University of Queensland (P.B., M.D., P.L., J.H., S.L.C.,
G.E.O.M.) Centre for Molecular and Cellular Biology
Ritchie Research Laboratories, B402A St Lucia, 4072 Queensland,
Australia
University of Southern California (Y.H., V.S.)
Institute of Genetic Medicine No. 140 Los Angeles, California
90033
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ABSTRACT |
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INTRODUCTION |
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Cofactors function as bridges between DNA-binding proteins and the
basal transcriptional machinery. The identification of corepressors
(i.e. N-CoR/RIP13 and SMRT/TRAC) that interact with the
thyroid hormone receptor, retinoic acid receptor, and vitamin D
receptor (TR, RAR, and VDR) has shed some light on the mechanism of
transcriptional repression (by classical nuclear receptors) in the
absence of ligand (12, 13, 14). The C-terminal receptor interaction domain
(RID) of the corepressors interacts with the ligand binding
domain/DE region of unliganded receptors. This interaction
induces a series of protein-protein interactions that repress
transcription. The corepressors contain two interaction domains that
interact independently and synergistically with the nuclear receptors
(15, 16). These corepressors interact with Sin3 and recruit the histone
deacetylases (HDAc-1 or Rpd-3/HDAc-2) that lead to hypoacetylation of
the histones. This deacetylation leads to conformational changes that
stabilize the nucleosome structure, and limit the accessibility of the
chromatin to the transcriptional machinery (17, 18, 19). The
N-CoR/Sin3/HDAc complex mediates transcriptional repression from a wide
variety of other non-receptor-mediated pathways including the
bHLH-LZ proteins, Mad/Mxi, that mediate repression of myc
activities and tumor suppression (18), retinoblastoma-mediated
repression of E2F action (20, 21), and the oncoproteins PLZF-RAR
(22, 23) and LAZ3/BCL6 (24), which are involved in acute promyelocytic
leukemia and non-Hodgkin lymphomas, respectively. These receptor- and
non-receptor-mediated pathways share common attributes: they are
converted in response to environmental stimuli from the repressive to
the operative condition and function in the management of
differentiation and cell division.
N-CoR and the splice variants (RIP13a and RIP131) mediate
transcriptional repression by the orphan nuclear receptors, Rev-erbA
(15, 25), RVR (15, 25), and chick ovalbumin upstream promoter
transcription factor II (COUP-TF II) (26). The carboxy-terminal
regions of N-CoR and RIP13 that encode the receptor interaction domains
are almost identical. There is one major difference between the RIP13s
and N-CoR. The first 1016 amino acids of N-CoR that encode repression
domains 1 and 2 are replaced by 10 unique amino acids at the
amino-terminal end of the RIP13s (Ref. 16 and Fig. 1
). However, the RIP13s contains seven copies
of a repeated motif, G-s-l-s/t-q-G-t-P, which is associated with
repressor activity. Interaction of the Rev-erb
/ß orphan nuclear
receptors with the corepressors requires an intact ligand-binding
domain (15, 25, 26), and physical association between the corepressors
and the nuclear receptors is dependent on two corepressor interaction
regions, located in helix 3 and helix 11, that probably form a
corepressor interface in three-dimensional space (25).
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The function of N-CoR and the multiprotein corepressor complex in the process of cellular differentiation, tissue-specific transcription, and coupled phenotypic acquisition remain to be resolved. This study focuses on the functional role of N-CoR during myogenesis, a well characterized paradigm of mammalian differentiation, and investigates the mechanisms used by N-CoR in the repression of MyoD activity (a master regulatory protein) and regulation of differentiation.
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RESULTS |
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To examine the expression levels of N-CoR, RIP13a, and RIP131 mRNA
in muscle cells, Northern analysis of RNA isolated from proliferating
C2C12 myoblast cells was undertaken. The cDNA probe used in the
Northern analysis corresponded to the carboxy-terminal RID of N-CoR, a
region that is highly conserved between all three transcripts
(i.e. N-CoR, RIP13a, and RIP13
1; Fig. 1A
). It was
observed that only one
9-kb mRNA transcript was expressed in
proliferating C2C12 myoblasts. This
9-kb band corresponds to N-CoR
mRNA. The N-CoR variants, RIP13
1 and RIP13a, should produce mRNA
transcripts around 4.5 kb (16); however, transcripts of this size were
not detected, indicating that C2C12 cells only express N-CoR mRNA (Fig. 1A
) or that the RIP13s were very rarely expressed.
