Skeletal muscle development involves coordinated expression
of transcription factors that control specification of mesodermal
progenitors to the muscle fate and differentiation of committed
myoblasts into myotubes (Stockdale, 1992). The development of
skeletal muscle is directed by four myogenic regulatory factors
(MRFs): MyoD, myf-5, MRF4 and myogenin (Sabourin and
Rudnicki, 2000; Yun and Wold, 1996). MyoD and myf5 are
expressed in proliferating myoblasts and influence lineage
restriction, whereas myogenin, a target of MyoD, is induced
upon differentiation (Rudnicki and Jaenisch, 1995). The MRFs
heterodimerize with ubiquitous E-proteins, bind to conserved E-
box sequences (CANNTG) in the promoter of muscle-specific
genes, and control transcription (French et al., 1991). The
transcriptional activity of MyoD is influenced by its interaction
with an array of coactivators and corepressors (McKinsey et al.,
2001). The repressor protein histone deacetylase 1 (HDAC1)
was reported to interact with MyoD and deacetylate it,
consequently suppressing the transcriptional activity of MyoD
(Mal et al., 2001). Activator proteins, histone acetylases (HATs),
on the other hand, are known to stimulate MyoD-dependent
transcription by engaging histone acetylases p300 and PCAF,
which, in turn, promote acetylation of MyoD itself and increase
its affinity for target-gene promoters (Puri et al., 1997; Sartorelli
et al., 1999). It is well known that the positive transcription
elongation factor b (P-TEFb) complex, which consists of cdk9
and cyclin T1 (or the minor forms T2a and T2b), mediates the
transcription of RNA polymerase II (Pol II) genes (Fu et al.,
1999; Peng et al., 1998). P-TEFb phosphorylates the C-terminal
domain (CTD) of the largest subunit of Pol II at serine 2 of a 52-
tandem heptapeptide; this phosphorylation is required for
transcription to change from the abortive to productive phase of
transcriptional elongation (Price, 2000; Zhou et al., 2000).
We and others have demonstrated that the mouse cardiac
lineage protein 1 (CLP-1) (Huang et al., 2004), and its human
homolog HEXIM1 (Schulte et al., 2005), function as
transcriptional repressors. In HeLa cells, P-TEFb exists in
equilibrium between active and inactive forms by way of
association and dissociation of HEXIM1 from the P-TEFb
complex (Nguyen et al., 2001; Yang et al., 2001). Our laboratory
knocked out the CLP-1 gene in mice, which resulted in lethality
in late fetal stages (E17–E18) due to hypertrophic growth of the
embryonic hearts (Huang et al., 2002; Huang et al., 2004).
Subsequently, studies in our laboratory demonstrated that
introduction of CLP-1 heterozygosity +/– in the background of
cardiac-specific cyclin T1 overexpression enhanced RNA Pol II
phosphorylation at serine 2 (Espinoza-Derout et al., 2009).
Skeletal muscle differentiation, characterized by silencing of
the proliferative genes and up-regulation of muscle-specific
genes, is prominently influenced by MyoD, which is known to
target more than 300 genes controlling several subprograms of
skeletal muscle gene expression (Bergstrom et al., 2002). The
reported link between MyoD and P-TEFb prompted us to
examine whether CLP-1 is involved in control of P-TEFb activity
during the transition of myoblasts to myotubes. In this study, we
report that CLP-1 associates with MyoD and HDAC proteins in
the early phase of differentiation of C2C12 myoblasts and
conclude that the CLP-1/MyoD/HDAC complex is crucial for
control of P-TEFb activity and in regulation of skeletal muscle
Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and
dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b
(P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting
a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown
skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/– cells failed to differentiate
when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases
(HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial
to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC
complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest
a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression,
a requirement for myoblasts to exit the cell cycle and transit to myotubes.
Key words: CLP-1, HEXIM1, MyoD, P-TEFb, Skeletal muscle
CLP-1 associates with MyoD and HDAC to restore
skeletal muscle cell regeneration
Josephine Galatioto, Eduardo Mascareno and M. A. Q. Siddiqui*
Department of Cell Biology, Center for Cardiovascular and Muscle Research, State University of New York Downstate Medical Center, Brooklyn,
New York, NY 11203, USA
*Author for correspondence (email@example.com)
Accepted 3 August 2010
Journal of Cell Science 123, 3789-3795
© 2010. Published by The Company of Biologists Ltd
Journal of Cell Science
Association of CLP-1 with P-TEFb and MyoD in C2C12 cell
To investigate the role of CLP-1 in skeletal muscle cell differentiation,
we first examined its expression in C2C12 skeletal muscle cells,
which represent a highly suitable model for analysis of myogenic
differentiation. In high serum (10% FBS) growth medium, C2C12
myoblasts proliferate until they reach confluency. Differentiation
into multinucleated myotubes is triggered by switching to low serum
(2% horse serum) differentiation medium. As shown in Fig. 1A,
CLP-1 was present in C2C12 cells in growth medium (growth stage
G) and in differentiation medium at 24 hours (differentiation stage
D1) and 72 hours (differentiation stage D3). Myogenin, a known
marker for differentiation, is expressed At D1 and D3 but not in G.
