JCS ePress online publication date 1 September 2009
Research Article
3481
The small chromatin-binding protein p8 coordinates
the association of anti-proliferative and pro-myogenic
proteins at the myogenin promoter
Ramkumar Sambasivan1,*,‡, Sirisha Cheedipudi1,*, NagaRekha Pasupuleti1,§, Amena Saleh1,
Grace K. Pavlath2 and Jyotsna Dhawan1,¶
1
Centre for Cellular and Molecular Biology, Hyderabad, 500 007 India
Department of Pharmacology, Emory University, Atlanta, GA 30322, USA
2
*These authors contributed equally to this work
‡
Present address: CNRS URA 2578, Department of Developmental Biology, Institut Pasteur, 75724 Cedex 15, Paris, France
§
Present address: Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, USA
¶
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 2 July 2009
Journal of Cell Science 122, 3481-3491 Published by The Company of Biologists 2009
doi:10.1242/jcs.048678
Summary
Quiescent muscle progenitors called satellite cells persist in adult
skeletal muscle and, upon injury to muscle, re-enter the cell
cycle and either undergo self-renewal or differentiate to
regenerate lost myofibers. Using synchronized cultures of
C2C12 myoblasts to model these divergent programs, we show
that p8 (also known as Nupr1), a G1-induced gene, negatively
regulates the cell cycle and promotes myogenic differentiation.
p8 is a small chromatin protein related to the high mobility
group (HMG) family of architectural factors and binds to
histone acetyltransferase p300 (p300, also known as CBP). We
confirm this interaction and show that p300-dependent events
(Myc expression, global histone acetylation and posttranslational acetylation of the myogenic regulator MyoD) are
all affected in p8-knockdown myoblasts, correlating with
Key words: p8 (Nupr1), G1, Myoblast, MyoD, Gene trap, RNAi,
Satellite cell, p300, p68, Ddx5, Yeast two-hybrid
Introduction
Myogenic differentiation is an ordered process during which
determined myoblasts withdraw from the cell cycle and, under
instructions from a set of myogenic transcription factors, the
expression of muscle-specific genes within these cells is sequentially
activated. Differentiation proceeds by fusion of mononucleated
precursors into multinucleated myotubes, which assemble the
specialized contractile cytoskeleton of skeletal muscle. Whereas
cell-cycle arrest is essential for differentiation, muscle cells can also
withdraw into a reversible quiescent state in which tissue-specific
gene expression is suppressed. These alternate ‘out-of-cycle’ states
(permanent vs temporary) might result from coordination vs
uncoupling of the programs of cell-cycle exit and differentiation.
The mechanisms that regulate this divergence are poorly understood,
and are relevant to an understanding of how a balance of precursor
and differentiated cells arises during muscle formation and
regeneration.
Much of our understanding about the molecular nature of
crosstalk between the cell cycle and myogenesis comes from
analysis of the C2C12 myogenic cell line (Olson, 1992; Halevy et
al., 1995; Andres and Walsh, 1996). Inhibitory interactions between
cell-cycle activators and lineage-specific transcription factors
underlie the mutually exclusive programs of proliferation and
differentiation (reviewed by Wei and Paterson, 2001; Lassar et al.,
1994). Of the muscle regulatory factors [MRFs; a family of basic
helix-loop-helix (bHLH) transcription factors that orchestrate
myogenesis], the determination factors MyoD and Myf5 are
expressed in proliferating, undifferentiated myoblasts (reviewed by
Kitzmann and Fernandez, 2001), whereas myogenin is induced
during early differentiation (Wright et al., 1989; Andres and Walsh,
1996), activated in culture by serum withdrawal. Cell-cycle
inhibitors reinforce the action of differentiation-promoting MRFs.
For example, induction of the cyclin-dependent kinase inhibitor
(CDKI) p21 (a transcriptional target of MyoD) in myogeninexpressing cells (Halevy et al., 1995; Andres and Walsh, 1996) is
followed by a cascade of muscle-specific gene activation and fusion
into multinucleated post-mitotic myotubes (reviewed by
Buckingham, 2001). By contrast, exit of undifferentiated myoblasts
into G0 is accompanied by loss of MyoD expression, lack of
myogenin and p21 induction, and, consequently, absence of musclespecific gene induction (Milasincic et al., 1996; Kitzmann et al.,
1998; Sachidanandan et al., 2002; Dhawan and Helfman, 2004).
G0 is reversible, and the G0-G1 transition in myoblasts is
accompanied by reactivation of MyoD expression (Gopinath et al.,
2007) (reviewed by Dhawan and Rando, 2005). Thus, cell-cycle
re-entry correlates with the re-emergence of the capacity to
differentiate, but differentiation itself requires additional signals.
Competence for differentiation is confined to the G1 phase of
the myoblast cell cycle (Clegg et al., 1987). This phase, prior to
the retinoblastoma (Rb)-controlled restriction point, is the period
repression of MyoD target-gene expression and severely
defective differentiation. We report two new partners for p8
that support a role in muscle-specific gene regulation: p68
(Ddx5), an RNA helicase reported to bind both p300 and MyoD,
and MyoD itself. We show that, similar to MyoD and p300, p8
and p68 are located at the myogenin promoter, and that
knockdown of p8 compromises chromatin association of all four
proteins. Thus, p8 represents a new node in a chromatin
regulatory network that coordinates myogenic differentiation
with cell-cycle exit.
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when cells are responsive to environmental cues (Planas-Silva and
Weinberg, 1997). Thus, cells must be in cycle in order to respond
to extracellular differentiation signals; arrested cells cannot. The
molecular logic for this restriction is that MyoD expression is
suppressed in G0 (Milasincic et al., 1996; Kitzmann et al., 1998).
MyoD expression is regulated by multiple factors, including Pax3
and Pax7 (reviewed by Buckingham 2001), serum response factor
(SRF) (Carnac et al., 1998; L’Honore et al., 2003; Gopinath et al.,
2007), and the transcriptional co-activator p300 (also known as
CBP), a histone acetyl transferase (HAT) (reviewed by McKinsey
et al., 2001; McKinsey et al., 2002). Post-translational modifications
of MyoD also modulate its ability to promote differentiation. In
particular, p300-HAT-dependent acetylation of MyoD is crucial
(Polesskaya et al., 2001; Puri et al., 1997b; Dilworth et al., 2004),
whereas deacetylation of MyoD by HDAC1 silences MyoD
transcriptional function (Mal et al., 2001).
Although occupancy of its target promoters is the rate-limiting
step for transcriptional activation by MyoD, remodeling of
chromatin at these sites is also essential for efficient transcription
(Gerber et al., 1997; de la Serna et al., 2005). MyoD-HDAC
(histone deacetylase) complexes repressing muscle-specific
promoters in growing myoblasts are replaced by MyoD-HAT
complexes in differentiation conditions, leading to increased
accessibility and robust transcriptional activation (Mal and Harter,
2003; Yuan et al., 1996; Puri et al., 1997b; McKinsey et al., 2001).
p300-HAT activity is therefore required not only for acetylation
of MyoD protein but also for histone acetylation at the target
promoters of MyoD.