To gain some insight into the role played by N-CoR during the process
of myogenesis, we initially investigated the expression pattern of
N-CoR mRNA during the conversion of mouse myoblast C2C12 cells into
multinucleated myotubes. Proliferating C2C12 myoblasts were induced to
biochemically and morphologically differentiate into postmitotic
multinucleated myotubes by serum withdrawal in culture over a 24- to
72-h period. The transition from a nonmuscle phenotype to a contractile
phenotype is associated with the activation/expression of 1) the
myoD gene family (myoD, myogenin, myf-5, and
MRF-4), and 2) a structurally diverse group of genes that encode a
functional sarcomere responsible for contraction. Concomitant with
these events is terminal cell cycle exit, characterized by the
repression of cyclin D1 and the activation of the cell cycle inhibitor,
p21. Total RNA was isolated from proliferating myoblasts (PMB),
confluent myoblasts (CMB), and postmitotic myotubes after 4, 8, and
24 h of serum withdrawal and examined by Northern blot analysis.
N-CoR mRNA was abundantly expressed in myoblasts; however, this
transcript is suppressed, relative to 18S rRNA, as myoblasts exit the
cell cycle and fuse to form differentiated multinucleated myotubes that
have acquired a muscle-specific phenotype (Fig. 1B). Down-regulation of
N-CoR mRNA correlated with the recent observations that the mRNAs
encoding the three orphan nuclear receptors, Rev-erbA
(29), RVR
(30), and COUP-TF II (31), are repressed during myogenesis. These
orphans have been demonstrated to antagonistically regulate myogenesis,
repress myoD mRNA expression, abrogate the induction of
myogenin and p21 mRNA after serum withdrawal, and interact
with N-CoR to repress transcription (15, 25, 26). Concomitant with this
decrease in N-CoR mRNA was the induction of myogenin and p21 mRNAs,
relative to 18S rRNA, which confirmed that these cells had terminally
differentiated (Fig. 1B
). The repression of cyclin D1 and the inhibitor
of differentiation (Id) (relative to the equivalent levels of 18S rRNA)
confirmed that these cells were exiting the cell cycle. The expression
of MyoD mRNA in myoblasts and myotubes confirmed the myogenic nature of
these cells (Fig. 1B
). The differential expression of N-CoR mRNA
suggested that this cofactor may regulate the process of
differentiation and/or the maintenance of the proliferative cycle.
N-CoR Expression in Myogenic Cells Blocks the Induction of
Myogenin and p21 mRNA and Suppresses MyoD mRNA
The Northern analyses demonstrated that repression of the N-CoR
mRNA correlate with the biochemical and morphological differentiation
of myogenic cells, concomitant with the acquisition of a contractile
phenotype. To further delineate the role played by N-CoR in myogenesis
and to identify the target(s) of this corepressor in muscle, we
proceeded to examine the effect of exogenous N-CoR expression on C2C12
cell differentiation.
Hence, C2 muscle cells were cotransfected with plasmids encoding
full-length N-CoR (pSG5-N-CoR) and the G418 resistance gene, Neomycin
(pCMV-NEO). Stable transfectants were then isolated as a polyclonal
pool of G418-resistant colonies (comprised of >20 individual
resistant colonies). For reference, this pool of cells is denoted as
C2:N-CoR. To examine the effect of exogenous N-CoR expression on
factors involved in determination (e.g. MyoD), cell cycle
regulation (e.g. cyclin D1 and p21), and differentiation
(myogenin), we again isolated total cytoplasmic RNA from C2:N-CoR and
normal C2C12 cells before and 72 h after serum withdrawal. After
blotting, the RNA was probed with 18S rRNA, MyoD, myogenin,
cyclin D1, and p21-labeled cDNAs (Fig. 2A).