MyoD is expressed in both myoblasts and myotubes, as expected.
Next, CLP-1 association to the P-TEFb complex was analyzed
by immunoprecipitation with anti-cdk9 antibody followed by
western blotting with anti-CLP-1 antibody (Fig. 1B). There was a
prominent level of association of CLP-1 with P-TEFb in early
differentiation (D1), whereas in both proliferative cells (G) and the
terminally differentiated cells (D3) it was negligible. Thus, it
appeared that there was a clear shift in P-TEFb equilibrium to the
CLP-1 bound state in C2C12 cell differentiation, which suggests
that CLP-1-mediated regulation of P-TEFb activity might have a
role in the transition of C2C12 cells from growth to differentiation.
Because the kinase activity of P-TEFb is influenced by the
association of CLP-1, one might envision that association of CLP-
1 to the P-TEFb/MyoD complex will regulate MyoD-mediated
transcriptional activity. We coimmunoprecipitated with MyoD
antibody using lysates from proliferating C2C12 myoblasts and
from cells induced to differentiate for 24 hours and 72 hours. We
observed that there was association between MyoD and CLP-1 in
the differentiation stage D1, whereas it was markedly reduced in
G and D3 (Fig. 1C).
HDACs are implicated in the regulation of skeletal myogenesis
by their interaction with MyoD and in regulating MyoD-mediated
gene transcription (Mal et al., 2001). HDAC1 binding to MyoD is
implicated in blocking the function of MyoD in initiating the
myogenic program. To examine whether MyoD association with
HDAC1 occurs in growth and/or differentiation conditions,
coimmunoprecipitations were performed on proliferating C2C12
cells and on differentiating C2C12 cells at D1 and D3 (Fig. 1D). The
results show that MyoD binds to HDAC1 and PCAF in both growth
and differentiation conditions, albeit at a relatively lower level in
growth conditions. The association of MyoD with HATs and HDACs
suggests that both activation and suppression mechanisms coexist,
perhaps in directing distinct promoters toward differentiation.
Association of CLP-1 with HDACs in C2C12 cell
We then examined the interaction of CLP-1 with HDACs in C2C12
cells using immunoprecipitation with antibodies against HDAC1,
HDAC3 and HDAC5, and western blotting for the presence of
CLP-1 and cdk9. As seen in Fig. 2A, CLP-1 associates with class I
HDAC1 and HDAC3 and with class II HDAC5, preferentially at
the D1 stage of differentiation. HEXIM1 was previously reported
to bind directly to HDAC3 in HeLa cells (Fu et al., 2007). Also,
upon western blotting with anti-cdk9 antibody, we observed that
association of cdk9 is highest in D1 (Fig. 2A). The pattern of CLP-
1 association with HDACs was distinct from HDAC protein
expression, as seen by direct western blotting (Fig. 2A, bottom right
panel). It is known that class II HDACs, which includes HDAC5,
translocate from the nucleus to the cytoplasm, whereas Class I
HDACs are restricted to the nucleus. To ascertain whether CLP-1
association to HDAC5 is nuclear or cytoplasmic, we fractionated
C2C12 cells and immunoprecipitated with anti-HDAC5 antibody.
Western blotting with CLP-1 antibody showed that CLP-1 was
present in both the nuclear and cytosolic fractions, whereas cyclin
T1 and MyoD were present in nuclei only, as expected (Fig. 2B).
Coimmunoprecipitation analysis showed that the CLP-1/HDAC5
complex was localized only in the nuclear fraction (Fig. 2B).
CLP-1 associates with MyoD and HDAC on the cyclin D1
In order to gain mechanistic insights into how CLP-1 regulates
skeletal muscle differentiation, we hypothesized that CLP-1
associates with MyoD and HDAC and is involved in
downregulation of cell-cycle genes, for example cyclin D1, to
allow expression of differentiation-specific genes. We examined
association of this inhibitory complex to the cyclin D1 promoter
because cyclin D1 is a cell cycle regulatory protein whose levels
were reported to change in response to HEXIM1 (Ogba et al.,
2008). Cyclin D1 protein in C2C12 cells is expressed only under
growth conditions, as shown by western blotting (Fig. 3A).
