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
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