Comparisons of C2:N-CoR to native C2C12 cells showed that the level of
myoD mRNA in the presence and absence of serum was significantly
reduced in the C2:N-CoR cell line. Furthermore, the induction of the
p21 and myogenin mRNAs after serum withdrawal was completely ablated in
these cells (Fig. 2A
). Interestingly, exogenous expression of N-CoR did
not effect the repression of cyclin D1 after serum withdrawal,
relative to the equivalent levels of 18S rRNA (Fig. 2A
). The lack of
the tissue-specific bHLH master regulators, MyoD and myogenin, and the
cyclin-dependent kinase inhibitor, p21Waf-1/Cip-1, in the
C2:N-CoR cells after 72 h of serum withdrawal correlates with the
inability of this cell line to differentiate morphologically (Fig. 2A
).
In summary, the data above suggest that N-CoR regulates the
transcription/expression of the muscle-specific HBH genes (that are
auto-regulated by their own expression) and functions as a negative
regulator of myogenic differentiation.
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C3H10T1/2 cells were transiently transfected with pSG5-MyoD in
combination with either pSG5 (vector/vehicle only) or pSG5-N-CoR. As
expected, cells transfected with pSG5-MyoD were found to be positively
stained with the MHC antibody (Fig. 2B). In contrast, cells that were
cotransfected with both MyoD and N-CoR did not undergo myogenic
conversion (Fig. 2B
). The experiments presented above suggest that the
cofactor, N-CoR, is involved in the inhibition of myoD-mediated
myogenic conversion of pluripotent C3H10T1/2 cells. This repression of
MyoD action is independent of the silencing of its gene expression in
this system; hence, we suggest that N-CoR directly regulates the action
of MyoD by posttranscriptional mechanisms. In summary, we propose that
N-CoR functions as corepressor of the myogenic specific, bHLH protein,
MyoD.
N-CoR Inhibits MyoD-Mediated Transactivation
The experiments presented above suggest that N-CoR directly
regulates MyoD action in vivo by a mechanism that is
independent of MyoD gene expression. To confirm this, we examined the
effect of N-CoR expression on MyoD-mediated transactivation in the GAL4
hybrid system. In these assays the activity of MyoD is independent of
its binding to its cognate binding motif, the E-Box (CANNTG). If N-CoR
directly regulates MyoD transcriptional activity, then the potential of
GAL4-MyoD to transactivated gene expression should be greatly reduced
in this assay (Fig. 3A).
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We similarly examined the ability of N-CoR to repress the
activity of Gal4-MyoD in myogenic C2C12 cells. Again, N-CoR silenced
GAL4-MyoD-mediated transactivation (Fig. 3C). Subsequently, we
investigated whether N-CoR could repress MyoD-dependent E-box-driven
transcription in myogenic cells. To achieve this, we used a 4RE-tkLUC
reporter construct, in which four copies of the right E-box of the
muscle-specific MCK enhancer (a cognate binding site for MyoD) are
placed upstream of a minimal thymidine kinase (tk) promoter linked to
the LUC reporter gene. This reporter has been demonstrated to
facilitate MyoD-dependent transactivation. C2C12 muscle cells were
cotransfected with the 4RE-tkLUC reporter in combination with either
the pSG5 or pSG5-N-CoR vectors. Cotransfection of C2C12 cells with
pSG5-N-CoR significantly repressed (
5-fold) the expression of the
4RE promoter in myogenic cells Fig. 3D
). In summary, we observed that
MyoD-mediated transactivation in the presence and absence of its
cognate binding motif is silenced in muscle and nonmuscle cells by the
cofactor, N-CoR. We hypothesized that N-CoR could function as a
corepressor of MyoD activity/function.
We used an alternative approach to confirming the functional role of
the N-CoR complex in the regulation of MyoD activity and to gain
insight into the mechanism of N-CoR-mediated repression. We examined
the ability of the histone deacetylase inhibitor, trichostatin A (TSA,
100 ng/ml), to regulate MyoD-mediated transactivation. We initially
examined the affect of TSA treatment on the activity of Gal-MyoD in
myogenic C2 cells. We observed that TSA treatment increased the
activity of MyoD by approximately 6-fold (Fig. 3E). Furthermore,
consistent with the role of the corepressor-HDAc-1 complex in the
regulation of MyoD activity, TSA treatment blocked the ability of N-CoR
to repress the MyoD-dependent reporter, 4RE (Fig. 3F
). Our data
indicated that one mechanism by which N-CoR represses MyoD-mediated and
-dependent transcription involves the recruitment of histone
deacetylases.
