(Received for publication, June 18, 1996, and in revised form, October 8, 1996)
From the Department of Pharmacology, University of Missouri, School of Medicine, Columbia, Missouri 65212
We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor, ITF-2b, that interacts with the transcriptional inhibitor Id3/HLH462. The interaction was confirmed in vitro, and inside intact myogenic C2C12 cells. As expected, overexpression of either Id3/HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD. However, when overexpressed simultaneously, ITF-2b and Id3/HLH462 counteracted each other's inhibitory effect to produce a reduced overall inhibition. Moreover, while ITF-2b inhibited the creatine kinase promoter, it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site. Further investigation indicated that the ITF-2b/MyoD heterodimer bound to its specific DNA binding site in vitro, and the DNA binding was effectively blocked by Id3/HLH462. Additional analysis revealed the presence of transcripts for both the activating (ITF-2a) and inhibitory (ITF-2b) isoforms in differentiating C2C12 cultures, suggesting that both isoforms might participate in regulating the differentiation process. Taken together, this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated.
The basic helix-loop-helix (bHLH)1
family of transcription factors have been shown to play an important
role in regulating muscle-specific gene expression (1, 2). Myogenic
bHLH factors heterodimerize with ubiquitously expressed bHLH proteins
(referred to as E proteins) and transactivate gene expression through
binding to consensus DNA binding sites (E boxes) in the promoter region of various muscle-specific genes (3, 4). In contrast, the Id family
proteins, which contain an HLH domain but lack the DNA-binding basic
region (5-8), interact preferentially with the E protein family
members (8) and sequester them from dimerization with the
tissue-specific bHLH factors (5-8), thereby inhibiting gene expression
(5). More recent studies have also demonstrated the existence of a new
group of bHLH proteins that contain the basic and the HLH domain but
nevertheless function as transcriptional inhibitors. One of these
inhibitory bHLH proteins, BETA3, interacts with E proteins to form
heterodimers that are incapable of DNA binding (9). Recently, an
alternatively spliced variant of the E protein ITF-2, ITF-2b, was
identified and shown to inhibit the MyoD-mediated transactivation of
the cardiac -actin promoter (10), but the exact mechanism
responsible for this inhibition has not yet been directly tested. Here
we reported the independent and accidental cloning by the yeast
two-hybrid system of a full-length ITF-2b cDNA while we were
searching for protein factors that interact with the Id protein,
Id3/HLH462. We have confirmed that Id3/HLH462 and full-length ITF-2b
physically interacted with each other in vitro and
complemented each other in a mammalian two-hybrid assay in the myogenic
C2C12 cell line. Interestingly, although ITF-2b exhibited a
dose-dependent inhibitory effect on the muscle creatine kinase (MCK) promoter in C2C12 cells, it functioned as a transactivator on an artificial promoter containing three tandem E box sites. Moreover, when both Id3/HLH462 and ITF-2b were co-expressed, their combined inhibitory effect on the MCK promoter was attenuated. Gel
shift studies revealed that the ITF-2b/MyoD heterodimer formed a strong
DNA binding complex comparable with that formed by the MyoD/E47
heterodimer, but the formation of both heterodimer-DNA complexes was
effectively inhibited by Id3/HLH462. Analysis of ITF-2 and Id3/HLH462
gene expression revealed that transcripts of both activating (ITF-2a)
and inhibitory (ITF-2b) isoforms were present initially in C2C12 cells
placed under differentiation-promoting conditions, while the expression
of Id3 was high in proliferating cells and declined as the culture
differentiated. The potential implication of the expression pattern of
ITF-2b and its interaction with Id3/HLH462 in the regulation of
terminal myogenesis was discussed.
An Id3/HLH462 cDNA containing the complete open reading frame was removed from the pHLH462 plasmid (ATCC, Rockville, MD) with EcoRI/XhoI digest and subcloned into the EcoRI/SalI site of the GAL4 DNA binding vector, pGBT9 (Clontech, Palo Alto, CA) to form pGBTHLH. An 11-day mouse embryo cDNA library in pGAD10 vector (Clontech) was screened according to the manufacturer's protocol to select for genes whose products interact with Id3/HLH462.
Cell CultureMouse myogenic C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and gentamycin as described previously (11). Cell differentiation was induced by changing to DMEM + 5% horse serum.
