We used satellite cells derived from intact myofibers to investigate the impact of pharmacological blockade of p38 and IGF1/Pi3K on muscle regeneration. Upon culturing intact myofibers in mitogen rich medium (growth medium – GM), satellite cells delaminate and proliferate. Subsequent cell-to-cell contact together with mitogen withdrawal (differentiation medium – DM) promotes their differentiation into multinucleated myotubes. Pharmacological blockade of the p38 kinases α and β, by SB203580 (SB), or the IGF-1/Pi3K/AKT pathway, by the Pi3K inhibitor LY294002 (LY), was achieved by the continuous exposure to the inhibitors throughout the transition from myofiber-associated satellite cells to myoblasts cultured in DM. Inhibition of either pathway prevented the expression of muscle-specific genes and precluded satellite cell fusion into myotubes with comparable efficiency (). The same effects were also observed in established muscle cell lines, such as C2C12 cells (Suppl. Fig. 1A and B
) and in primary cultures of human skeletal myoblasts (data not shown
). However, the impact of SB and LY on the cell cycle machinery was different; only p38 blockade prevented the down-regulation of cyclin A that typically occurs during myotube formation (). Furthermore, the undifferentiated myoblasts derived from the exposure to SB or LY during the incubation in DM showed striking morphological differences; SB-treated satellite cells appeared as elongated myoblasts, while LY-treated cells displayed rounded morphology () Both phenotypes could be readily distinguished from the typical morphology of normal, satellite cells. Comparable morphological effects were observed after simultaneous downregulation of individual p38 α and β or AKT 1 and 2 kinases, respectively, by RNA interference (RNAi) (Suppl. Fig. 1C, D, E and F
). Thus, despite the shared ability to prevent the initiation of the differentiation program, pharmacological or genetic blockade of p38 and Pi3K resulted in biochemical and morphological differences, which likely reflect discrete chromatin signatures at specific loci. Consistent with this notion, we detected two unique patterns of histone acetylation at muscle loci in SB- vs LY-treated satellite cells () that reflect the assembly of two distinct chromatin-bound complexes with different enzymatic activity. Only Pi3K blockade prevented the local increase in histone acetylation on the regulatory elements of muscle genes (
), despite the global histone acetylation did not change in LY-treated satellite cells ().
Pharmacological blockade of p38 and Pi3K in satellite cells leads to distinct undifferentiated phenotypes
The results presented in suggest that the Pi3K/AKT and p38 pathways target different components of the muscle transcriptosome, and that the selective inhibition of either cascade can generate two populations of undifferentiated myoblasts showing distinct cellular phenotypes and discrete patterns of chromatin modification at muscle loci.
Based on this hypothesis, we carried out an extensive chromatin immunoprecipitation (ChIP) analysis to compare the assembly of the myogenic trancriptosome in myoblasts induced to differentiate in the presence of SB vs LY. We employed C2C12 myoblasts for this analysis, as they provided ample material necessary for ChIP. The chromatin of myoblasts in GM or DM, with and without SB or LY, was immunoprecipitated with antibodies against different components of the muscle transcriptosome and specific histone modifications. Consistent with Pi3K/AKT and p38 being parallel cascades delivering distinct external cues to discrete components of the myogenic transcriptosome, the composition of chromatin-bound complexes on myogenin promoter and muscle creatine kinase (MCK) enhancer was different in myoblasts exposed to SB or LY. SB selectively prevented the recruitment of the SWI/SNF complex, as previously reported (Simone et al 2004a
). LY precluded the recruitment of p300 and PCAF acetyltransferases, and the consequent hyperacetylation on muscle promoters/enhancers (). As hyperacetylation of MyoD increases its DNA binding affinity (Sartorelli et al. 1999
), it was not surprising that in the absence of acetyltransferases, the majority of MyoD was hypoacetylated and hence unable to bind chromatin (). By contrast, chromatin binding of MEF2A/C and SWI/SNF was not affected by LY treatment (). The relative occupancy of Brg1, p300, PCAF, MyoD and the acetylation status of histone H3 were also quantified by real time PCR in independent experiments, which confirmed the differences reported above (Suppl. Fig. 2A and B