N-CoR Directly Interacts with MyoD: The Amino-Terminal Repression
Domain (RD1) of N-CoR Mediates the Interaction and Is Required for
Repression of Myogenesis
We postulated that N-CoR could be functioning to repress MyoD
activity/function by directly interacting with MyoD. To determine this,
we used the in vitro glutathione-S-transferase
(GST) pull-down assay, in which glutathione agarose-immobilized GST and
GST-MyoD proteins were incubated with in vitro35S-radiolabeled full-length N-CoR (Fig. 4A). This assay clearly showed that N-CoR and
MyoD directly interact in vitro (Fig. 4B
).
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To examine whether the repression domain of N-CoR is critical for the
regulation of myogenesis and bHLH gene expression in vivo,
we examined the effect of exogenous RIP131 expression in muscle
cells. RIP13
1 is an N-CoR variant that is almost identical to N-CoR,
except it lacks the first 1017 amino acids of N-CoR, which encode the
two amino-terminal repression domains and facilitate the interaction
with MyoD. The biochemical profile of C2C12 cells that express
exogenous RIP13
1 mRNA (denoted C2:RIP13
1) was compared with
normal C2C12 cells via Northern analysis of mRNAs that encode the key
regulators and markers of myogenic differentiation. Total RNA from both
C2:RIP13
1 and normal C2 cells was isolated from PMB, CMB, and
myotubes after 4, 8, 24, and 72 h of serum withdrawal. RNA samples
were blotted and probed with 32P-labeled cDNA encoding 18S
rRNA, MyoD, Myogenin, Cylin D1, ß-actin, p21cip-1/waf-1,
and N-CoR. It should be noted that the C2:RIP13
1 cell line expresses
the exogenous RIP1
1 mRNA at levels similar to or greater than the
endogenous N-CoR transcript. When compared with normal C2C12 cells, the
C2:RIP13
1 cells exhibited a similar biochemical profile of mRNA
expression before and after serum withdrawal (Fig. 4D
). Specifically,
they expressed MyoD before and after serum withdrawal. The myogenin and
p21 mRNAs were induced after serum withdrawal, whereas the N-CoR,
ß-actin, and cyclin D1 mRNAs were all repressed. This demonstrated
that the phenotype of these cells was not affected by RIP13 expression.
This strongly suggested that the affect of N-CoR on myogenic
differentiation is mediated by the amino-terminal repression domain of
the cofactor, which specifically interacts with MyoD. Furthermore, this
region is lacking in the RIP13 variants, which have no affect on
myogenesis after stable transfection, and suggests that the change in
phenotype induced by N-CoR expression is not due to the selection
process.
In summary, the data suggest that the amino-terminal region of the corepressor, N-CoR, which encodes the repression domain, directly interacts with MyoD in a functional manner to regulate its activity during myogenesis.
The bHLH Region of MyoD Mediates the Interaction with N-CoR: The
bHLH Domain Functions as a Repressor
To delimit the region within MyoD (see Fig. 5A) that interacts with N-CoR, we examined
the ability of a number of GST-MyoD fusion chimaeras containing
functional subdomains of MyoD and immobilized on glutathione agarose
beads [i.e. GST-MyoD (aa 1318), GST-N terminal MyoD (aa
1100), GST-bHLH (102161), and GST-C terminal MyoD (aa 162318)]
to interact with the 35S-labeled N-CoR (see Fig. 5A
). As
expected, full-length MyoD interacted with N-CoR. The bHLH region of
MyoD linked to GST also interacted strongly with in vitro
translated N-CoR (Fig. 5A
). In contrast, the GST-MyoD N-terminal and
C-terminal regions did not support any significant interaction with
N-CoR. This suggests that the bHLH domain of MyoD (aa 109162) is
required for N-CoR binding.
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This set of experiments demonstrate that N-CoR directly interacts with the bHLH region of MyoD and that the bHLH region of MyoD encodes a minimal repressor domain and further supports the notion that N-CoR functions as a transcriptional corepressor of the MyoD protein.