Chromatin immunoprecipitation (ChIP) assay revealed that the
inhibitory complex was associated with the promoter of the MyoD-
target cyclin D1 gene. DNA was amplified using primers flanking
two putative E-box sequences, the MyoD target-site, within the
3790Journal of Cell Science 123 (21)
Fig. 1. Association of CLP-1 with cdk9 and MyoD is dynamic in C2C12
cell differentiation. Whole-cell lysates were collected from growth medium
(G) and differentiation medium at 24 hours (D1) and at 72 hours (D3).
(A)Equal amounts of protein were subjected to SDS-PAGE followed by
immunoblotting. Myogenin (MyoG) served as a marker of differentiation.
GAPDH served as a loading control. (B)Immunoprecipitation (IP) of C2C12
cell lysates with anti-cdk9 antibody, and western blotting with anti-CLP-1
antibody. Western blot with anti-cdk9 antibody served as a control of total
immunoprecipitated protein. (C)IP of C2C12 cells with anti-MyoD antibody
and western blotting with anti-CLP-1 antibody. Western blot with anti-MyoD
antibody served as a control of total immunoprecipitated protein. (D)IP using
C2C12 cell lysates with anti-MyoD antibody and western blotting with anti-
HDAC1 and anti-PCAF antibodies. Direct western blots of HDAC1 and PCAF
served as input controls. In all cases, IP with antibody alone served as a control
for immunoreactivity (Ab). Data shown represent one of three separate
Journal of Cell Science
cyclin D1 promoter (insert size 188 bp). Complexes containing
CLP-1/MyoD/HDAC were associated with the cyclin D1 promoter
at MyoD-target binding sites, preferentially in differentiating muscle
cells (Fig. 3B). MyoD association was maximal in D1 and D3.
CLP-1, HDAC1 and HDAC3 had little association in G as
compared with D1. The association of HDAC5 was reduced in G
and D3. These results are consistent with our immunoprecipitation
experimental data above.
To examine regulation of cyclin D1 expression by CLP-1, we
performed transient transfection assays in C2C12 cells under growth
and differentiation conditions. There was a drop in cyclin D1
promoter activity in differentiation conditions compared to growth
conditions (Fig. 3C). Co-transfection with MyoD in differentiation
medium did not appear to have any effect on cyclin D1 expression.
However, co-transfection of MyoD and CLP-1 caused inhibition
of cyclin D1 expression (Fig. 3C). Co-transfection with MyoD,
CLP-1 and HDAC5 further reduced Cyclin D1 reporter activity.
MyoD has been reported to act as a transcriptional activator as well
as inhibitor (Bergstrom et al., 2002). In C2C12 skeletal muscle,
MyoD inhibited the cyclin D1 promoter, and there was a synergistic
effect when negative regulators CLP-1 and HDAC5 were added.
Knock down of CLP-1 inhibits C2C12 cell differentiation
To further investigate the putative function of CLP-1 in early
myogenic differentiation, we chose to knock down CLP-1 in C2C12
cells by homologous recombination. C2C12 cells were first
transfected with a CLP-1 replacement gene-targeting vector (Huang
CLP-1 associates with MyoD
Fig. 2. CLP-1 associates with HDACs at the onset of C2C12 cell
differentiation. (A)Immunoprecipitation (IP) of C2C12 cell lysates from
growth (G) and differentiation medium at 24 hours (D1) and at 72 hours (D3)
with antibodies specific for HDAC1, HDAC3 and HDAC5, and western blot
with anti-CLP-1 and anti-cdk9 antibodies. IP with antibody alone served as a
control for immunoreactivity (Ab). Western blot with anti-CLP-1 and anti-
GAPDH antibodies served as input controls. Data represent one of three
separate experiments for each antibody. Bottom right panel: western blot of
total cell lysates with anti-HDAC1, anti-HDAC3 and anti-HDAC5 antibodies.
Western blot with GAPDH served as input control. (B)Left panel: nuclear
(Nuc) and cytoplasmic (Cyto) fractions of C2C12 cells in differentiation
medium at 24 hours were subjected to direct western blotting with antibodies
specific for CLP-1, cyclin T1, MyoD and GAPDH. Right panel: lysates were
subjected to IP with anti-HDAC5 antibody and western blotting with anti-
CLP-1 antibody. Direct western blot of CLP-1 served as an input control.
Fig. 3. Analysis of the cyclin D1 promoter in C2C12 cell differentiation.
(A)Analysis of C2C12 cells in growth medium (G), and differentiation medium
at 24 hours (D1) and 72 hours (D3) by western blotting with antibodies specific
for CLP-1, myogenin and cyclin D1. GAPDH served as a loading control.