In this study, we analyzed the expression and function of p8 [also
known as Nupr1 and Com1 (Ree et al., 2000)], a small nuclear
protein related to the HMGA1 family of chromatin architectural
factors. We identified p8 in a gene-trap screen for loci induced in
synchronized myoblasts (Sambasivan et al., 2008). However, this
p300-binding phospho-protein has been implicated in the stress
response (Vasseur et al., 2004), growth control (Malicet et al., 2003;
Vasseur et al., 2002a; Vasseur et al., 1999), tumorigenesis (Vasseur
et al., 2002b; Iovanna, 2002), metastasis (Ito et al., 2003) and cardiac
hypertrophy (Goruppi et al., 2007). Here, we report a functional
analysis of p8 in C2C12 myoblasts using RNA interference (RNAi),
overexpression and yeast two-hybrid analysis. Our findings
implicate p8 in the negative regulation of the cell cycle, and in the
coordination of chromatin and transcriptional regulators to promote
an early step in myogenic differentiation.
Results
Suspension culture in methylcellulose medium generates
homogeneous G0-arrested populations, whereas allowing the G0
cells to reattach leads to rapid and synchronous re-entry into G1
(Milasincic et al., 1996; Sachidanandan et al., 2002). This system
facilitates the identification of G1-regulated genes, opening a
window into the myogenic events that might be coupled to this
phase. In a previous study, we used a gene-trap approach in
combination with flow cytometry to screen for genes that were
induced in G0 but suppressed during the subsequent S phase
(Sambasivan et al., 2008; Sebastian et al., 2009). The nuclear
protein p8 (Mallo et al., 1997) was one of 15 genes identified by
this strategy. Expression of lacZ reporter RNA in the p8 genetrap clone gtQ39 was induced >fourfold in G0-synchronized
myoblasts as compared with asynchronously growing myoblasts,
as was the endogenous p8 mRNA in parental C2C12 cells (Fig.
1A).
Fig. 1. Expression of p8 during the cell cycle. (A) Northern blot analysis of
total RNA isolated from growing myoblasts (Mb) and arrested myoblasts (G0)
of the p8 gene-trap clone gtQ39 (top panels) and parental C2C12 cells (bottom
panels). In gtQ39 cells, the β-galactosidase (βgal) probe detects a gene-trap
fusion transcript (exon 1 of p8 fused to the βgal-coding sequences) that is
upregulated ~fivefold in G0. Expression of endogenous p8 RNA in parental
C2C12 cells (detected using a p8-specific probe) is also induced >fivefold by
arrest. 28S rRNA and L7 RNA serve as loading controls. (B) p8 expression is
further activated during the G0-G1 transition, reaching 20-fold induction in
mid-G1. G0, northern blot analysis of arrested myoblasts 48 hours after
induction of arrest; R2-24, G0-synchronized cells reactivated into the cell
cycle for the indicated number of hours. Histone mRNA reports the proportion
of cells in S phase. (C) MyoD expression during a timecourse of reversible
arrest as in B. Expression of the MyoD transcript is induced in G1, later than
p8 expression.
p8 gene expression is transiently induced in G1
p8 encodes a small DNA-binding protein related to the HMGA1
family and has been implicated in the control of cell proliferation
(Vasseur et al., 2002a; Malicet et al., 2003; Malicet et al., 2006).
To address a possible role for p8 in growth control of muscle cells,
we analyzed its expression during exit from reversible arrest (Fig.
1B). To monitor cell-cycle status, we used the S-phase-specific
protein histone H2B – expression was absent in G0, induced at 14
hours of reactivation and peaked at 24 hours, consistent with timing
of the S phase (Sachidanandan et al., 2002). By contrast, p8 mRNA,
although expressed in G0, was strongly upregulated early during
cell-cycle re-entry (2 hours), peaking at 6 hours (early G1) but
returned to basal levels by 14 hours, well before the peak of S phase.
This repression prior to S phase despite strong transient induction
in G1 accounts for the recovery of p8 in the original screen. Notably,
the peak of p8 expression precedes the induction of MyoD
expression in G1 (Fig. 1C). Taken together, these results are
consistent with a role for p8 during G0-G1.
p8 negatively regulates the myoblast cell cycle
To address the function of p8 in muscle cells, we used a knockdown strategy, using RNAi with short hairpin RNAs (shRNAs) (Yu
et al., 2002) targeted against the p8 mRNA. Of the four shRNAs
tested (p8sh1-p8sh4), growing myoblasts of the p8sh2 and p8sh4
pools exhibited a ~60% reduction in the steady-state level of p8
mRNA compared with the vector control pool (Fig. 2A). The p8sh1
pool, which showed unperturbed levels of p8 mRNA, was used as
an additional control.
Quantitative real-time reverse transcriptase (RT)-PCR analysis
of a timecourse of activation from 2 to 24 hours in two independent
pools showed suppression of the p8 transcript levels throughout the
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p8 function in reversibly arrested myoblasts
Fig. 2. p8 negatively regulates the cell cycle: precocious S-phase entry in p8-knockdown myoblasts. (A) Endogenous p8 mRNA levels analyzed by northern
blotting in asynchronous cultures of pools generated by the expression of four independent shRNAs. p8sh2 and p8sh4 pools showed significantly reduced p8 RNA
levels compared with control cells transfected with empty vector ‘C’, whereas the p8sh1 pool showed no reduction and subsequently served as a control shRNA
pool. (B) Quantification of p8 mRNA levels during a timecourse of reactivation in two independent knockdown pools relative to control cells expressing GFP
shRNA: quantitative real-time RT-PCR reveals suppression of p8 mRNA throughout the timecourse (control cell mRNA levels at each time point=1). (C) p8 protein
levels are suppressed in p8-knockdown cells (p8-sh) vs GFP-sh control cells (con-sh) in mid-G1 (6 hours after reactivation). (D) Flow cytometric cell-cycle
analysis of p8-knockdown cells (p8sh2 pool, p8sh4 pool, p8sh4 clone 12) and control cells containing empty vector. DNA content associated with each phase of the
cell cycle of asynchronously growing myoblasts (Mb) and G0-synchronized populations reactivated for 6, 12 or 24 hours (R6, R16, R24) is shown. Note that the
majority of control cells are still in G1 at R16, whereas the knockdown cells have already progressed into S phase (arrowheads), with a reduction in the G1 peak.
(E) Quantification of the proportion of cells in S phase from data shown in D. Distribution of cells during asynchronous growth and arrest is not affected in the p8knockdown lines (Mb and R6 points). However, all shRNA pools enter S phase precociously at 16 hours of reactivation, ahead of control cells at 24 hours.