To further demonstrate that the bHLH region mediates the repressive
activity in vivo, we examined the impact of deletions of the
basic or HLH domain in the context of full-length MyoD linked to GAL4
to regulate the transcription of the MH100-tk-LUC reporter. Consistent
with previous experiments, the bHLH region repressed transcription, in
contrast to the N-terminus of MyoD (GAL4-MyoD 'N') [Sartorelli
et al. (8) demonstrated that the N-terminal region encodes
an activation domain.] Interestingly, internal deletion of the basic
or HLH domains in the context of full-length MyoD compromised its
ability to repress the MH100-tk-LUC reporter (Fig. 5F). Interestingly
TSA treatment blocked the ability of Gal-MyoD-bHLH to repress the
MH100LUC reporter (Fig. 5G
), consistent with a mechanism that
involves the recruitment of histone deacetylases by N-CoR.
MyoD Shares Common Corepressor(s) with RAR : Expression of RAR
in the Absence of Ligand Squelches the Endogenous Corepressors
Retinoic acid receptor- (RAR
) in the absence of ligand binds
N-CoR and represses transcription (12, 13). The RAR ligand,
all-trans-RA, transforms RAR
from a transcriptional
repressor to a transcriptional activator. This transition is
characterized by the ligand-driven dissociation of N-CoR from RAR
.
To demonstrate that MyoD and RAR
share a common corepressor in
vivo (i.e. N-CoR), we performed squelching experiments.
Initially, JEG-3 cells were cotransfected with the MH100tk-LUC reporter
and GAL-MyoD bHLH in the presence and absence of the retinoic acid
receptor (Fig. 6A
). If MyoD and RAR
share
a common corepressor in vivo, then overexpression of RAR
should, at least in part, alleviate GAL-MyoD bHLH-mediated repression
of MH100tk-LUC basal transcription. We observed that overexpression of
RAR
(in the absence of ligand) reversed GAL-MyoD bHLH-mediated
repression, suggesting that RAR
and MyoD indeed share a common
corepressor in vivo (Fig. 6A
). In contrast, when the
experiment was repeated in the presence of the RAR ligand,
all-trans-RA [10-7 M] (Fig. 6B
),
overexpression of RAR
did not alleviate GAL-MyoD bHLH-mediated
repression (Fig. 6B
). Consistent with the RAR
not binding with N-CoR
(in the presence of ligand) and unable to squelch GAL-MyoD, bHLH
mediated repression. These results were consistent with the
demonstration that N-CoR binds and regulates the activity of the
hierarchical transcription factor, MyoD.
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DISCUSSION |
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The Cofactor, N-CoR, Represses Myogenic Differentiation
Specifically, our investigation demonstrated that the expression
of the mRNA encoding the cofactor, N-CoR, is repressed as proliferating
myoblasts exit the cell cycle and form postmitotic myotubes.
Furthermore, we observed that exogenous N-CoR expression (driven by an
SV40 promoter) in myogenic cell lines inhibited morphological
differentiation and resulted in lower steady state levels of MyoD mRNA
and a failure to activate myogenin and p21 mRNA expression after serum
withdrawal. These data demonstrate for the first time that N-CoR
functions as a physiologically relevant regulator of myogenesis and
provides direct evidence for the developmental role of the corepressor
during mammalian differentiation. Consistent with these observations,
our study showed that N-CoR could repress MyoD-mediated myogenic
conversion of pluripotential 10T1/2 cells. This suggests that N-CoR
functions as a corepressor of MyoD-mediated events.
Puri and co-workers (9, 11) and Sartorelli et al. (8) and others (10, 32) have demonstrated that exogenous p300 and PCAF expression potentiates MyoD-mediated transcription, stimulates differentiation, and promotes p21 expression and cell cycle arrest. Furthermore, the coactivators, p300 and PCAF, are abundantly expressed in differentiated muscle cells and promote MyoD-mediated conversion of pluripotent C3H10T1/2 cells (8, 11). These observations are consistent with our studies on the expression and function of the corepressor, N-CoR, in muscle and its contrasting effects on myogenesis. The opposing effects of p300/PCAF and N-CoR/Sin3B/HDAc-1 on MyoD-mediated events are a corollary to the opposing actions of the p300/CBP-PCAF-SRC [SRC-1/N-CoA-1, TIF-2/GRIP-1, RAC-3/ACTR] coactivator complex and the N-CoR/Sin3/HDAc corepressor complex on nuclear receptor-mediated transcriptional regulation (12, 16, 17, 35, 36) and similar effects mediated by the p300/CBP coactivator complex and the RB/histone deacetylase-corepressor complex on E2F1-mediated transcription (20, 21, 37, 38).