). Interestingly, p38 α kinase was detected on the chromatin of target genes only in conditions permissive for differentiation (DM), regardless of the presence of SB or LY (). In these conditions the large majority of p38 α is phosphorylated (Wu et al. 2000
), suggesting the MKK3/6-dependent phosphorylation of p38 could be an important signal for chromatin recruitment. The presence of p38 on the chromatin of muscle genes in SB-treated cells is consistent with this scenario, as SB blocks the catalytic activity of p38α, without altering the phosphorylation status of this kinase. Notably, the effect of Pi3K blockade on the composition of chromatin-bound complexes appeared to be restricted to those muscle genes, whose expression was inhibited by LY. Indeed, p300 recruitment and histone hyperacetylation on the promoter of Atrogin-1, a catabolic gene repressed by IGF1 pathway (Stitt et al. 2004
; Sandri et al. 2004
), was not reduced, but rather increased in LY-treated myoblasts (Suppl. Fig. 2C and D
Inhibition of Pi3K prevents the recruitment of acetyltransferases and MyoD on chromatin of muscle-regulatory genes
The comparison of the effects produced by SB vs LY emphasizes the notion that these two cascades converge on the chromatin of muscle genes to target distinct components of the myogenic transcriptosome. The presence of the SWI/SNF complex in association with a transcriptional activator (MEF2) on the chromatin of muscle genes in LY-treated myoblasts raises the question of whether this complex is competent to remodel the chromatin at these loci. To address this issue we performed an endo-nuclease accessibility assay and found that, despite the different composition of chromatin-bound complexes in SB- and LY-treated myoblasts, both treatments precluded chromatin remodeling (). This evidence demonstrates that the MEF2-SWI/SNF complex detected on the regulatory elements of muscle genes in LY-treated cells is not competent to remodel the chromatin.
Taken together these data reveal a functional interdependence between the p38 and Pi3K pathways at the chromatin level. The p38 pathway directs the recruitment of the SWI/SNF complex, but the ability of this complex to remodel the chromatin is conferred by the IGF-1/Pi3K pathway, via engagement of MyoD/acetyltransferases and consequent local hyperacetylation. In this regard, it is difficult to discriminate the role of MyoD as an essential factor for the recruitment of acetyltransferases (Yuan et al. 1996
; Eckner et al. 1996
; Puri et al. 1997a
; Sartorelli et al. 1997
) or as an independent regulator of SWI/SNF remodeling activity (Gerber et al. 1997
; de la Serna et al. 2001
Previous reports have documented the ability of MEF2 to form distinct complexes with transcriptional repressors (Miska et al. 1999
; Lu et al. 2000
). Consistently, immunoprecipitated MEF2A/C co-purified significantly higher deacetylase activity in LY-treated myoblasts, as compared to untreated or SB-treated myoblasts (
), and HDAC4 was found to be associated to MEF2A/C and SWI/SNF on the chromatin of muscle genes in LY-treated myoblasts (
). The reduced histone acetylation observed at the regulatory elements of muscle genes following Pi3K inhibition raised the possibility that these lysines were instead methylated. Recruitment of Polycomb-associated methyltransferase Ezh2 was previously reported to mediate silencing of muscle genes in undifferentiated myoblasts (Caretti et al. 2005). Indeed, the presence of the methyltransferase Ezh2 and its enzymatic effect - tri-methylation of H3 lysine K27 - was detected on the promoter of myogenin and the MCK enhancer in LY-treated myoblasts (Suppl. Fig. 3A
). This finding suggests that the pharmacological blockade of Pi3K converts the conformation of the chromatin at muscle loci from permissive to repressive. The drastic changes in the components of the chromatin-bound complexes generated by LY treatment, which lead to histone lysine methylation, rather than acetylation and chromatin remodeling, also suggest that, in the absence of the IGF1 signaling, the MEF2-SWI/SNF complex associated with co-repressors could impose repressive chromatin modifications. We next tested whether the inhibition of the myogenic program by IGF1-Pi3K or p38 blockade is a stable or reversible epigenetic event. Suppl. figure 3B
shows that the effects of SB and LY on muscle differentiation were readily reversible. These data indicate that is possible to generate a dynamic population of myoblasts with different chromatin conformation at muscle loci by pharmacological manipulation of signaling pathways.