(F) Precocious histone expression in p8-knockdown cells confirms FACS analysis. Northern blot shows asynchronously growing myoblasts (‘G’), arrested
myoblasts (‘A’) and synchronized cells reactivated into the cell cycle for 6 hours (‘R6’). Induction of p8 mRNA at R6 was specifically attenuated in p8sh4
knockdown cells. Histone mRNA reports for S phase and shows accelerated expression in the p8sh4 pool. Induction of expression of MyoD mRNA during G1 reentry is also blunted in the p8-knockdown cells. (G) Quantification of normalized levels of histone and p8 mRNA in knockdown pool p8sh4 compared with p8sh1
control cells. Note the reciprocal relationship between control and knockdown cells with respect to p8 and histone mRNAs specifically at R6.
cell cycle but especially in mid-late G1 (Fig. 2B). p8 protein
expression was also inhibited: Fig. 2C shows suppression in p8sh4knockdown cells at the time of peak expression in late G1.
To monitor the consequences of p8 knockdown, the timing and
extent of arrest in methylcellulose suspension culture and
reactivation following reattachment were analyzed using flow
cytometry. Growing cells of control (p8sh1) and knockdown [p8sh2
and p8sh4 pools and a clone derived from the p8sh4 pool (sh4 clone
12)] lines were compared with cells reactivated from arrest for
different periods. Asynchronous populations of control and both
p8-knockdown lines showed a similar cell-cycle profile (Fig. 2D).
At 6 hours after reactivation from arrest, 90% of cells in both control
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and knockdown pools exhibited a G1 DNA content. However, at
16 hours of reactivation, whereas only 6% of control cells had
entered S phase, significantly more knockdown cells had done so
(14%, 25% and 50% in p8sh4-clone 12, p8sh4 pool and p8sh2 pool,
respectively) (Fig. 2E). Control pools showed a substantial S-phase
population only at 24 hours after reactivation. Taken together, these
observations implicate p8 in the negative regulation of the cell cycle
during G1.
To assess the effects of p8 knockdown on gene expression in
G1, RNA was isolated from growing cells, G0-arrested cells and
reactivated (mid-G1) cells of the control (p8sh1) and knockdown
(p8sh4) pools. Northern blot analysis (Fig. 2F) showed a marked
attenuation of p8-mRNA induction in the p8sh4 pool during G1.
When compared with the respective G0 sample, p8 mRNA
expression was induced tenfold in G1 in control cells, but not
induced at all in the p8sh4 pool (quantified in Fig. 2G, lower panel).
Thus, as in Fig. 2B, the effect of RNAi on p8 expression was
strongest during the reactivation of myoblasts from G0 into G1.
Altering p8 expression affected both cell-cycle- and myogenicmarker expression. In control cells, S-phase-specific histone H2B
expression was suppressed during G0 as expected and was yet-tobe induced at 6 hours of reactivation, consistent with the peak of
S phase at >16 hours (Fig. 2F). By contrast, in knockdown cells
(p8sh4 pool), histone mRNA was not as severely downregulated in
G0 as in control cells and, by 6 hours of reactivation, knockdown
cells had started to re-express histone mRNA, suggesting precocious
entry into S-phase (quantified in Fig. 2G). Similar results were seen
with the p8sh2 pool and p8sh4 clone 12 (not shown). Reduced p8
expression correlated with more rapid re-expression of histone
mRNA, consistent with faster re-entry into S phase, implying a role
for p8 in negative control of the G1-S transition in myoblasts. MyoDmRNA induction in G1 was mildly reduced (twofold) in the p8knockdown pool. Thus, a G1-induced transcript (MyoD) and an Sphase-specific transcript (histone) showed altered levels and timing
in p8-knockdown myoblasts.
p8-knockdown cells fail to differentiate
Myogenic differentiation is coupled to the G1 phase of the cell cycle
(Clegg et al., 1987; Kitzmann et al., 1998). During differentiation,
endogenous p8 expression was mildly induced at a stage when
expression of myogenin was already activated (Fig. 3A), consistent
with a role in myogenic-gene activation. Knockdown of p8 using
either p8sh2 or p8sh4 led to reduced p8 transcript levels throughout
the timecourse of differentiation (Fig. 3B). To explore the role of
p8 in myogenesis, cells were incubated in low serum for 3 days.
Whereas control cells expressed myosin heavy chain and fused to
form multinucleated myotubes (fusion index 35-40%), p8knockdown cells showed no fusion at all and only 9% of p8sh2
and 2.5% of p8sh4 cells expressed the muscle-specific myosin.
Thus, compromising p8 expression also affects differentiation
competence (Fig. 3C). To investigate the timing of the differentiation
defect, control (shGFP) and knockdown (p8sh4) myoblasts were
cultured in differentiation conditions for 12 hours and expression
of early markers monitored. p8 protein levels were reduced in the
p8sh4 cells (Fig. 3D). Interestingly, whereas MyoD protein levels
were unaffected, induction of its target myogenin was strongly
suppressed in knockdown cells. Expression of the CDKI p21,
another target of MyoD with an important role in coupling arrest
to differentiation, was also suppressed. However, expression of
another CDKI, p27 (a marker of arrest that is not a direct MyoD
target), was not affected, consistent with the ability of the p8knockdown cells to exit the cell cycle (Fig. 2D).
Taken together, these observations are consistent with a role for
p8 in regulating the ability of MyoD to activate its transcriptional
targets.
Overexpression of p8 arrests the cell cycle and inhibits
muscle-marker expression
To confirm the role of p8 in the control of myoblast growth and
differentiation, we used ectopic expression of a human p8-FLAG
construct in C2C12 myoblasts (Fig. 4). Whereas attenuation of p8
Fig. 3. p8 positively regulates myogenesis: knockdown
myoblasts are severely differentiation-defective. (A) p8
expression is mildly but progressively induced during
differentiation. Growing cultures (Mb) were shifted to
differentiation medium for 6-72 hours and total RNA
analyzed. The induction of myogenin, an early myogenic
marker, indicates the progress of differentiation.
(B) Quantitative real-time RT-PCR analysis of p8 mRNA
suppression during a timecourse of differentiation in two
independent knockdown lines, p8sh2 and p8sh4. Values
represent the fold reduction of p8 mRNA levels in p8sh2 and
p8sh4 cells relative to levels in GFP-sh control cells (n=3).
(C) GFP-sh control (con), p8sh2 pool and p8sh4 cells were
maintained in differentiation medium for 3 days and
immunostained for the muscle-specific myosin heavy chain
(Alexa Fluor 594). Control cells differentiated efficiently
(fusion index 34%), whereas both p8-knockdown lines
showed some myosin-positive mononucleated ‘needles’ but
no fusion (fusion index 0%). (D) Control (shGFP) and p8sh4
knockdown cells (sh4) were incubated in differentiation
medium for 12 hours and analyzed by immunoblotting for
p8 protein, myogenic markers (MyoD, myogenin) and cellcycle inhibitors (p21, p27). Desmin was used as a loading
control.
p8 function in reversibly arrested myoblasts
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differentiation. p300 also represses the transcription of Myc, an
essential regulator of the G0-G1 transition (Kolli et al., 2001;
Baluchamy et al., 2003). Thus, similar to p8, p300 promotes
myogenesis and slows the cell cycle. To investigate whether the
phenotype of p8-knockdown myoblasts (rapid S-phase entry and
depressed MyoD function) is consistent with altered p300-dependent
activities, we used three assays. First, we used quantitative RT-PCR
to assess the levels of Myc expression during the synchronized cell
cycle (Fig. 5A). In p8-knockdown cells, the expression of Myc
mRNA was induced to higher levels and was sustained into late
G1, consistent with the accelerated S-phase entry as well as
inhibition of myogenesis (Miner and Wold, 1991).