The repression of N-CoR mRNA is also consistent with the observations
that the mRNAs encoding the three orphan nuclear receptors
[Rev-erbA, RVR, and COUP-TF II] are abundantly expressed in
proliferating myoblasts and repressed during myogenesis. Exogenous
expression of these orphans has been demonstrated to antagonistically
regulate myogenesis, repress MyoD mRNA expression, induce
cyclin D1, abrogate the induction of myogenin and p21 mRNA
after serum withdrawal, and interact with N-CoR to repress
transcription (15, 25, 26, 29, 30, 39, 40).
The Corepressor, N-CoR, Directly Regulates the Activity of MyoD
We demonstrated that the 4RE-tkLUC MyoD-dependent reporter was
specifically inhibited by N-CoR expression and that the activity of
GAL-MyoD was repressed by N-CoR overexpression. Interestingly, this
suggested that N-CoR functioned as a corepressor in the presence or
absence of the cognate binding site for MyoD (the E-box). Our
experiments suggest that one of the mechanisms involved in this process
of repression was the direct interaction between MyoD and N-CoR.
Additional experiments demonstrated that the interaction was mediated
by the bHLH domain of MyoD and the repressor domain of N-CoR. The
importance of this repressor domain-dependent interaction in the
regulation of myogenesis by N-CoR was strikingly demonstrated by the
observation that exogenous RIP13 expression, in absolute contrast to
exogenous N-CoR expression, did not affect myogenic differentiation or
cell cycle withdrawal. RIP13 is identical to N-CoR, but it lacks the
N-terminal region encoding repressor domains 1 and 2, which mediate the
interaction with MyoD. This highlighted the functional significance of
the repressor region within N-CoR. Transfection experiments
demonstrated that the bHLH region functioned as a repressor in the GAL4
hybrid system, and squelching experiments verified that the bHLH region
of MyoD and the retinoic acid receptor shared a common corepressor(s),
N-CoR. These observations were further reinforced by the use of the
histone deacetylase inhibitor, TSA, which significantly increased the
transcriptional activity of myoD and ablated N-CoR-mediated repression
of MyoD-dependent gene expression in myogenic cells.
The bHLH Region Is a Crucial Target during Myogenesis
The targeting of the bHLH region in MyoD is quite interesting in
the light of several observations. First, the pocket proteins, pRb,
p107, and p130, which interact with many classes of transcription
factor and viral oncoproteins, participate in the induction and
maintenance of the postmitotic state (41, 42, 43, 44). Second, these pocket
proteins, in their hypophosphorylated forms, specifically and directly
interact with the bHLH region of the myoD gene family and
mediate cell cycle withdrawal and the activation of the myogenic
program (43, 44). Moreover, myoD-mediated myogenic
conversion and growth arrest of fibroblastic cells is dependent on
functional pRB or other pocket-like proteins (43). Third, a number of
groups have recently demonstrated that the silencing of E2F-dependent
gene expression involves a complex between E2F, hypophophorylated RB
protein, and the histone deacetylases (20, 21, 38). It has been
suggested that active transcriptional repression by hypophosphorylated
RB involves a modification of chromatin structure, which leads to the
repression of E2F-dependent gene expression and cellular proliferation.
Control of myoblast differentiation is also regulated by factors that
effect the phosphorylation of the RB. RB activity is controlled by cell
division kinase (cdk) complexes with the cyclins (reviewed in Ref. 45).
The cyclins and cdks inhibit muscle-specific transcription via the
hyperphosphorylation of RB, which leads to the activation of
E2F1 and cellular proliferation (46, 47). The activity of cdks is
regulated at the level of synthesis of the subunit partners
(e.g. cyclins) of the complex and by binding of inhibitors
(e.g. p21Cip1/Waf1) (reviewed in Ref.
45). The critical role of these cell cycle regulators in myogenesis has
been demonstrated by 1) the inhibition of myogenesis by forced
expression of cyclin D1 by mechanisms dependent/and independent of pRB
hyperphosphorylation and MyoD phosphorylation (46, 47, 48) and 2) the
ectopic expression of p21 in growing myoblasts results in cell cycle
arrest (4, 6, 49).