Transient TransfectionA cDNA containing the complete
open reading frame of ITF-2b, corresponding to nucleotides 506-2613 of
the published sequence (10) was removed from pGAD10 by EcoRI
digest and subcloned into the pEMSV mammalian expression vector (12).
An XhoI fragment of Id3/HLH462 from pHLH462 was
blunt-end-ligated into the blunted EcoRI site of pEMSV
vector to form the Id3 expression plasmid. The p3ESVCAT reporter
construct was made by inserting three copies of the E box DNA binding
site (5) into the BglII site of a pCAT-Control plasmid
(Promega, Madison, WI) with the 3 SV40 enhancer sequence removed by
HincII digest. The MCKCAT reporter gene construct contained
a 3300-base pair promoter region of the muscle creatine kinase gene
(13). Various amount of constructs as described in the individual
figure legend was transfected into C2C12 cells cultured on 100-mm
dishes by calcium phosphate (14), and the cells were incubated for 3 days in differentiation-permissive medium (DMEM + 5% horse serum)
after transfection before harvesting. Chloramphenicol acetyltransferase
(CAT) assay was done essentially as described (14).
The ITF-2b cDNA
containing the complete open reading frame was removed as a
SalI restriction fragment from the pGAD10 vector and
subcloned into the XhoI site of pBlueScript (Strategene, La Jolla, CA) to form pBSITF-2b. The FLAGHLH462 plasmid was made by
polymerase chain reaction to insert an antigenic FLAG epitope (15) to
the N-terminal of the full-length Id3/HLH462 protein. FLAGHLHH2 was
made by removal of a PstI/NarI fragment from the HLH462 plasmid to partially delete the second helix domain. All proteins were made using the TNT®-coupled in vitro
transcription/translation reticulocyte lysate system (Promega) and
labeled with [3H]leucine. Immunoprecipitation was carried
out in buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.2% Triton X-100, using the M2 anti-FLAG
antibody (Eastman Kodak Co.)
A fragment containing the complete open reading frame of ITF-2b was removed from the pGAD10 vector by EcoRI digestion and cloned into the EcoRI site of the mammalian expression vector pVP16 (Clontech). An Id3/HLH462 fragment containing the entire protein coding region was cloned into the EcoRI/SalI site of the pM vector containing the GAL4 DNA binding domain (Clontech). C2C12 cells were transfected with either or both of these vectors together with the G5CAT mammalian reporter plasmid to determine their interactions in intact cells. The assay was performed according to the manufacturer's protocol.
Gel Shift AssayThe assay was carried out as described (5) using in vitro translated proteins produced by TNT®-coupled transcription and translation. The oligonucleotide used in the assay corresponds to the consensus MEF1 binding site (13).
RT-PCR Analysis of mRNA Expression of ITF-2a, ITF-2b, and Id3/HLH462Total RNAs were isolated from differentiated C2C12
cells at day 0, 2, 4, 6, 8 by the guanidinium
isothiocyanate-phenol-chloroform extraction method (16). First strand
cDNA was made by reverse transcription at 37 °C for 2 h and
used for PCR amplification using primers that are specific to ITF-2a,
ITF-2b Id3, or myogenin. Amplification was first performed with
-actin primers to normalize the amount of cDNA present in each
preparation, and the normalized values were used to adjust for the
amount of templates used in the PCR reaction with the gene specific
primers. The primer sets specific to each gene for PCR were listed as
follows.
ITF-2a: | 5![]() ![]() |
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ITF-2b: | 5![]() ![]() |
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Id3/HLH462: | 5![]() ![]() |
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Myogenin: | 5![]() ![]() |
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A typical PCR profile used was: 94° 30 s,
59 °C 30 s, 72 °C 1 min for 27 cycles for ITF-2a and ITF-2b,
25 cycles for Id3/HLH462 and myogenin, and 20 cycles for -actin,
followed by extension at 72 °C for additional 10 min.
We have used the yeast two-hybrid interaction cloning strategy to
identify genes whose products can interact with Id3/HLH462. Out of
about 4 × 106 yeast clones co-transformed with GBTHLH
plasmid and the pGAD10 mouse embryonic cDNA library, 95 clones grew
in the absence of histidine, among which 51 clones showed
-galactosidase activity. Further analysis confirmed 32 clones that
showed Id3/HLH462-dependent galactosidase expression.