Second, to determine the acetylation status of histones, we used
western blotting with antibodies specific for histone modifications
carried out by p300. A mild global reduction of histone acetylation
[18% reduction of acetylated H3K18 (H3K18-Ac)] was observed
in p8-knockdown cells, suggesting that p8 is a positive regulator
of the HAT activity of p300 (Fig. 5B).
Finally, to determine the acetylation status of MyoD protein, we
immunoprecipitated MyoD, followed by immunoblotting with a
pan-acetyl lysine antibody (Fig. 5C). For equal amounts of MyoD
Fig. 4. Overexpression of p8 leads to cell-cycle exit with suppression of
myogenic markers. (A,B) C2C12 myoblasts were transfected with control
EGFP plasmid or FLAG-tagged human p8, and BrdU incorporation as well as
the expression of myogenic and cell-cycle markers determined by
immunofluorescence analysis (A). In the topmost panel, transfected cells
(EGFP or hp8-FLAG) are red (Alexa Fluor 594), and BrdU+ nuclei are green.
In all other panels, transfected cells are green and the endogenous markers
(MyoD, p27, myogenin and p21) are red. At least 250 transfected cells were
counted in each of two independent experiments and the results quantified B.
expression correlated with accelerated S-phase entry, overexpression
of p8 completely inhibited BrdU incorporation, confirming that p8
negatively regulates the myoblast cell cycle. Whereas p21 was not
affected, cells expressing p8-FLAG showed induction of p27,
consistent with the inhibition of DNA synthesis. However, MyoD
expression, which was seen in ~50% of control EGFP-transfected
myoblasts, was strongly repressed in p8-FLAG cells, and myogenin
expression was not activated 24 hours after serum withdrawal. Thus,
although endogenous levels of p8 are required for myogenesis (Fig.
3), sustained overexpression of this chromatin factor leads to arrest
in a state that is refractory to differentiation.
Molecular phenotype of p8-knockdown myoblasts is consistent
with altered p300 function
p8 is related to the HMGA1 (formerly HMG I/Y) family (Encinar
et al., 2001) of chromatin architectural proteins and binds to the
HAT p300 in a complex that regulates glucagon gene expression
(Hoffmeister et al., 2002). In addition to histones, p300 acetylates
MyoD (Polesskaya et al., 2000) and serves as its co-activator (Yuan
et al., 1996; Puri et al., 1997a), promoting MyoD function in
Fig. 5. p300-dependent events are affected in p8-knockdown cells: increased
Myc expression and decreased acetylation of histones and MyoD. (A) Myc
transcripts were quantified using real-time RT-PCR in RNA isolated from p8knockdown (sh-p8) and control (sh-gfp) cells. Asynchronously growing
myoblasts (‘G’), G0-arrested myoblasts (‘A’), synchronously reactivated
myoblasts at 6 or 12 hours after cell-cycle re-entry (R6 and R12) were
analyzed. Values represent normalized fold differences in Myc mRNA levels
[mean ± s.d. (n=6)]. (B) Analysis of total histone acetylation in p8-knockdown
(shp8) and control (shgfp) cells. Total protein was probed with histoneacetylation-specific antibodies that detect modifications characteristic of p300
activity. Blots were reprobed with an antibody that recognizes all forms of H3
to determine the percent of protein that was acetylated. Data shown are
representative of three independent experiments and show up to 18% reduction
in global histone-3 (H3) acetylation. (C) Analysis of MyoD acetylation status
in p8-knockdown (shp8) and control (shGFP) cells. MyoD was
immunoprecipitated from nuclear protein extracts with anti-MyoD polyclonal
antibody (lanes marked +) and acetylation detected by immunoblotting with a
pan-acetyl lysine antibody. Negative controls (lanes marked –) used equal
amounts of rabbit IgG. Reprobing of the same blot with MyoD antibody was
used to determine the percent of immunoprecipitated protein that was
acetylated. Data shown are representative of three independent experiments.
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protein, acetylation of MyoD in p8-knockdown myoblasts was only
38% of the level seen in control myoblasts. As p300 is known to
acetylate MyoD as well as bind p8, our data are consistent with the
model that lowering p8 expression compromises the function of
p300 in post-translational modification of MyoD. Taken together,
these data demonstrate that p8 regulates molecular events that are
known targets of p300, consistent with p8 functioning through a
p300-dependent mechanism.
p8 interacts with two other pro-myogenic p300-binding
proteins: the RNA helicase p68 and the myogenic regulator
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Journal of Cell Science
To gain further insight into the function of p8, we used a yeast twohybrid strategy to screen for proteins that interact with full-length
mouse p8 (N.P., A.S. and J.D., unpublished). Among the candidates
for a role in p8 function in myoblasts was p68 (Ddx5), another
p300-binding protein (Fig. 6A). This DEAD-box RNA helicase is
reported to complex not only with p300 (Rossow and Janknecht,
2003), but also with MyoD and SRA, a non-coding RNA (Caretti
et al., 2006). Silencing of p68 suppresses myogenesis via
mechanisms that are thought to involve altered chromatin
remodeling by the ATP-dependent SWI-SNF Brg1 complex.
Interestingly, p8 features in the list of genes that are downregulated
by p68 RNAi (Caretti et al., 2006), suggesting that p68 is an
upstream activator of p8. Indeed, p68 induction during G1 precedes
the activation of p8 (data not shown).
To confirm the interaction between p8 and p68 we used two
further tests. First, we used a mammalian two-hybrid assay. Fulllength mouse p8 and p68 cDNAs were cloned into pBIND and
pACT, respectively, and transfected into C2C12 cells along with
the pluc5 reporter construct (Fig. 6B). Luciferase activity was
induced twofold in the presence of both constructs, indicating a
weak but reproducible interaction in mammalian cells. Second, we
used an in vitro binding assay. Purified His-tagged mouse p8 protein
was able to pull down p68 from C2C12 myoblast lysate (Fig. 6C).
We also confirmed that p8 interacts with p300 in this assay (Fig.
6C). Notably, neither p68 nor p300 expression was affected in the
p8-knockdown cells (Fig. 6D).
The ability of p8 to affect MyoD acetylation as well as to interact
with two important chromatin regulators that are also known to bind
MyoD suggested the possibility of a direct interaction between p8 and
MyoD. Therefore, we tested whether p8 could directly bind MyoD. As
with p68 and p300, His-tagged p8 could specifically pull down MyoD
from myoblast lysates (Fig. 6C). The specificity of the interaction
between p8 and either MyoD, p68 or p300 was underscored by the
inability of His-p8 to pull down histone H3 (Fig. 6C).
Fig. 6. Interaction of p8 with the RNA helicase
p68 and p300. (A) Yeast two-hybrid analysis: the
PJ69-4A yeast strain was co-transformed with the
p8-GAL4-DNA-binding domain fusion construct
and p68-GAL4-activation domain fusion
construct obtained in the yeast two-hybrid screen.