(B)ChIP was performed on C2C12 cells using antibodies specific for MyoD,
CLP-1, HDAC1, HDAC3 and HDAC5. Nonspecific IgG and anti-actin
antibody served as negative controls. Precipitated DNA was amplified by PCR
for regions of the cyclin D1 gene corresponding to the 5? upstream promoter
region encompassing two E-box sequences. Input DNA represents 10% of total
chromatin used in each reaction. Primers specific to GAPDH were used before
(input) and after immunoprecipitation as a control to monitor
immunoprecipitation specificity. Data represent one of three separate
experiments. (C)Luciferase assay in C2C12 cells in growth media (GM) and
for 24 hours in differentiation media (DM). Cells were co-transfected with
cyclin D1-luciferase plasmid along with expression plasmids encoding MyoD,
CLP-1 and HDAC5. Renilla luciferase expression was used for normalization.
Luciferase activities for cyclin-D1 in differentiation media is expressed relative
to the mean value derived from cells co-transfected with cyclin D1-luciferase
and Renilla luciferase in growth media. Luciferase activity for + MyoD, +
MyoD + CLP-1 and + MyoD + CLP-1, + HDAC5 in differentiation media are
expressed relative to the mean value derived from cells co-transfected with
cyclin D1-luciferase and Renilla luciferase in differentiation media.
Experiments were repeated three times. Bars show mean + s.e.m., *P<0.05.
Journal of Cell Science
et al., 2004) and then selected for neomycin resistance. Single
clones were examined for expression of the recombinant allele by
PCR (Fig. 4A). Individual clones were also assayed for CLP-1
protein level by western blot (Fig. 4B). The CLP-1 protein level
was lower in CLP-1 heterozygote (+/–) C2C12 cells than in wild-
type (+/+) C2C12 cells, as expected. When C2C12 CLP-1 +/– cells
were challenged to differentiate by switching to low-serum medium,
the cells were unable to differentiate, as indicated by the absence
of myogenin protein in D1 and D3 (Fig. 4C). The CLP-1 +/– cells
continued to proliferate, as indicated by the presence of proliferating
cell nuclear antigen (PCNA) and cyclin D1 in differentiation
conditions (Fig. 4C). The analysis was quantified using
densitometry and the results depicted graphically (Fig. 4C, bottom
panel). To confirm the differentiation deficiency, we performed
immunofluorescence analysis on cells in growth and differentiation
medium (Fig. 4D). Under differentiation conditions, wild-type
C2C12 cells fused into multinucleated myotubes. By contrast,
when challenged to differentiate in low-serum medium, C2C12
CLP-1 +/– cells remained mononucleated and maintained a
nondifferentiated phenotype. We also assessed the proliferation
and differentiation rate of these cells through immunofluorescence
with anti-PCNA and anti-myosin heavy chain antibodies under
growth conditions and after differentiation for 24, 48, and 72
hours. Stained cells were counted and compared to total cells, and
the results depicted graphically (Fig. 4E). In wild-type cells, PCNA
was only expressed under growth conditions, whereas in CLP-1
+/– C2C12 cells, it was also expressed in differentiation media.
Also, in differentiation conditions wild-type cells begin to form
tubes, expressing myosin heavy chain at 24 hours in differentiation
media, whereas heterozygote cells did not express myosin heavy
chain except for a minor expression at 72 hours.
Analysis of CLP-1-associated proteins in CLP-1 –/– mouse
Because the above data on association of CLP-1 with MyoD and
HDAC were obtained in in vitro cultured C2C12 cells, we
addressed the question whether such functional association exists
in vivo, in the animal. We used embryonic day 12 (E12) embryos
3792 Journal of Cell Science 123 (21)
Fig. 4. C2C12 CLP-1 +/– cells are differentiation deficient. (A)PCR using DNA from C2C12 CLP-1 +/+ cells and one CLP-1 +/– clone. Primers generate a 457
base pairs product for the CLP-1 gene and a 383 base pairs product for the mutated allele. M denotes DNA size ladder. Data are shown for one representative clone.
(B)Western blot of C2C12 CLP-1 +/+ cells and C2C12 CLP-1 +/– cells in growth medium probed with anti-CLP-1 antibody. GAPDH served as a loading control.
Western blot is shown for one representative clone. The analysis was performed on three independently isolated C2C12 CLP-1 +/– cell clones, and means are
depicted graphically + s.e.m.; GAPDH was used for normalization (*P<0.05). (C)Western blot of lysates from C2C12 CLP-1 +/+ and +/– cell cultures in growth
medium (G) and differentiation medium at 24 hours (D1) and at 72 hours (D3), probed with anti-CLP-1, anti-myogenin, anti-PCNA, anti-cyclin D1 and anti-MyoD
antibodies. GAPDH served as a loading control. The analysis was performed three times, and means are depicted graphically + s.e.m.; GAPDH was used for
normalization (*P<0.05, **P<0.01). (D)Immunofluorescence of C2C12 CLP-1 +/+ and C2C12 CLP-1 +/– cells in growth and differentiation medium using actin
stain (green) and co-stained with anti-CLP-1 (red) or DAPI nuclear stain (blue). This analysis was performed on three independently isolated C2C12 CLP-1 +/–
cell cultures. (E)Graphical analysis of immunofluorescence of C2C12 CLP-1 +/+ (WT) and +/– cells in growth media (GM) and differentiation media (DM) for 0,
24, 48 and 72 hours. Antibodies to PCNA and myosin heavy chain were used, and DAPI was used as a nuclear stain. At each time point, cells were counted
(n500) and the ratio of cells expressing PCNA or myosin heavy chain versus total cells is depicted graphically (n3).