Sequence analysis revealed that all are members of the E protein family
(Table I). One of these clones appeared to correspond to
a novel splicing variant of ITF-2, ITF-2b, recently isolated by the
Mcburney's laboratory and reported to exhibit the unusual property of
being able to inhibit the MyoD-mediated transactivation of
-cardiac
actin gene expression (10). The clone that we isolated contains the
complete reading frame of ITF-2b fused to the GAL4 transactivation
domain at nucleotide position 506 of the published sequence and ends in
the 3
-untranslated region at position 2613 (10).
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Since the apparent interaction of two putative inhibitory HLH proteins
was unexpected, we wanted to confirm the association of the two
proteins under other assay conditions. First, we examined by
co-immunoprecipitation experiment to see if in vitro
translated Id3/HLH462 and ITF-2b proteins could interact with each
other without the attached GAL4 DNA binding and transactivation domains used in the yeast two-hybrid assay. Using an antibody directed against
the FLAG antigenic epitope, we showed that ITF-2b was co-precipitated
with the FLAG-tagged Id3/HLH462 protein. A second helix deletion mutant
of Id3 (FLAGHLHH2), which exhibited a dramatically reduced
inhibitory effect on MCK gene expression,2
co-precipitated ITF-2b to a much lower extent (Fig.
1A). An unrelated protein, luciferase, was
not precipitated at all by the anti-FLAG antibody. The result indicates
that the interaction of ITF-2b and Id3 is specific and may be dependent
on an intact HLH domain in Id3. The relatively small amount of ITF-2b
co-precipitated in comparison with the amount added may reflect the
inefficiency of the in vitro assay system or steric
hindrance by the precipitating antibody.
As a further confirmation of the ability of ITF-2b to associate with Id3, we also analyzed the interaction between these two proteins using the mammalian two-hybrid systems. As shown in Fig. 1B, full-length ITF-2b fused to the VP16 transactivation domain was able to complement an Id3/GAL4 DNA binding domain fusion protein to transactivate a GAL4-regulated promoter in transfected C2C12 cells. The resulting CAT activity was 50-200-fold higher than what was obtained with expression vectors lacking one or the other of the interacting proteins and reached about 20% the level obtained with the covalently linked Gal4-VP16 fusion protein. Thus, the two inhibitory proteins could associate inside mammalian cells with affinity that is consistent with a specific noncovalent interaction.
Next we want to confirm that ITF-2b could indeed inhibit
muscle-specific gene expression as reported previously (10) and to
examine the potential consequence when two mutually interacting inhibitory HLH proteins were co-expressed in the same cell. We first
demonstrated that both Id3/HLH462 and ITF-2b could inhibit the ability
of MyoD to transactivate the expression of a muscle-specific creatine
kinase (MCK) promoter-regulated CAT reporter gene in a
dose-dependent manner (Fig. 2).
Interestingly, when ITF-2b and Id3/HLH462 were co-expressed, the
inhibitory effect on MCK gene expression was attenuated relative to
what was observed when each protein was expressed by itself. This
apparent mutual interference of inhibitory activity presumably was due
to the formation of Id3/HLH462 and ITF-2b complexes that were no longer
capable of inhibiting MCK gene expression.
In order to determine the mechanism through which ITF-2b might inhibit
MCK promoter activity, we examined whether ITF-2b protein could form
homo- or heterodimers that bind to the consensus MyoD/E protein binding
site (E box). As shown in Fig. 3, only a faint DNA-protein complex was detectable when the labeled oligonucleotide was
incubated with in vitro translated ITF-2b protein alone,
suggesting that ITF-2b either could not homodimerize efficiently or
that the homodimer could not bind DNA well. When ITF-2b was
co-incubated with the activating E protein, E47, the mobility of the
shifted complexes that were formed resembled those formed by ITF-2b and E47 homodimers, suggesting that the ITF-2b/E47 heterodimer might bind
DNA with relatively low affinity. That ITF-2b-E47 heterodimers were
formed was supported by the fact that the presence of the ITF-2 protein
resulted in a much reduced level of apparent E47 homodimer/DNA
complexes as compared with that seen with E47 alone. In contrast to
these weak interactions, ITF-2b/MyoD heterodimers formed a very strong
DNA complex comparable to the E47/MyoD heterodimer DNA complex, but the
formation of both complexes was still efficiently blocked by
Id3/HLH462. Taken together, our data indicate that ITF-2b did not
inhibit MyoD function because of failure to bind DNA.