The data presented in suggest that the IGF1/Pi3K signaling affects the composition of the complexes bound to the chromatin of muscle genes both by promoting the recruitment of positive regulators (MyoD and acetyltransferases) and by preventing the engagement of co-repressors (histone deacetylases and methyltransferases). Given the importance of recruiting MyoD and acetyltransferases to enable the chromatin remodeling activity of SWI/SNF, we investigated the mechanism by which IGF1/Pi3K signaling promotes formation of the MyoD/acetyltransferase complex on the chromatin of muscle genes.
Interactions between MyoD and p300 are regulated at multiple levels (Puri and Sartorelli 2000
). We first investigated the role of the IGF-1/Pi3K signaling in promoting physical interactions between MyoD and acetyltransferases in muscle cells, by co-immunoprecipitation studies with endogenous proteins. An association of MyoD with p300 and PCAF was detected in myoblasts induced to differentiate, and this complex was specifically disrupted by Pi3K inhibition (). Simultaneous downregulation of indivudual kinases p38 α and β or AKT 1 and 2, respectively, by RNAi was also exploited to definitively assess the contribution of these kinases in regulating MyoD-p300 interaction. Although downregulation of either p38 α and β or AKT 1 and 2 () impaired the expression of muscle genes in differentiating myoblasts (), only donwregulation of AKT1 and 2 prevented the association between endogenous MyoD and p300 (). The interaction between MyoD and p300 was further measured by a mammalian two-hybrid system, by using a Gal4-luciferase reporter co-transfected with Gal4MyoD and p300 fused with the acidic activation domain VP16 (VP16p300). Exposure to IGF-1 in a serum-free medium stimulated the interaction between Gal4MyoD and VP16p300, and this effect was prevented by LY (
). By using two truncated versions of p300 (N-terminal aa1-744 and C-terminal aa871-2377), we established that the IGF-1 responsive region maps to the C-terminus (). This finding is in agreement with the reported observation that the IGF1/Pi3K/AKT pathway promotes interaction between bHLH transcription factors and p300 during neurogenesis (Vojtek et al. 2003
). Other works have reported on the ability of AKT to phosphorylate p300 (Huang and Cheng 2005
). Indeed, two putative AKT consensus sites are present at the C-terminus of p300, within the MyoD interaction domain - the CH3 region. One consensus site (RRLS) maps to serine 1734 and is preferential for AKT1. The second phospho-acceptor site maps to serine 1834 (RRRMASM) and can be phosphorylated both by AKT1 and 2 (
). These sites are conserved in both p300 and CBP in different species. However, whether MyoD-p300 interaction is regulated via direct phosphorylation of p300 by AKT or by any other downstream kinase(s) has not been addressed by previous studies.