Journal of Cell Science
and adult wild-type skeletal muscle for western blotting. In adult
muscle, CLP-1 and cdk9 levels were very low compared to
embryonic tissue (Fig. 5A, input). Likewise, CLP-1 association
with cdk9 and MyoD was not observed in adult muscle (Fig. 5A).
Embryos at day 12 contain myoblasts as well as myotubes,
whereas in the adult there are only myotubes in which CLP-1
cooperation with cdk9 and MyoD is no longer necessary. Next,
we examined CLP-1 wild type, +/– and –/– E12 embryos by
western blot for expression of the components of P-TEFb and
MRFs. CLP-1 was not expressed in –/– embryos, as expected,
whereas CLP-2 (a homolog of CLP-1) was expressed (Fig. 5B).
Cdk9, cyclin T1 and Pol II levels remained steady (Fig. 5B).
Skeletal muscle marker MyoD was down in CLP-1 +/– and –/–
embryos, and the differentiation markers myogenin and myosin
heavy chain were also decreased markedly (Fig. 5C). Next,
immunohistochemical analysis of E12 embryos was performed
with anti-CLP-1 and anti-myogenin antibodies (Fig. 5D). Embryos
were sectioned coronally, and the neural tube (Fig. 5D top left)
was used for orientation and localization of the myotome. In
wild-type embryos, myogenin was clearly localized adjacent to
the neural tube and this colocalized with CLP-1, which is
ubiquitously expressed. In CLP-1 –/– embryos, myogenin is still
expressed, yet the expression is at a lower level. These findings
suggest that the absence of CLP-1 in the knockout mice might
lead to reduced myogenesis, which might partly be responsible
for embryonic lethality. It was also clear that CLP-2 does not
compensate for the loss of CLP-1 because CLP-1 –/– mice die at
E17 or E18, despite CLP-2 expression.
Mechanistically, eukaryotic cell development and its maintenance
are attributed to a critical balance between transcriptional activators
and inhibitors and their effect on specific genes. At the
transcriptional level, it is evident that the P-TEFb complex plays a
role in expression of tissue and developmental stage specific genes.
Current data support the notion that the active recruitment of P-
TEFb is crucial for the expression of these genes, but it is not
known how P-TEFb is specifically recruited and what cellular
factors cooperate with P-TEFb. A great deal of information is
available on the molecular composition of the P-TEFb complex
and its role in transcription elongation. However, there is limited
knowledge on the role of P-TEFb in skeletal muscle gene control,
and the involvement of CLP-1 is totally unknown.
In this study, we have examined the role of CLP-1 in skeletal
muscle cell differentiation using C2C12 mouse myoblast cells.
We noted that CLP-1 association with P-TEFb is dynamic and is
maximal during the early phase of differentiation, implying that
CLP-1 is functionally connected with the transition phase of
myoblasts to myotubes. A recent report also examined HEXIM1
association with P-TEFb in C2C12 cells and found that HEXIM1
dissociated from P-TEFb 30 minutes after switching to low-serum
medium and the association was shown to be restored at 2 hours
(Nojima et al., 2008). However, myogenin is known to be
expressed only after 12 hours in low-serum medium (Simone et
al., 2002). We therefore used 24 hours as the early phase D1, and
72 hours as the fully established phase D3 of C2C12 cell
CLP-1 associates with MyoD
Fig. 5. Analysis of expression of P-TEFb and MRFs in
mouse skeletal muscle. (A)Immunoprecipitation (IP) of
skeletal muscle lysates from wild-type E12 embryo and
adult, with anti-cdk9 and anti-MyoD antibodies and
western blotting with anti-CLP-1 antibody. IP with
antibody alone served as a control for immunoreactivity.
Western blot of total protein served as an input control.
GAPDH served as a loading control. (B)Western blotting
of skeletal muscle tissue lysates from E12 mouse embryos
wild-type (WT), heterozygote (+/–) and homozygote (–/–)
for the CLP-1 allele with anti-CLP-1, anti-CLP-2, anti-
cdk9, anti-cyclin T1 and anti-RNA polymerase II (Pol II)
antibodies. GAPDH served as a loading control.