We suggest that, during cell cycle exit and the activation of the myogenic program, the multiprotein corepressor complex that functions to repress the activity of MyoD begins to play a critical role in mediating the effects of RB on E2F-dependent gene expression and the inhibition of cell cycle-related transcription.
Coactivators and Corepressors, Critical Regulators of Cellular
Proliferation and Myogenic Differentiation
This study contributes to an emerging and complex story about the
crucial role of cofactors in myogenesis. Whether the domains involved
and the subsets of cofactors used during myogenesis display specificity
with respect to the 1) class of transcription factor involved, 2)
target gene(s), and 3) cell cycle remains to be resolved. The evidence
to date supports the notion that the coactivator and corepressor
complexes function in both the proliferative phases and postmitotic
states of myogenesis. It has been observed that the coactivator complex
p300 mediates E2F-dependent S-phase gene expression, DNA synthesis, and
cell cycle progression (37). This process is dependent and
mediated by the phosphorylation of RB or the other pocket proteins by
the cyclin/cdk pathway. During withdrawal from the cell cycle and
myogenic differentiation, a number of key events take place: 1) pRB
becomes hypophosphorylated and recruits histone deacetylase (in a
pocket domain-dependent fashion) to directly repress E2F-dependent gene
expression (20, 21, 38); 2) the hypophosphorylated pRB and MyoD
directly bind to each other through an interface that involves the
pocket and the bHLH regions, consistent with the effects of MyoD on the
myogenic pathway and cell cycle withdrawal, respectively (43); and 3)
concomitant with the above events, p300 and PCAF directly interact with
MyoD and potentiate myogenic differentiation and permanent withdrawal
from the cell cycle (8, 9, 10, 11, 32). Our study demonstrates that the N-CoR
corepressor complex can prevent activation of p21 and myogenesis;
moreover, it can directly mediate the repression of MyoD function (see
Fig. 7).
|
Conclusion
We propose that this study provides a paradigm for corepressor
function in other systems of differentiation and highlights that the
regulation of mammalian differentiation by hierarchical classes of
transcription factors involves a fine balance between coactivator and
corepressor complexes that are tightly linked to the regulation of the
cell cycle.
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MATERIALS AND METHODS |
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Luciferase Assays
Luciferase activity was assayed using a Luclite kit (Packard
Instruments, Meriden, CT) according to the manufacturers
instructions. Briefly, cells were washed once in PBS and resuspended in
150 µl of phenol red-free DMEM and 150 µl of Luclite substrate
buffer. Cell lysates were transferred to a 96-well plate, and relative
luciferase units were measured for 5 sec in a Wallac Trilux 1450
microbeta luminometer.
Construction of Stable Cell Lines
C2C12 cells were stably transfected at approximately 40%
confluence using the DOTAP (Boehringer Mannheim)-mediated
procedure as described previously (29). Briefly, a 1 ml DNA/DOTAP
mixture (containing 20 µg of pSG5-N-CoR or RIP13, 1.5 µg of
pCMV-NEO, 75 µl of DOTAP, 75 µl of DOSPER in 20 mM
HEPES, 150 mM NaCl, pH 7.4) was added to the cells in a
150-mm dish and 20 ml of fresh culture medium. The cells were then
grown for a further 24 h to allow cell recovery and for high-level
pCMV-NEO expression before selection. Stable transfectants were
isolated after 714 days selection in DMEM supplemented with 20% FCS
and 400 µg/ml G418.
C3H10T1/2 Cell Myogenic Conversion
To determine whether pSG5-NCoR was capable of repressing the
SV40 promoter 0.3 µg, 1 µg, and 3 µg of either pSG5 or pSG5-N-CoR
were cotransfetced with the pGL-3-Control/SV40-LUC reporter in
C3H10T1/2. For the myogenic conversion assay, C3H10T1/2 fibroblasts
were grown to 60% confluence in 12-well dishes. Cells in each well
were transfected by the DOTAP/DOSPER (Boehringer Mannheim)-mediated procedure, using 1 µg of pSG5-MyoD in
combination with either 1 µg of pSG5-N-CoR or pSG5 carrier DNA to
total 2 µg per transfection. Twenty four hours after transfection,
fresh medium, 10% FCS in DMEM, was added until 100% confluent, after
which the media was changed to DMEM plus 2% horse serum. Cells were
grown under these conditions for 6 days, with media changes occurring
every 2 days. Immunostaining was then performed using a monoclonal
antibody directed toward the fast isoform of the major thick filament
protein, skeletal myosin heavy chain (Sigma Chemical Co.,
St. Louis, MO; clone MY32). This procedure is described in detail
elsewhere (8).