Pi3K/AKT-dependent interactions between MyoD and the p300 C-terminus during myogenic differentiation
We monitored the phosphorylation pattern of endogenous p300 in myoblasts after metabolic labeling with 32P orthophosphate and immunoprecipitation with anti-p300 antibodies. This analysis showed minimal fluctuations of global p300 phosphorylation along the transition from myoblasts to myotubes. However, Pi3K inhibition by LY significantly reduced the phosphorylation of p300 in myoblasts incubated in differentiation medium (DM) (). This evidence further indicates the contribution of the IGF1/Pi3K pathway to p300 phosphorylation during myogenic differentiation. To precisely map the AKT phosphorylation sites in the p300 C-terminal, we expressed a HA-tagged truncated version of p300 (HA-1640-1840) in 293 cells along with constitutively active, myristoylated (myr) AKT1 or with inactive kinase death (kd) AKT1. After metabolic labeling with 32P orthophosphate, the p300 HA-1640-1840 fragment was immunoprecipitated with anti-HA antibodies and subjected to phospho-peptide mapping (). The two prominent spots detected in cells transfected with AKT1myr, but not in cells expressing AKT1 kd, were further subjected to phospho-aminoacid analysis, which revealed the presence of phospho-serines (). The migration pattern of the phospho-peptides () and their confirmed identity by phospho-aminoacid analysis () are consistent with AKT-dependent phosphorylation of p300 on serines 1734 and 1834. Note that ectopic expression of AKT1myr correlated with the activation of endogenous AKT2 (data not shown), suggesting a reciprocal activation and functional synergy between these two kinases. To confirm that these serines are phosphorylated in vivo during myoblast differentiation, we transfected the p300 C/H3 (p300 HA-1640-1840) fragment in C2C12 cells and monitored its phosphorylation after metabolic labeling with 32P orthophosphate. An increased phosphorylation was observed in myoblasts induced to differentiate (, compare lane 1 and 2, and , compare lane 1 and 2). The 32P incorporation was abrogated by either Pi3K inhibition with LY () or by replacement of the two serines with non-phosphorylatable alanines (p300 CH3 S1734/1834A) ().
Pi3K/AKT-dependent phosphorylation of the C/H3 domain of p300 during myogenic differentiation
The phosphorylation of serine 1834 of endogenous p300 was further studied in human skeletal myoblasts (HSKM) using ser1834 phospho-specific antibodies (Liu et al. 2006
). Immunofluorescence studies showed an increased nuclear staining in multinucleated, differentiated myotubes, as compared to undifferentiated myoblasts. Treatment with LY, but not SB, drastically reduced the nuclear accumulation of phosphoserine 1834 p300, although both compounds inhibited the formation of myotubes (Suppl. Fig. 4A
). The changes in phosphorylation of serine 1834 of p300 in the conditions described above were confirmed by western blot on nuclear extracts (Suppl. Fig. 4B
We next addressed the functional impact of AKT-mediated phosphorylation of p300 C-terminal serines 1734 and 1834 on p300-MyoD interaction, by using the mammalian two-hybrid-based assay shown in . We compared the interaction of Gal4MyoD and the p300 C/H3 fragment, either wild type (VP16p300 C/H3 wt) or phospho-mutant (VP16p300 C/H3 S1734/1834A), fused to VP16. An interaction between Gal4MyoD and the VP16p300 C/H3 wt was observed in response to differentiation cues and was inhibited by LY. In the same conditions, the VP16-p300C/H3 S1734/1834A double mutant failed to interact with Gal4MyoD (). Consistent with the role of AKT in promoting the association between MyoD and p300 via phosphorylation of the C/H3 domain of p300, co-transfection of the constitutively active form of AKT1myr, but not the AKT1 kd, was sufficient to promote interaction between Gal4MyoD and the VP16-p300 C/H3 wt, but not the VP16-p300C/H3 S1734/1834A mutant (). The impact of each phosphoacceptor site on MyoD-p300 interaction was further analyzed by comparing the ability of full length Flag-tagged p300 wild type (wt) and single mutants (S1734A and S1834) to interact with endogenous MyoD, upon transient transfection in C2C12 myoblasts. shows that although these mutants are expressed at comparable levels (bottom panel), only p300 wt was found associated with MyoD (top panel). The relative impact of AKT1 and 2 on p300-MyoD interactions was also addressed by using mouse embryonic fibroblasts (MEFs) derived from AKT1 or AKT2 null mice (Liu et al. 2006