Transformants were grown on plates lacking Trp,
Leu and Ade (–TLA), to confirm activation of
the adenine reporter gene (pink colonies).
Activation of the β-gal reporter was confirmed on
plates supplemented with X-gal (–TL+X-gal,
blue colonies). Four independent colonies are
shown. Interaction between Drosophila Trithorax
and GAGA factor was used a positive control and
the empty vectors were used as negative controls.
(B) Mammalian two-hybrid analysis: p8 and p68
were cloned into the mammalian two-hybrid
vectors pBIND (BD vector) and pACT (AD
vector), respectively, transfected individually or
together into C2C12 myoblasts along with the
reporter gene pluc5 (luc) and luciferase activity
measured and normalized to a co-transfected lacZ
gene. ‘E’ refers to the respective empty vector in
control transfections. Co-transfection of p68AD
and p8BD showed a ~twofold induction over
negative controls. The positive control (Id-BD +
MyoD-AD, not shown) yielded a value of
626±4.8 relative light units (rlu). (C) In vitro
pulldown assay: His-tagged mouse p8 was
purified from E. coli using Ni-agarose beads
(Ni+p8), incubated with C2C12 nuclear extract,
and the bound fraction displayed on SDS-PAGE
followed by immunoblotting with antibodies
against p8, p68 or p300. One tenth of the input
extract (‘I’) was loaded to verify the size of the
precipitated proteins. Negative controls for each
pulldown experiment included extract incubated
with beads without p8 protein (Ni) and p8 beads
without extract (Ni+p8). The specificity of the
pulldown of p68, p300 and MyoD from the cell extract by His-p8 was determined by absence of pulldown of the very abundant nuclear protein histone 3 (H3,
lower panel). Data are representative of three independent experiments. (D) Western blot analysis confirms that p300 and p68 protein levels are not altered in p8knockdown myoblasts. (Fig. 3D shows that MyoD levels are not significantly affected by p8 knockdown.) (E) Co-immunoprecipitation analysis confirms that p8
interacts with MyoD. Mouse MyoD-YFP was co-transfected into HEK293 cells along with human p8-FLAG, immunoprecipitated with anti-FLAG antibody and
western blotted with either anti-MyoD or anti-FLAG. IgG denotes the antibody heavy chain. Data are representative of three independent experiments.
p8 function in reversibly arrested myoblasts
To confirm the interaction between p8 and MyoD, we
coexpressed MyoD-YFP and FLAG-p8 in HEK293 cells.
Immunoprecipitation of p8 with anti-FLAG also recovered MyoD,
providing further evidence for their interaction. Thus, p8 interacts
with three proteins already reported to individually interact with
each other – p300, p68 and MyoD.
Journal of Cell Science
p8 associates with chromatin at the myogenin promoter and
regulates the association of p68, p300 and MyoD
Direct interaction of p8 with three chromatin-binding proteins and
transcriptional activators, two of which (MyoD and p300) are known
to bind the myogenin promoter, suggested that p8 might also be
associated at this element. The myogenin promoter is a key genomic
target of mechanisms that regulate differentiation. Interestingly, not
only were MyoD and p300 detected at this site, but p8 itself and
the RNA helicase p68 also bound to this early differentiation
promoter (Fig. 7). Furthermore, the association of all four proteins
was reduced in p8-knockdown cells. Whereas reduced association
of p8 could be attributed to lower levels of p8 protein in knockdown
cells (Fig. 2C; Fig. 3D), reduced association of the three binding
partners of p8 is not due to lower expression levels (Fig. 3D; Fig.
6D). Thus, occupancy of the myogenin promoter by all three p8interacting proteins – MyoD, p300 and p68 – was dependent on p8
expression and chromatin association.
Taken together, our findings demonstrate that, in myoblasts, p8
physically binds to three other transcriptional modulators and coactivators with a common chromatin target – the early musclespecific myogenin promoter – and regulates not only their chromatin
association but also the post-translational modification and function
of MyoD, a key activator of this promoter. Thus, p8 is a cell-cycleregulated molecule that orchestrates the functioning of an important
early regulatory hub during myogenic differentiation (Fig. 8).
Discussion
In this study, we investigated the function of p8, a small chromatinbinding protein whose expression peaks in G1 (Sambasivan et al.,
2008). We establish that p8 inhibits cell-cycle progression and
regulates myogenic differentiation. We confirm that p8 binds the
HAT p300, and report two new interactions with proteins also known
to bind p300: the RNA helicase p68 and the myogenic regulator
3487
MyoD. We show that p8 is required for MyoD acetylation, itself
associates with the myogenin promoter, and regulates association
of MyoD, p300 and p68 with this promoter. Our findings suggest
that p8 orchestrates the action of chromatin factors that converge
to regulate an early muscle-specific promoter, and represents a new
node in which control of myogenic differentiation intersects with
the cell cycle.
p8 negatively regulates the cell cycle: control of G1 kinetics
and the transition to differentiation
p8 has been previously implicated in growth control: knockout
fibroblasts show reduced cell-cycle time (Vasseur et al., 2002a;
Malicet et al., 2003), suggesting that this chromatin-binding protein
inhibits cell proliferation. In myoblasts, as in fibroblasts, p8 acts
as a brake on the cell cycle. Altering p8 expression in opposite
ways has reciprocal effects on DNA synthesis: reducing p8
expression accelerated the kinetics of G1 progression to S phase,
whereas ectopic expression of p8 led to arrest in G0-G1. These
results suggest that not only induction of p8 in early-mid G1, but
its repression prior to S phase, are essential for normal cell-cycle
kinetics. The effects of p8 on G1 kinetics might be mediated by
induction of the G1 CDKIs p27 and p21, and by negative control
of Myc, a major regulator of progression that acts on a large number
of cell-cycle promoters (Gartel and Shchors, 2003; Wanzel et al.,
2003). Sustained expression of Myc is also known to suppress
differentiation (Miner and Wold, 1991).
The ‘shrinking’ of G1 caused by reduced p8 expression is
associated with a loss of tissue-specific gene activation, suggesting
that p8 might control events in G1 that bridge the programs of
proliferation and differentiation. MyoD plays a key role at this
intersection by two mechanisms: transcriptional induction of cellcycle inhibitors (Halevy et al., 1995; Magenta et al., 2003) and
myogenic activators, as well as direct binding of Cdk4 to inhibit
phosphorylation of Rb (Zhang et al., 1999b), promoting
differentiation-coupled arrest. In this context, our finding that p8
not only negatively regulates the G1-S transition but also positively
modulates MyoD function suggests participation in the mechanism
that restricts myogenic differentiation to the G1 phase.
p8-knockdown myoblasts failed to differentiate owing to the
absence of key early differentiation regulators, namely myogenin
and p21. However, p8 overexpression in growing myoblasts did
not enhance the myogenic program. This is probably an indirect
consequence of arrest in early G1 prior to MyoD activation,
blocking the development of differentiation competence.