GST Pulldowns
GST and GST fusion proteins were expressed in Escherichia
coli (BL21) and purified using glutathione-agarose affinity
chromatography as described previously (55). The GST fusion proteins
were analyzed on 10% SDS-PAGE gels for integrity and to normalize the
amount of each protein. The Promega Corp. (Madison, WI)
TNT-coupled transcription-translation system was used to produce
[35S]methionine-labeled MyoD and N-CoR proteins that were
visualized by SDS-PAGE. In vitro binding assays were
performed with glutathione agarose beads (Sigma Chemical Co., St. Louis, MO) coated with approximately 500 ng GST fusion
protein and 230 µl [35S]methionine-labeled protein in
200 ml of binding buffer containing 100 mM NaCl, 20
mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonident
P-40, 5 µg of ethidium bromide, and 100 µg of BSA. The reaction was
allowed to proceed for 12 h at room temperature with rocking. The
affinity beads were then collected by centrifugation and washed five
times with 1 ml of binding buffer without the ethidium bromide or BSA.
Beads were resuspended in 20 µl of SDS-PAGE sample buffer and boiled
for 5 min. The denatured proteins were run on a 10% SDS-PAGE gel that
was subsequently treated with Amersham Amplify fluor
(Amersham, Arlington Heights, IL), dried, and
autoradiographed.
Plasmids
GAL-MyoD, GAL-MyoD N-terminal, GAL-MyoD HLH, GAL-MyoD
basic,
GAL-MyoDbHLH, 4REtk-LUC, G5E1b-LUC, MH100-LUC, pSG5-N-CoR, and CMX-RAR
are described elsewhere (8, 17). pSG5-MyoD was constructed by excising
MyoD cDNA from the pEMSVscribe (Moloney Sarcoma Virus)-based expression
vector pEMSV-MyoD and recloning into the EcoRI site of the
pSG5 vector.
RNA Extraction, Northern Hybridization, and Probe Preparation
Total RNA was extracted by the acid guanidinium
thiocyanate-phenol-chloroform method. Northern blots, random priming,
and hybridizations were performed as described previously (56). The
actin probes used have been described by Bains et al. (57).
The mouse myogenin (58) and MyoD (59) cDNAs were excised from the
pEMSVscribe (Moloney Sarcoma Virus)-based expression vectors. Mouse
Cyclin D1 was excised from pGEX-3X-CYL1 (60), and mouse p21 was excised
from pCMW35, an unpublished clone encoding mouse p21 from the
Vogelstein laboratory. HDAC1 was excised from pBluescript-HDAC1 using
SalI restriction digest. mSin3BLF was excised
from pcDNA3.1-mSin3BLF with HindIII.
cDNA probes were radioactively labeled by random priming. DNA fragment
(50100 ng) was boiled with 20 ng random primers (pdN6;
Pharmacia Biotech, Piscataway, NJ). The DNA was then
incubated at room temperature overnight with RPB (50 mM
Tris, pH 7.5, 10 mM MgCl2, 200 µM
dATP, dGTP, dTTP), 10 µl [-32P]-dCTP (Bresatec Ltd.,
Thebarton, Australia), and 2 U of Klenow polymerase. Probes were
purified using a NICK column (Pharmacia Biotech) according
to the manufacturers instructions. Quantitation of 18S rRNA was
performed using the following 25-mer oligonucleotide,
5'-CATGGTAGGCACGGCGACTACCATC-3'. To label the 18S rRNA oligonucleotide
probe, 100 ng of the 25-mer were incubated at 37 C for 2 h with 1
µl polynucleotide kinase (PNK) (Boehringer Mannheim), 2
µl 10x PNK buffer (Boehringer Mannheim), and 3 µl
32P-
dATP in a total volume of 20 µl. The probe was
purified on a NAP column (Pharmacia Biotech) as directed
by the manufacturers.
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FOOTNOTES |
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Received for publication January 11, 1999. Revision received February 25, 1999. Accepted for publication March 11, 1999.
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