). Employment of AKT 1 or 2 null cells eliminates the caveat intrinsic to the RNAi downregulation, consisting in the presence of residual, minimal expression level of the targeted isoform. We first monitored the binding of Gal4MyoD and VP16-p300 C/H3 wt in AKT1 and AKT2 deficient MEFs, as compared to wt MEFs. Absence of either AKT1 or AKT2 drastically impaired Gal4MyoD-VP16p300 interactions (), further indicating that phosphorylation of p300 by both AKT1 and AKT2 is required for complex formation. In keeping with this conclusion, the interaction between endogenous p300 and ectopically expressed MyoD was detected in wt MEFs and in MEFs from MKK3/6 null mice, in which endogenous p38 cannot be activated (Brancho et al. 2003
), but not in AKT1 null or AKT2 null MEFs (Suppl. fig. 5A
). Consistently, the recruitment of MyoD and p300 to the chromatin of muscle genes was selectively impaired in MyoD-converted AKT1 or AKT2 null MEFs (), while Brg1 was detected on the chromatin of these cells, but not in MyoD-converted MKK3/6 null MEFs (
Replacement of two potential AKT-target serines with non-phosphorylatable alanines or genetic ablation of one AKT isoform impairs the interaction between the p300 and MyoD during myogenic differentiation
Cooperation at the chromatin level between the Pi3K/AKT-mediated recruitment of MyoD and acetyltransferases and the p38-directed recruitment of SWI/SNF on the regulatory sequences of muscle genes was further explored by functional assays that combined deliberate activation of p38 with inactivation of AKT chromatin effectors. The p38 pathway was activated in C2C12 myoblasts by the ectopic expression of the constitutively active form of its upstream kinase, MKK6EE-HA, via adenoviral delivery (). These conditions resulted in the forced expression of muscle genes despite culture conditions non-permissive for differentiation (GM), and either p38 or Pi3K blockade was sufficient to prevent MKK6EE-induced muscle differentiation (). While the effect of SB is expected, as this compounds blocks the downstream MKK6EE effector p38, the effect of LY was independent on the activation status of p38 (). Consistent with the notion that the p38 and the IGF1/Pi3K/AKT pathways proceed as parallel cascades, which converge on different chromatin-bound complexes, the ability of LY to prevent MKK6EE-mediated activation of the myogenic program correlates with the local hypoacetylation and impaired chromatin remodeling at muscle loci (). We next performed experiments aimed at establishing a direct relationship between MKK6EE-mediated activation of muscle gene expression and Pi3K/AKT-directed recruitment of p300 to the chromatin of muscle genes. In one experimental setting we compared the effect of the cytoplasmic inhibitor of the Pi3K/AKT pathway (LY) with that of the inhibitor of the Pi3K/AKT nuclear target p300 (the LysCoA) (Lau et al. 2000
) on MKK6EE-dependent activation of the differentiation program in myoblasts. shows that microinjection of LysCoA at concentrations previously reported to inhibit p300 enzymatic activity (Lau et al. 2000
; Polesskaya et al. 2002) was as efficient as LY in preventing the expression of myosin heavy chain (MyHC) in myoblasts injected with MKK6EE. These results establish a functional link between the p38 pathway and the integrity of the enzymatic activity of the Pi3K/AKT chromatin sensor p300. However, they do not address the specific role of AKT-mediated phosphorylation of p300 in the functional cooperation between the p38 pathway and p300. To this aim, we have compared the effect of over-expression of the truncated form of the C/H3 p300 (1640-1840), either wild type (wt) or the phospho-mutant (mt) ser1734/1834ala, on the ability of MKK6EE to promote the myogenic conversion in MyoD-expressing fibroblasts cultured in GM (). Increasing concentrations of C/H3 (1640-1840) wild type countered the ability of MKK6EE to promote myogenic conversion, but the effect was minimal with the phospho-mutant (). This result suggests that the dominant negative effect of C/H3 (1640-1840) fragment on muscle differentiation relies on the presence of AKT phosphorylation sites. Increasing concentrations of C/H3 (1640-1840) likely buffer the kinase availability of AKT toward endogenous p300. This evidence further supports the functional interdependence at the chromatin level between different enzymes, which are recruited into the myogenic transcriptosome by different signal-activated kinases, via direct phosphorylation.