Partners of p8: p300, p68 and MyoD
Fig. 7. p8 regulates the association of p68, p300 and MyoD with the myogenin
promoter. Chromatin immunoprecipitation analysis of myogenin-promoter
occupancy in control and knockdown myoblasts 24 hours after shift to
differentiation medium (mean ± s.d., n=4; P-values: *0.05; **0.005). All four
proteins (p8, p68, p300, MyoD) are substantially depleted from the myogenin
promoter in p8-knockdown myoblasts, whereas H3 association is unchanged.
Although loss of p8 association reflects the specific knockdown of p8 protein,
expression levels of p300, p68 and MyoD are unaltered (see Fig. 6D and Fig.
3D, respectively).
p8 shares 35% amino acid identity with the HMGA1 (formerly
HMGI/Y) chromatin architectural factors (Encinar et al., 2001) and
can regulate the activity of different transcription factors, such as
Smad (Garcia-Montero et al., 2001), p53 (Clark et al., 2008), Jun
and other established AP1 effectors (Goruppi et al., 2007). p8 also
binds to p300 and Pax2-transactivation domain interacting protein
(PTIP) to regulate the activity of Pax2A and Pax2B on the glucagon
promoter (Hoffmeister et al., 2002).
In myoblasts, interactions with other chromatin proteins also point
to a mechanism by which p8 influences tissue-specific gene
expression. We show that p8 binds three proteins: the HAT p300,
the RNA helicase p68 and MyoD. The role of p300 in the control
of myogenesis and the cell cycle is well established. p300 influences
two activities that are de-regulated in p8-knockdown myoblasts:
MyoD function (both by acetylating MyoD and by acting as its co-
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Journal of Cell Science 122 (19)
Journal of Cell Science
Fig. 8. Model for p8 function at the myogenin promoter. p8 might
act to nucleate a group of chromatin-binding and transcription
factors – p68, p300 and MyoD – required for the activation of the
myogenin promoter. All three proteins share anti-proliferative and
pro-myogenic effects with p8 and are shown to interact, suggesting
the possibility that they act as a complex. However, our data do not
distinguish between the alternative models shown here: a complex
in which p8 co-interacts with all three factors (A), or individual
interactions of p68, p300 or MyoD with p8 (B). Because strong
expression of p8 marks early-mid G1, this regulatory node might
participate in restricting competence for myogenic differentiation
to the G1 phase of the cell cycle.
activator via HAT activity) (Polesskaya et al., 2001; Yuan et al.,
1996; Puri et al., 1997a; Sartorelli et al., 1997; Magenta et al., 2003),
and Myc expression (Kolli et al., 2001; Baluchamy et al., 2003).
Our study confirms the direct interaction of p8 with p300 and
establishes a crucial role for p8 in p300 function, because common
as well as cell-type-specific p300-dependent activities are altered
when p8 expression is compromised.
The identification of the RNA helicase p68 as a partner of p8 in
an unbiased interaction screen expands the scope of this small
chromatin-binding factor, because interactions are reported between
p68 and p300 (Rossow and Janknecht, 2003), as well as between
p68 and MyoD (Caretti et al., 2006). In conjunction with the noncoding RNA SRA, p68 is essential for muscle differentiation.
Although ubiquitously expressed, both p300 and p68 are particularly
relevant to myogenesis because they enhance MyoD function.
Pharmacological inhibition or genetic ablation of p300 HAT activity
(Polesskaya et al., 2001; Roth et al., 2003) or silencing of p68
(Caretti et al., 2006) all phenocopy the p8 knockdown: the
expression of myogenin and myosin heavy chain as well as
myoblast fusion competence are compromised, much as when
MyoD itself is inactivated (Yablonka-Reuveni, et al., 1999).
The mechanism by which p8 affects differentiation probably
involves reduced acetylation by p300 of MyoD; p300 modifies three
lysines near the bHLH domain of this protein. Acetylation leads to
increased binding to p300 and enhances MyoD activity. Similar to
MyoD, p8 is acetylated by p300, which leads to enhanced
transcriptional activity of associated tissue-specific factors such as
Pax2 (Hoffmeister et al., 2002). Analysis of p8 secondary structure
predicts a HLH motif (Goruppi et al., 2007) towards the C-terminus
(positions 46-71), suggesting the potential to dimerize with other
HLH proteins, including MyoD (Murre et al., 1989; Benezra et al.,
1990). However, the importance of the HLH motif is unresolved
because our preliminary studies suggest that overexpression of a
mutant p8 lacking helix 2 mimics wild-type p8 in inhibiting the
cell cycle as well as MyoD and myogenin expression (S.C.,
unpublished).
p8 might not interact with DNA directly but probably associates
with chromatin via transcriptional activators and co-activators
(Goruppi et al., 2007). In addition, p8 knockdown might
compromise the function of p300 and/or p68 on other targets. For
example, p300 acetylates Rb (Nguyen et al., 2004) and MEF2
(Ma et al., 2005), both of which are crucial for myogenic
differentiation. Altered chromatin remodeling might also play a
role, because MyoD normally recruits p300 to its target promoters
(Giacinti et al., 2006; Wilson and Rotwein, 2006) and p68
interacts with HDAC1 (Wilson et al., 2004), influencing the
recruitment of the Swi-Snf chromatin-remodeling protein Brg1
and the transcription machinery onto MyoD target promoters
(Caretti et al., 2006).
p8: a crucial link in a pro-myogenic and anti-proliferative
chromatin network?
Our findings suggest that four proteins – p8, p68, p300 and MyoD
– participate in a common mechanism to modulate myogenesis via
chromatin remodeling at the myogenin promoter. As shown in this
report for p8, depleting either p300, p68 or MyoD (Baluchamy et
al., 2003; Kolli et al., 2001; Caretti et al., 2006; Rudnicki et al.,
1993; Yablonka-Reuveni et al., 1999) also leads to rapid
proliferation and defective differentiation. Thus, p300, p68, MyoD
and p8 are all anti-proliferative and pro-myogenic, interact with
each other and with a common chromatin target, and similar effects
on the cell cycle and muscle differentiation seem to result by
compromising each of their functions individually. Taken together,
these findings suggest that the chromatin architectural protein p8
regulates myogenic differentiation via the effects of the chromatin
modulators p300 HAT and p68, and the myogenic determination
factor MyoD. Because p300, p68 and p8 are all expressed in G0
myoblasts (Sindhu Subramaniam and J.D., unpublished) but MyoD
is absent (Milasincic et al., 1996; Kitzmann et al., 1998;
Sachidanandan et al., 2002), the composition of p8-containing
complexes might vary in quiescent, proliferating and differentiating
muscle cells.
p8 is a candidate coordinator of the G1 differentiation control
point
p8-overexpressing cells could be staged as G0 or early G1, because
MyoD expression – characteristic of mid-G1 – is absent. Therefore,
arrest of the cell cycle by p8 led to an uncoupling of myogenesis
from cell-cycle exit. p8 seems to regulate the pace of G1 progression
such that MyoD expression is activated appropriately and MyoD
protein is sufficiently acetylated.