(C)Western blotting of skeletal muscle tissue lysates with
anti-MyoD, anti-myogenin and anti-myosin heavy chain
(MHC) antibodies. GAPDH served as a loading control.
Data represent one of three separate experiments with one
embryo per experiment. Relative MyoD, myogenin and
MHC levels are depicted graphically as means + s.e.m.
GAPDH was used for normalization (*P<0.05).
(D)Immunohistochemical analysis of coronal section of
CLP-1 +/+ and CLP-1 –/– E12 mice. Sections were stained
with DAPI (blue), anti-CLP-1 antibody (red) and anti-
myogenin antibody (green). Merge of CLP-1 and
myogenin is depicted in the fourth panel (yellow). In
diagram above, NT denotes location of the neural tube, and
red square denotes relative localization of section. Images
were captured at 10? magnification.
Journal of Cell Science
By reducing CLP-1 expression in C2C12 cells, we saw an arrest
in the transition of skeletal myoblast to myotubes, implying that
CLP-1, and possibly its association with components of P-TEFb, are
obligatory to this transition. Because the CLP-1 knockout mice died
at E17 to E18, CLP-2 expression does not substitute CLP-1 function
in mice, despite high homology between the C-termini of the two
proteins. Although the CLP-1 heterozygosity in C2C12 cells was
sufficient to arrest differentiation in cell culture, the CLP-1 +/– mice
survive. One might speculate that during embryonic development
some compensatory molecule that is not available in the cell line
acts as a substitute for CLP-1. Furthermore, embryonic growth
undergoes defined phases of development, whereas the C2C12 cells
are immediately challenged to differentiate and therefore possibly
bypass the regulatory subprograms of gene expression. During early
embryonic stages CLP-1 +/– embryos appear smaller than their
wild-type litter mates, but after birth they appear phenotypically
similar. There might very well be a skeletal muscle deficiency, as
evidenced by reduced skeletal muscle markers during development,
a phenomenon that needs to be examined in the future.
In this report, we also showed that MyoD and HDACs associated
with CLP-1 maximally at 24 hours. This would suggest that perhaps
both CLP-1 and HDACs are recruited by MyoD to offer an
environment optimal for repression of proliferative genes and
allowing the transition of myoblasts to myotubes. At this junction,
one would expect that MyoD must also associate with HAT proteins
in a separate protein complex to facilitate the promotion of muscle-
specific gene expression. In support of this notion, Mal and Harter
(Mal and Harter, 2003) have shown that once myoblasts undergo
differentiation, MyoD actively engages in recruitment of HAT
proteins at skeletal muscle promoters. In our present study, we
noted that MyoD associates with HDAC1 and PCAF in the
differentiation stage. The role of MyoD as a repressor or activator
can be dictated by its association with interacting proteins (HDAC,
HAT). Recent reports have examined the effect of HDAC and HAT
association with the P-TEFb complex. It was shown that cdk9 is
acetylated, but whether this acetylation enhances or inhibits the
kinase activity is not settled. Fu and colleagues (Fu et al., 2007)
showed that cdk9 was deacetylated by HDAC3, which reduced the
association of cdk9 with activator Brd4 and promoted the
interaction of P-TEFb with HEXIM1. Sabo and colleagues (Sabo
et al., 2008) showed that cdk9 acetylation by PCAF reduced kinase
function and P-TEFb transcriptional activity. The cyclin T1
component of P-TEFb can also be acetylated, which results in a
decrease in HEXIM1-bound P-TEFb and an increase in cdk9
kinase activity (Cho et al., 2009). In our study, we observed that
CLP-1 associates with HDAC1, HDAC3 and HDAC5 in skeletal
muscle and that this association is specifically enhanced in the
early differentiation phase. CLP-1 associated with HDAC in skeletal
muscle might act to counter the activity of acetylated cyclin T1 or
cdk9. We speculate that inactive P-TEFb bound by CLP-1 is
actively recruited to silence specific gene promoters.
In summary, we have presented evidence that CLP-1 interacts
with MyoD, and hypothesize that CLP-1 associated with MyoD is
required for downregulation of proliferative genes and to steer the
myoblast-to-myotube transition. HEXIM1 expression has been
reported to correlate with estrogen receptor-inducible cyclin D1
protein expression and binding to the E2-responsive region on the
cyclin D1 promoter (Ogba et al., 2008). Using ChIP assay, we
localized CLP-1 and its associated proteins, MyoD and HDACs, on
the cyclin D1 promoter in C2C12 cells, which occurs preferentially
in the differentiation stage. On the basis of our results, we speculate
that MyoD guides CLP-1 to MyoD-target DNA, which is in complex
with HDAC proteins. We believe that both suppression and activation
of MyoD activity are likely to co-exist in C2C12 cells at the onset
of differentiation, to act on distinct promoters. The signaling that
triggers specific interaction of these molecules is not known. Future
studies to characterize the fundamental regulatory events mediated
by CLP-1 would provide further insights into the physiological role
of CLP-1 in skeletal muscle cells. Our data collectively highlight a
distinct function of CLP-1 in cell-cycle exit, suggesting that regulation
of P-TEFb activity by CLP-1 is likely to play a pivotal role in
targeting skeletal muscle cells toward differentiation.