Unlike ITF-2b, the human ITF-2 protein (also called E2-2) with the
first 49 amino acid missing from its N-terminal was reported to
cooperate with MyoD to transactivate an artificial reporter gene,
(MEF1)4CAT, containing four tandem MyoD binding MEF1 sites (4). Since the inhibitory activity of ITF-2b appeared to reside in the
N-terminal first 83 amino acids (10), it was assumed that the
differential activity of the two proteins was due to the absence of the
49 amino acids. To test this assumption, we also examined the effect of
the full-length ITF-2b protein on the MyoD-mediated transactivation of
a reporter gene containing three copies of the MEF1 sites (3ESVCAT).
Surprisingly, whereas Id3/HLH462 remained fully inhibitory on this
reporter gene, the full-length ITF-2b protein was no longer inhibitory
but instead enhanced the transcriptional activity of MyoD (Fig.
4). It thus appeared that ITF-2b-MyoD heterodimerization
did not always result in a transcriptionally silent DNA binding
complex.
While the preceding data provided clear evidence for the biochemical
function of the ITF-2b protein, the biological relevance of this
protein to the muscle differentiation process could not be inferred
from these results alone. To explore this question further, we have
analyzed the mRNA expression patterns of both the activating and
"inhibitory" ITF-2 isoforms (ITF-2a and ITF-2b) by RT-PCR during
the in vitro differentiation of the myogenic C2C12 cells.
Results of this assay indicated that both transcripts were absent in
proliferating cells but became detectable as the cells were placed into
differentiation-permissive conditions (Fig. 5). In
contrast, the Id3 gene was expressed in proliferating C2C12 cells and
down-regulated as the cultures were allowed to differentiate. Whether
both Id3 and ITF-2b proteins are co-expressed in the same cells at some
point during the differentiation process is an interesting question
that would need to be confirmed when appropriate antibodies become
available.
We reported in this manuscript the cloning of an inhibitory form of the murine ITF-2 protein, ITF-2b, by virtue of its interaction with another transcriptional inhibitory protein Id3. The interaction was confirmed by in vitro co-immunoprecipitation analysis and DNA binding assay, by functional complementation in the mammalian two-hybrid system and by mutual antagonism in the MCK reporter gene assay. Moreover, our data revealed that the inhibitory function of ITF-2b was evident on the MCK reporter gene but not with an artificial promoter regulated by the MEF1 (E box) DNA binding site. Interestingly, ITF-2b mRNA was not detectable in proliferating myoblasts but became observable in cells initiating the differentiation process.
One possible explanation for the rather complex behavior of the ITF-2b
protein is that the inhibitory domain in ITF-2b might prevent the
myogenic factor-ITF-2b protein heterodimer from interacting with
transcription factors bound to other regulatory elements in the MCK
enhancer that are required for maximal MCK activation. Such
interactions might be dispensable in the case of the artificial promoter regulated solely by multiple E boxes. The inhibitory effect on
the cardiac -actin promoter could be explained if similar interactions were required for its full activation. Thus depending on
the constellation of factors that are needed to activate the expression
of a particular gene, ITF-2b might exert either inhibitory or
activating influences in a promoter-specific manner. It would be very
interesting to know whether ITF-2b will inhibit or activate other
muscle-specific genes as well as other tissue-specific genes that
depend on bHLH proteins for transcriptional activation (17).
The reason for the enigmatic increase in the transcripts of the inhibitory ITF-2b isoform when cells were first exposed to differentiation-inducing conditions is not clear. One possibility is that it might serve to prevent cells from initiating the differentiation process prematurely in response to small fluctuations in the extracellular environment. This inhibitory effect would be overcome when the expression levels and activities of the myogenic regulatory factors and the activating E proteins became sufficiently elevated and the levels of the inhibitory Id proteins declined. Alternatively, the expression of the ITF-2b isoform in differentiating muscle cells might allow different muscle-specific genes to be regulated independently in a promoter-specific manner. Which if either of these speculative models are accurate can only be borne out by further investigations. Taken together, our data presented here painted a more complex picture concerning the regulatory interactions between the HLH proteins than previously recognized. The complete implication of these interactions remains to be elucidated.
We acknowledge Xianwu Li for help with the yeast two-hybrid cDNA library screening and Melissa Nevils for help with the RT-PCR.