Functional interdependence at the chromatin level between the IGF1 and the p38 pathways
Our results illustrate a model of cooperation between insulin/IGF1-activated signaling and the p38 pathway during skeletal myogenesis. In this model, IGF1-activated Pi3K/AKT signaling promotes the recruitment of MyoD and acetyltransferases to the chromatin of muscle genes, an event necessary to enable the chromatin remodeling activity of the p38-recruited SWI/SNF complex. Furthermore, our data suggest another, apparently distinct activity of IGF1 signaling, which is to displace or prevent the association of histone deacetylases and methyltransferases within the muscle transcriptosome. Selective interruption of IGF1/Pi3K/AKT signaling in conditions permissive for differentiation (DM), in which the p38 pathway is active, results in the formation of a complex containing MEF2 associated with co-repressors (i.e HDAC4, Ezh2), instead of co-activators (acetyltransferases) (depicted in
). Indeed, MEF2-associated class II HDACs (Lu et al. 2000
) and Ezh2-directed lysine methylation (Caretti et al. 2004
) were reported to contribute to muscle gene silencing in myoblasts. Interestingly, the SWI/SNF complex can mediate repression of transcription, instead of activation, when associated to co-repressors (de la Serna et al. 2006
; Marenda et al. 2004
; Martens and Winston 2002
). Further studies should establish whether p38-directed phosphorylation is involved in recruitment of SWI/SNF into repressive complexes. Likewise, it will be important to elucidate the role of IGF1 signaling in the chromatin re-distribution of histone deacetylases and methyltransferases.
Fig. 7 Illustration of the composition of muscle transcriptosome on the chromatin of muscle genes that reflects different chromatin status and distinct cellular phenotypes generated in response to Pi3K or p38α/β inhibition, as compared to untreated (more ...)
The terminal effectors of IGF1 signaling to the chromatin of muscle genes are AKT 1 and 2 kinases, which phosphorylate the C-terminal region of the acetyltransferase p300 at the onset of differentiation. The individual contribution of AKT 1 and 2 to the myogenic program is currently unclear, with works reporting on the essential role of AKT2, which is upregulated in differentiating myoblasts (Kaneko et al. 2002
), and works reporting on the central role of AKT 1 in skeletal myogenesis (Wilson and Rotwein 2007
). Our data indicate that AKT 1 and 2 are both necessary to phosphorylate p300 and promote interaction with MyoD and possibly other muscle specific transcription factors. Although the amino-acidic sequence of one p300 phospho-acceptor site (serine 1734) appears to be a preferential consensus for AKT 1 (), our results indicate the requirement of both kinases for optimal phosphorylation of p300 C-terminal and the consequent interaction with MyoD. Genetic studies in MEFs deficient for either AKT 1 or 2 show that one kinase cannot compensate for the absence of the other ( and Suppl. Fig. 5A
). Likewise, elimination of either phosphoacceptor site impairs the ability of p300 to interact with MyoD, as revealed by complementary assays (). Conversely, expression of the constitutively active AKT 1 (AKT 1 myr) alone led to the phosphorylation of both serines 1734 and 1834, and this correlates with the activation of endogenous AKT 2 (data not shown). These results suggest that cross-activation between these two kinases might take place to achieve a cooperative effect on phosphorylation of common substrates (i.e. p300), although the biochemical basis for such synergy needs to be elucidated by future studies.
How does p300 C-terminal phosphorylation regulate the interaction with MyoD? Several mechanisms can cooperatively mediate this effect. The C/H3 domain is the region of p300 that mediates the interaction with MyoD (Yuan 1996
, Eckner et al. 1996
; Sartorelli et al. 1997
). AKT-mediated C/H3 phosphorylation can induce/stabilize this interaction. MyoD and histone acetylation are additional events that promote MyoD/p300 binding and chromatin recruitment (Sartorelli et al. 1999
; Polesskaya et al. 2001
). As AKT-mediated phosphorylation was reported to stimulate the enzymatic function of p300 (Huang and Chen 2005
), it is possible that an increased acetyltrasferase activity toward MyoD contributes to AKT-dependent interaction between MyoD and p300. Additionally, the C/H3 domain mediates interactions between p300 and pCAF (Yang et al. 1996
), and AKT-mediated phosphorylation of p300 appears to contribute to the recruitment of pCAF, which in turn acetylates MyoD, p300 and histones to further stabilize the p300/MyoD/pCAF complex and activate transcription (Puri et al. 1997b
; Sartorelli et al. 1999
; Dilworth et al. 2004
). Consistent with our results, others showed that inhibition of IGFII production in MyoD-converted fibroblasts precluded the engagement of p300 and PCAF on myogenin promoter (Wilson and Rotwein 2006
). However, in their experimental setting chromatin recruitment of MyoD was not affected and the consequence on SWI/SNF enzymatic activity was not addressed.