According to the ‘G1 model’ of cell-cycle regulation, decisions
to divide, arrest, differentiate or die are taken during this phase
(Pardee, 1989; Clegg et al., 1987; Riddle et al., 1979), when
environmental and intrinsic cues are assessed and integrated towards
an appropriate response. Cells lose their responsiveness to external
signals during the Rb-controlled restriction point at the G1-S
boundary (Planas-Silva and Weinberg, 1997). Classical studies
indicated that myogenic differentiation is only initiated during the
G1 phase (Clegg et al., 1987), whereas recent studies provide
evidence for a control point in G1 in which cell-cycle progression
and myogenic differentiation are linked (Kitzmann and Fernandez,
2001; Zhang et al., 1999a). However, although MyoD itself has
been implicated in this link, the molecular mechanisms by which
p8 function in reversibly arrested myoblasts
this restriction occurs are not clear. Our findings suggest that p8
contributes to the coupling of differentiation to the G1 phase of the
cell cycle via effects on post-translational modifications that affect
the transcriptional function of MyoD and the assembly of a cohort
of anti-proliferative and pro-myogenic regulators on an early
muscle-specific promoter. The identification of p8 as an interacting
partner of MyoD, and its association with p68 as well as p300,
expands the list of molecular players at this regulatory node,
providing a framework in which to dissect its mechanism.
Materials and Methods
Journal of Cell Science
Cell culture
C2C12 myoblasts (Yaffe and Saxel, 1977; Blau et al., 1983) were obtained from
Helen Blau (Stanford, CA) and a sub-clone A2 (Sachidanandan et al., 2002) used in
all experiments. Myoblasts were maintained in growth medium (GM: DMEM with
20% FBS). Differentiation was induced by incubating cultures in differentiation
medium (DM: DMEM with 2% horse serum), replaced daily for 3-5 days.
Differentiation is expressed as the fusion index (calculated as % of total nuclei present
in myotubes of >two nuclei).
Suspension culture was performed as described (Milasincic et al., 1996;
Sachidanandan et al., 2002). Briefly, sub-confluent cultures were harvested and
cultured as a single cell suspension (105 cells/ml) in DMEM containing 1.3% methyl
cellulose and 20% FBS. After 48 hours (>98% of cells arrested in G0), cells were
harvested by dilution and centrifugation. G0 cells were reactivated into the cell cycle
by replating in GM and harvested 6-24 hours later. Upon reattachment, G0 myoblasts
synchronously enter G1 at ~4-6 hours, with a peak of S phase at 24 hours
(Sachidanandan et al., 2002).
Immunofluorescence
Cells plated on coverslips were fixed with 3.5% formaldehyde/PBS and permeabilized
in PBS/0.2% Tween-20. Primary antibodies were diluted in PBS/10% HS/0.02%
Tween: anti-FLAG (Sigma) 1:1000; anti-MyoD (DAKO), anti-p21 (Santa Cruz), antip27 (Santa Cruz), anti-BrdU (BD Biosciences), anti-myogenin (Santa Cruz) all at
1:100; anti-myosin (A4-1025) 1:10 (Blau et al., 1983). Detection of incorporated
BrdU was performed after denaturation of DNA using 0.4 N HCl, 0.5% Tween, 0.5%
Triton X-100 as described (Sachidanandan et al., 2002). Secondary antibody was
goat anti-mouse Alexa Fluor 488 or 594 (Molecular Probes), 1:500. Secondaryantibody controls were negative; no cross-reactivity of secondary reagents was
detected. Nuclei were detected with Hoechst 33342 (1 μg/ml). Staining was recorded
on a CCD camera using an Olympus microscope equipped with epifluorescence or
on a Zeiss LSM510 Meta confocal microscope. Images were assembled using Adobe
Photoshop 6.0.
Northern blot analysis was performed as described (Sachidanandan et al., 2002)
using a probe spanning nucleotides 147-550 of mouse p8 mRNA. L-Process and
Image Gauge programs (Fuji) were used to quantify background-subtracted phosphorimager signals.
Western blot analysis was performed as described (Sachidanandan et al., 2002).
Antibodies were diluted in blocking buffer: anti-MyoD (Santa Cruz) 1:400; antimyogenin (Santa Cruz) 1:500; anti-p27 (BD Bioscience) 1:3000; anti-p21 1:1000;
p8 polyclonal antisera (kind gift of Juan Iovanna, INSERM, Marseille, France) 1:200.
HRP-conjugated secondary antibody (Bangalore Genei 1:10,000) was detected using
ECL (Amersham).
RNAi experiments
shRNA design
Potential targets for shRNA-mediated silencing of p8 expression were identified using
OligoRetriever (http://www.cshl.org/public/SCIENCE/hannon.html). Four different
shRNA hairpins were designed, each containing 21 b antisense and sense sequences
separated by a loop (5⬘-GAAGC-3⬘). For each shRNA construct, complementary
oligos (Microsynth, Switzerland) were annealed and ligated into the mU6 promoter
vector (Yu et al., 2002).
Generation of RNAi transfectants
C2C12 myoblasts were transfected (Lipofectamine, Invitrogen) with each of the
shRNA constructs plus a zeocin selection marker pKA23 at ratio of 4:1, and selected
in 200 μg/ml zeocin to generate stable pools. Individual clones were isolated by ring
cloning.
Sequences of shRNA template oligonucleotides
p8-knockdown shRNAs: P8SH-2a: 5⬘-TTTGGGCTGTCTTCCTAGCTCTGCCGAGCGGCAGAGCATGGAAGACAGCCTTTTT-3⬘; P8SH-2b: 5⬘-CTAGAAAAAGGCTGTCTTCCATGCTCTGCCGCTTCGGCAGAGCTAGGAAGACAGCC-3⬘; P8SH-4a: 5⬘-TTTGGGGCCAGGCTGTACTGATCATGAAGCATGATCTGTACACCCTGGCCCTTTTT-3⬘; P8SH-4b: 5⬘-CTAGAAAAAGGGCCAGGGTGTACAGATCATGCTTCATGATCAGTACAGCCTGGCCC-3⬘.
3489
Control shRNAs: P8SH1a: 5⬘-TTTGTCTTGCCTGTGTCTCCTTGTCGAAGCGACAAGGACTCACAGGCAAGATTTTT-3⬘; P8SH1b: 5⬘-CTAGAAAAATCTTGCCTGGAGTCCTTGTCGCTTCGACAAGGAGACACAGGCAAGA-3⬘; GFPSH1a: 5⬘-TTTGAACTTCAAGGTCCGCCACAACGAAGCGTTTTGGCGGACCTTGAAATTTTTTT-3⬘; GFPSH1b: 5⬘-CTAGAAAAAAATTTCAAGGTCCGCCAAAACGCTTCGTTGTGGCGGACCTTGAAGTT-3⬘.
Cell-cycle analysis
Flow cytometry was performed on a FACSCaliber (BD Biosciences), using CelQuest
software as described (Sachidanandan et al., 2002).
