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 post-translational acetylation of the myogenic regulator MyoD) are all affected in p8-knockdown myoblasts, correlating with 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.

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 myogenin-expressing 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 muscle-specific 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 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 gene-trap 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.

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 cell cycle but especially in mid-late G1 (Fig. 2B). p8 protein expression was also inhibited: Fig. 2C shows suppression in p8sh4-knockdown cells at the time of peak expression in late G1.

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 p8-knockdown 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 re-entry 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.

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 p8-knockdown 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 re-entry 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.

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 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 myogenic-marker expression. In control cells, S-phase-specific histone H2B expression was suppressed during G0 as expected and was yet-to-be 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. MyoD-mRNA induction in G1 was mildly reduced (twofold) in the p8-knockdown pool. Thus, a G1-induced transcript (MyoD) and an S-phase-specific transcript (histone) showed altered levels and timing in p8-knockdown myoblasts.

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 cell-cycle inhibitors (p21, p27). Desmin was used as a loading control.

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 cell-cycle inhibitors (p21, p27). Desmin was used as a loading control.

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%), p8-knockdown 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 p8-knockdown 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 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.

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.

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.

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 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 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.

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 p8-knockdown (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 histone-acetylation-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.

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 p8-knockdown (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 histone-acetylation-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.

p8 interacts with two other pro-myogenic p300-binding proteins: the RNA helicase p68 and the myogenic regulator MyoD

To gain further insight into the function of p8, we used a yeast two-hybrid 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).

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 p8-knockdown 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.

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 p8-knockdown 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.

To confirm the interaction between p8 and p68 we used two further tests. First, we used a mammalian two-hybrid assay. Full-length 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).

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.

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 p8-interacting 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 co-activators with a common chromatin target – the early muscle-specific 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-cycle-regulated 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 chromatin-binding 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 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.

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).

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 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 cell-cycle 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

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-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.

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.

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.

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 non-coding 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 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

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), anti-p27 (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. Secondary-antibody 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 phosphor-imager signals.

Western blot analysis was performed as described (Sachidanandan et al., 2002). Antibodies were diluted in blocking buffer: anti-MyoD (Santa Cruz) 1:400; anti-myogenin (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′.

Control shRNAs: P8SH1a: 5′-TTTGTCTTGCCTGTGTCTCCTTGTCGAAGCGACAAGGACTCACAGGCAAGATTTTT-3′; P8SH1b: 5′-CTAGAAAAATCTTGCCTGGAGTCCTTGTCGCTTCGACAAGGAGACACAGGCAAGA-3′; GFP - SH1a: 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 (TrpLeuAde) were re-tested on TrpLeuXgal+ 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 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

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′-ACGCCAACTGCTGGGTGCCA-3′, 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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