The importance of AKT-mediated phosporylation of p300 extends to the regulation of SWI/SNF chromatin remodeling activity, as in the absence of MyoD/acetyltransferases the chromatin of muscle genes was hypoacetylated and un-remodeled, despite the presence of Brg1 (). It is possible that histone acetylation is an event required for SWI/SNF to remodel chromatin, as nucleosomes containing acetylated histones are better predisposed for the enzymatic activity of of SWI/SNF (Chandy et al. 2006
). The presence of SWI/SNF on hypoacetylated chromatin is apparently in conflict with previous works reporting on SWI/SNF chromatin binding via interactions of the bromodomain with acetylated histones (Hassan et al. 2001
). However, according to a two-step model of SWI/SNF chromatin recruitment and activation, SWI/SNF can first be recruited by transcriptional activators, and then stabilized by interactions with acetylated histones. Local hyperacetylation (Chandy et al. 2006
) and the association with transcriptional activators (Gutierrez et al. 2007
) appear to further promote the chromatin remodeling activity of SWI/SNF. Thus, in conditions in which acetyltransferases and MyoD are not recruited to the chromatin of muscle genes (i.e. interruption of Pi3K/AKT signaling), SWI/SNF recruitment by MEF2 ( and Suppl. Fig. 5B
), in complex with deacetylases and methyltransferases ( and Suppl. Fig. 3A
), could result in an impaired remodeling activity of SWI/SNF ().
The interplay between two parallel signaling pathways at the chromatin levels may have a remarkable impact during the regeneration process. Both pathways are activated by regeneration cues and influence the ability of muscle progenitors to execute different stages of the regeneration program. The Pi3K/AKT pathway is elicited by locally released IGFs and promotes satellite cell proliferation, survival and differentiation (Mourkioti and Rosenthal 2005). The p38 pathway is activated in satellite cells, presumably in response to locally released inflammatory soluble factors or cell-to-cell interactions, and promotes cell cycle arrest (Puri et al. 2000
; Perdiguero et al. 2007
) and terminal differentiation (Zetser et al. 1999
; Wu et al. 2000
). The convergence of these two pathways at the chromatin level provides a mechanism for integration of regeneration cues to coordinate gene expression during cellular differentiation.
The different chromatin modifications observed in response to pharmacological inhibition of the p38 or the IGF1-activated pathways correlate with distinct cellular phenotypes in treated myoblasts. Pi3K blockade prevents the hyperacetylation of the chromatin of muscle genes, and leads to quiescence. p38 blockade promotes proliferation, which correlates with hypearcetylation at muscle genes (see illustration in ). The acetylation status of the chromatin at muscle loci has a relevant biological impact during regeneration. For instance, MyoD acetylation regulates satellite cell capability to regenerate injuried muscles (Dunquet et al. 2006
) and agents that increase histone hyperacetylation at muscle loci, such as deacetylase inhibitors, promote myogenesis in vitro
and muscle regeneration in vivo
, and revealed to be effective in the treatment of muscular dystrophies (Iezzi 2002
; Iezzi 2004
; Minetti 2006
). Furthemore, deacetylase inhibitors can induce AKT activation in satellite cells (CM and PLP unpublished observation) and promote AKT-mediated phosphorylation of p300 in other cell types (Liu et al. 2006
). Thus, the AKT signaling to chromatin-bound proteins, such as p300, could also contribute to the beneficial effects of deacetylase inhibitors on muscles regeneration.
Our results illustrate an example of convergence of distinct signaling pathways at the chromatin level, to coordinate the expression of genes implicated in satellite cell transition from quiescence to terminal differentiation. These data suggest potential pharmacological avenues for selective control of gene expression to manipulate muscle regeneration.