Determination of MyoD acetylation status
Immunoprecipitation of MyoD
Control or RNAi myoblasts grown to high density were incubated overnight in DM
containing sodium butyrate (1 mM) to inhibit histone decactylase activity. Nuclei
were isolated from ~107 cells using NP40 lysis buffer (10 mM Tris, pH 7.5, 5 mM
MgCl2, 10 mM NaCl, 1% NP40) containing protease inhibitors and extracted by
gentle Dounce homogenization in Dignam C buffer (20 mM HEPES, pH 8, 10%
glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA). Cleared nuclear protein
extracts (equal protein) were diluted in modified RIPA buffer, incubated with 5 μg
anti-MyoD monoclonal antibody (Dako) overnight, immune complexes captured using
protein-G beads, washed with cold PBS + 0.5% Triton X-100 and pelleted.
Western blot analysis
IP pellets were solubilized in 2⫻ Laemmli sample buffer and run on 10% SDS-PAGE.
Antibodies were diluted in blocking buffer: MyoD polyclonal (Santa Cruz) 1:400,
pan-acetyl lysine monoclonal (Upstate) 1:2000.
Human p8-FLAG overexpression construct
Full-length human p8 cDNA was obtained from A549 cells using RT-PCR using
primers with BamH1 and Xho1 restriction sites (5⬘-AATGACGGATCCATGGCCACCTTCCCACCAGCA-3⬘ and 5⬘-GAACTACTCGAGTCAGCGCCGTGCCCCTCGCT-3⬘) and cloned into pCMV-2b to generate an N-terminal FLAG-tag.
Quantitative real-time RT-PCR
Total RNA (1 μg) isolated from stable pools (p8sh or GFP-sh) was used to generate
cDNA (Clontech). 2 μl of cDNA (diluted 1:5) were mixed with 10 μl of SYBR Green
PCR Master Mix (Applied Biosystems) and analyzed in triplicate using a 7900HT
cycler (Applied Biosystems). Normalized fold differences of cycle thresholds
[2–(–ΔΔCt)] of Myc and p8 amplicons were calculated relative to a control L7 amplicon;
dissociation curves and sequencing were used to verify amplicons. Primers were:
Myc: 5⬘-GCGCAAAGACAGCACCA-3⬘ and 5⬘-GCGAGCTGCTGTCGTTGA-3⬘;
L7: 5⬘-GGAGCTCATCTATGAGAAGGC-3⬘ and 5⬘-AAGACGAAGGAGCTGCAGAAC-3⬘; p8: 5⬘-AGGACCTAGGCCTGCTTGAT-3⬘ and 5⬘-CTCTGCTTCTTGCTCCCATC-3⬘.
Yeast two-hybrid analysis
Mouse p8 cDNA (coding) was amplified from mRNA of G0 myoblasts by RT-PCR
using primers with EcoRI and SalI restriction sites (5⬘-ATCGAATTCGGCATAATGGCCACC-3⬘ and reverse 5⬘-ATGTCGACGTGCTGTCACTGCTGT-3⬘), and cloned into the pGBKT7 vector as bait for screening a mouse
7-day embryo Matchmaker cDNA library in pACT2 (Clontech), in Saccharomyces
cerevisiae PJ694A, according to the manufacturer’s instructions. Transformants
passing three rounds of selection on dropout plates (Trp–Leu–Ade–) were re-tested
on Trp–Leu–Xgal+ plates to confirm activation of the β-galactosidase reporter. Plasmids
were isolated from positive yeast clones, propagated in E. coli DH5α, verified by
re-transformation into yeast with the original p8-bait plasmid or control plasmids,
selected as above, and sequenced.
Mammalian two-hybrid analysis
Mouse p8 cDNA cloned into pBIND (Clontech; using the primers 5⬘-GCTGCACGGATCCTAATGGCCACCTTGCCACCA-3⬘ and 5⬘-GCATATTCTAGATGCTTGCACTGCTGTACGATT-3⬘) and full-length mouse p68 cDNA cloned into pACT using
the primers 5⬘-GCTGCACGGATCCGCATGTCGAGTTATTCTAGTGAC-3⬘ and 5⬘GCATATTCTAGATTGAGAATACCCTGTTGGCATG-3⬘) were introduced into
C2C12 cells along with p5luc reporter and luciferase assays performed on lysates
prepared 48 hours after transfection.
Protein pulldown assay
Mouse p8 cDNA was cloned into the EcoRI-SalI sites of pET28 (Novagen) and the
fusion protein with an N-terminal His-tag (His6mp8) purified from E. coli using Ni2+NTA resin (Qiagen). 50 μl bed volume of nickel-agarose bound to His6mp8 was
incubated in 100 μl of pulldown buffer (20 mM HEPES/KOH, pH 7.5, 100 mM KCl,
5 mM MgCl2, 0.5 mM EDTA, 0.05% NP-40, 1 mM DTT, 0.02% BSA, and protease
inhibitors), with 200 μg of C2C12 myoblast extract prepared by salt extraction of
isolated nuclei using Dignam C buffer as detailed above. After 3 hours of incubation
at 4°C, the nickel-agarose beads were washed 5⫻ with PBS before elution of bound
proteins with Laemmli sample buffer. One tenth of eluted material was subjected to
3490
Journal of Cell Science 122 (19)
western blotting with antibodies against p300 (clone RW128; Upstate), p68 (pAb204;
Upstate), MyoD (polyclonal, Santa Cruz); His-p8 (anti-His tag, Santa Cruz) or the
control Histone H3 (polyclonal, Abcam).
Co-immunoprecipitation assay
p8-FLAG and MyoD-YFP were co-transfected into HEK293 cells and cell extracts
subjected to immunoprecipitation with anti-FLAG antibody and western blotting with
anti-MyoD.
Chromatin immunoprecipitation assay
Journal of Cell Science
Control and p8 RNAi myoblasts were incubated in DM for 24 hours, chromatin
isolated, cross linked and subject to immunoprecipitation with antibodies against p8,
p68, p300, MyoD or H3. Antibodies to p300, p68, MyoD and H3 are as above; for
p8, a new polyclonal antibody raised against mouse p8 was used. The myogenin
promoter (–200 fragment containing the E box) was PCR amplified [primers: 5⬘GAATCACATGTAATCCACTGGA-3⬘ and 5⬘-ACGCCAACTGCTGGGTGCCA3⬘, and 5⬘-AGAGGGAAGGGGAATCACAT-3⬘ and 5⬘-CATTTAAACCCTCCCTGCTG-3⬘] from DNA recovered after reversal of cross-linking, using SYBR-green
QPCR reactions in an ABI 7900HT real-time cycler.
We thank M. Nagavalli for technical assistance, Nandini Rangaraj
for expert assistance with confocal microscopy, Juan Iovanna, Rakesh
Mishra, Helen Blau and Sanjeev Galande for generous gifts of reagents,
R. Mishra and members of his lab for help with the yeast two-hybrid
screen, and Tushar Vaidya and Veena Parnaik for comments on the
manuscript. This work was supported by fellowships from the
Government of India Council of Scientific and Industrial Research (R.S.
and S.C.), from UNESCO-MCBN (R.S.), and grants from the NIH
(G.K.P.) and the Government of India Department of Biotechnology
and the Wellcome Trust (UK) (J.D.). J.D. is an International Senior
Research Fellow of the Wellcome Trust. Deposited in PMC for release
after 6 months.
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