Materials and Methods
Polyclonal rabbit anti-CLP-1 antibody was generated to the peptide
HRQQERAPLSKFGD (Proteintech Group). Cdk9 (C-20, D-7), cyclin T1 (H-245),
GAPDH (FL-335), HDAC1 (H-11), HDAC3 (B-12), HDAC5 (H-714), Pol II (N-
20), myogenin (F5D), MyoD (5.8A, C-20), HEXIM2 (M-90), PCAF (E-8), PCNA
(PC10) and actin (C-2) antibodies were from Santa Cruz Biotechnology. Anti-
myosin heavy chain (MF20) antibody was from Developmental Studies Hybridoma
Bank. Anti-cyclin D1 antibody was from Abcam. HDAC antibody sampler kit was
from Cell Signaling Technology. ChIP Grade antibodies anti-HEXIM1 and anti-
MyoD (C-20x) were from Abcam and Santa Cruz Biotechnology, respectively.
C2C12 mouse myoblasts obtained from ATCC were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% FBS and penicillin/
streptomycin/amphotericin b (growth medium). To induce differentiation, growth
medium was substituted with differentiation medium (DMEM supplemented with
2% horse serum and penicillin/streptomycin/amphotericin b). Cells were incubated
at 37°C with 5% CO2.
CLP-1 allele replacement vector DNA (pKO-HR-CLP-1neo) (Huang et al., 2004)
was transfected into C2C12 cells at 10% confluency using Fugene 6 (Roche)
transfection reagent. Neomycin (G418) antibiotic was used to isolate resistant clones.
C2C12 cells heterozygous for the targeted CLP-1 allele were determined by PCR.
DNA was isolated and genotype determined by PCR using previously described
primers (Huang et al., 2004).
Cells were lysed in buffer A (Michels et al., 2004) supplemented with 1 mM
dithiothreitol, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, protease inhibitor cocktail
(Sigma) and RNasin (Promega). Lysate was subjected to “freeze” on dry ice, and
“thaw” at 37°C, followed by centrifugation at 16,000 g. Equal concentrations of
lysates were incubated with antibodies overnight at 4°C with rotation. For protein
capture, protein A/G plus agarose beads (Santa Cruz Biotechnology) were added at
4°C for 2 hours with rotation. After extensive washing with buffer A, bound proteins
were eluted by boiling in 1? SDS loading buffer.
Cells were lysed in cell fractionation buffer A (10 mM HEPES pH 7.4, 15 mM KCl,
2 mM MgCl2,0.1 mM EDTA) supplemented with 1 mM dithiothreitol, 1 mM PMSF,
10 mM NaF, 1 mM Na3VO4, protease inhibitor cocktail (Sigma), and RNasin
(Promega) with homogenization using a dounce homogenizer and monitored
microscopically with trypan blue. Samples were centrifuged (1500 g) at 4°C for 5
minutes and the supernatant collected as cytoplasmic extract. The pellet was incubated
with cell fractionation buffer B (10 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 0.5
mM EDTA, 0.5% NP-40) supplemented with 1 mM dithiothreitol, 1 mM PMSF, 10
mM NaF, 1 mM Na3VO4, protease inhibitor cocktail (Sigma) and RNasin (Promega).
Samples were subjected to “freeze” on dry ice, and “thaw” at 37°C, with vortexing.
After centrifugation (16,000 g) at 4°C for 10 minutes, the supernatant was collected
as nuclear extract.
Cells were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA,
1% Triton-X 100) supplemented with 1 mM dithiothreitol, 1 mM PMSF, 10 mM
NaF, 1 mM Na3VO4and protease inhibitor cocktail (Sigma).
Western blot analysis
Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane
in electroblotting buffer (20 mM Tris, 150 mM glycine, 20% methanol) for 1 hour.
The membranes were blocked in TBS-T with 5% nonfat dry milk and probed with
primary antibody in TBS overnight, followed by incubation with horse-radish-
peroxidase-conjugated secondary antibody for detection with enhanced
3794 Journal of Cell Science 123 (21)
Journal of Cell Science
C2C12 cells were seeded in six-well tissue culture dishes containing sterile glass
coverslips. Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at
room temperature. Cells were permeabilized in PBS containing 0.1% Triton X-100.
