shown in Fig. 6 were amplified by PCR and cloned into the pSG424 vector. The indicated amounts of each plasmid along
with pGal5E472CAT (5 µg/plate) and a CMV-gal (2 µg/plate) were
transfected into HeLa cells using a calcium phosphate precipitation procedure and assayed for
-galactosidase and CAT 2 days later.
[View Larger Version of this Image (16K GIF file)]
4, 1.5 × 10
5, and 1.9 × 10
6 M, respectively (60). These differences
are remarkably consistent with the values of 0.05, 1.5, and 24 ng of
-galactosidase obtained for these interactions in our study.
Similarly, Estojak et al. (63) reported that high,
intermediate, and low level interactions, determined in a
semi-quantitative yeast 2-hybrid system, accurately reflected
calculated Kd values (63). Other yeast 2-hybrid analyses have shown that Id1 and the class B factors MyoD and Scl/Tal-1
bind to the class A factors E2-2 and E12 (43, 44). Loveys et
al. (35) demonstrated the ability of Id3 to interact with E12,
E47, HEB, and MyoD both in yeast and in vitro, observing that Id3 interacted strongly with each E protein but with reduced ability to MyoD (35). They were also able to show that Id3 could repress E protein-mediated transcription from a multimerized
immunoglobulin E box reporter. Id1 and Id2 bind at low levels to MyoD
and very weakly, if at all, to Scl/Tal-1 as demonstrated by co-IP and
glutathione S-transferase-pull down analysis (28, 34, 41).
Mammalian 2-hybrid approaches have also demonstrated the ability of Id1 to interact with both MyoD and E12 and of Scl/Tal-1 to interact with
E47 (56, 64). These reports concur with the general observation that
class A-class B and Id-class A complexes are more stable than Id-class
B complexes. Our study not only confirms and extends previous reports
of HLH protein dimerization potentials but attempts to provide a
consistent comparison of a broad range of interactions.
*
This work was supported by National Institutes of Health
Grant HL33741 (to E. V. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Section of
Hematology/Oncology, Children's Hospital Medical Center, 3705 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-6796; Fax:
412-692-5723.
1
The abbreviations used are: bHLH,
basic-helix-loop-helix; CAT, chloramphenicol acetyltransferase; MCK,
muscle creatinine kinase; MRF, myogenic regulatory factor; IP,
immunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
CMV, cytomegalovirus.
2
K. Langlands, X. Yin, G. Anand, and E. V. Prochownik, unpublished data.
3
K. Langlands and E. V. Prochownik,
unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.