After blocking in 5% FBS in PBS, cells were incubated with primary antibody at
4°C overnight, followed by washes in PBS and incubation with secondary antibody
(1:200) conjugated to either Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) for 1
hour. For actin staining, cells were incubated for 20 minutes with 2 units of Alexa
Fluor 594/phalloidin (Invitrogen). Cell nuclei were stained with 4,6-diamidino-2-
phenylindole (DAPI). Imaging was captured at 40? magnification using a Zeiss
Axiokop microscope, Axio CamMRc camera and Axio vision software.
Chromatin immunoprecipitation assay
Proteins were crosslinked to DNA in culture medium using 1% formaldehyde (10
minutes, room temperature). Cells were lysed in 10 mM Tris pH 8.1, 1.5 mM MgCl2,
10 mM KCl and the nuclei sedimented by centrifugation (1500 g) for 5 minutes at
4°C. The pellet was resuspended in nuclear lysis buffer (50 mM Tris-HCl, 10 mM
EDTA, 1% SDS). Chromatin was sonicated five times, for 30 seconds each time,
generating DNA fragments of 250–1000 base pairs. DNA concentration was
quantified, and 10 mg of DNA was used per immunoprecipitation. Lysates were
diluted with ChIP dilution buffer (20 mM Tris pH 8.1, 150 mM NaCl, 2 mM EDTA,
0.01% SDS, 1% Triton X-100). Antibodies were added, and after rotation overnight
at 4°C, the immune complexes were collected by the addition of protein G agarose
beads/salmon sperm DNA (Millipore). After extensive washes, immune complexes
were eluted (1% SDS, 0.1 M NaHCO3) and crosslinking was reversed by the
addition of 190 mM NaCl overnight at 65°C. DNA was purified using a PCR
purification kit (Qiagen) and amplified by PCR. Sequence of PCR primers: cyclin
D1 5?-TATCCTGGAAGGGCGACTAA-3? and 5?-AATTCCAGC AAC AGC TCA -
AGA-3?;GAPDH primer control 5?-CGGTGCGTGCCCAGTTG-3? and 5?-GCG -
DNA was amplified for 18 cycles (annealing 60°C). Product was visualized on
2% agarose gel by ethidium bromide stain and an 8-bit digital camera.
Transient transfection and luciferase assay
C2C12 cells were plated in 12-well dishes and transiently transfected using
Lipofectamine 2000 (Invitrogen) with 2 mg of reporter gene construct, 0.5 mg
expression vector and 20 ng of thymidine kinase promoter driven Renilla luciferase
vector, which was used to normalize transfection efficiency. Human cyclin D1-
luciferase promoter was provided by Richard Pestell (Thomas Jefferson University,
Philadelphia, PA) (Albanese et al., 1995). Mouse MyoD expression vector was from
Addgene (plasmid 8399) provided by Andrew Lassar (Harvard Medical School,
Boston, MA). HDAC5 expression vector was provided by Eric Verdin (University
of California, San Francisco, CA) (Fischle et al., 1999). pcDNA6-CLP-1 expression
vector was previously created in our laboratory (Huang et al., 2002). At 24 hours
after transfection, cells were harvested, lysed, and cell lysates assayed using the
Dual-luciferase Reporter Assay system (Promega) and OptoComp I Luminometer
(MGM instruments). Statistical analysis was performed using a paired Student’s t-
test. P values of <0.05 were considered significant.
CLP-1 knockout mice have been described previously (Huang et al., 2004). All
experiments were performed in accordance with the Guidelines of the National
Institute of Health. Experimental protocols were reviewed and approved by the
Institutional Animal Care and Use Committee.
Embryos were fixed in 4% paraformaldehyde/PBS overnight at 4°C, washed in PBS,
and transferred to 30% sucrose overnight at 4°C. Tissue was embedded in M-1
matrix (Thermo Scientific) and 14 mm cryosections were collected. Slides were
blocked in 5% FBS in PBS, incubated with primary antibody overnight at 4°C,
washed in PBS, and incubated with secondary antibody conjugated to either Alexa
Fluor 488 or Alexa Fluor 594 for 1 hour. Cell nuclei were stained with DAPI.
Imaging was captured at 10? magnification using a Zeiss Axiokop microscope,
Axio CamMRc camera and Axio vision software.
For quantitative western blot analysis, films were scanned and the band signal
intensities determined using NIH ImageJ software. The densitometry values were
expressed as a fold level relative to the control, and standardized to corresponding
total GAPDH densitometry values obtained from the same sample. Statistical analyses
were performed using a paired Student’s t-test. P values of <0.05 were considered
This work was supported by NIH grant HL073399 (to M.A.Q.S.).
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CLP-1 associates with MyoD
Journal of Cell Science