During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatinmodifying enzymes to muscle-specific loci 1,2 . The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway 3-5 targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38α/β repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38α-β in vitro, and forced activation of p38α/β in myoblasts by expression of a constitutively active MKK6 (refs. 5-7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in-vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens1–7. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging due to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle (RV)8, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2 encoding plakophilin-29. The median age at presentation of ARVD/C is 26 years8. We used Yamanaka’s methods1,10 to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations11,12. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased β-catenin activity13 in cardiogenic conditions; yet these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor-gamma (PPARγ) activation underlie the pathogenesis of ARVD/C. By coactivating normal PPAR-alpha (PPARα)-dependent metabolism and abnormal PPARγ pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in-vitro model within two months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also displayed calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism plays a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified a novel inflammation-activated signalling in muscle stem (satellite) cells, by which the Polycomb Repressive Complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38alpha kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EzH2, the enzymatic sub-unit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. Anti-TNF antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signalling – p38alpha and EzH2 - invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signalling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signalling to Pax7 and satellite cell decision to proliferate or differentiate.

Transcriptional and posttranscriptional processes regulate expression of genetic networks in response to environmental cues. The extracellular signal-activated p38 MAP kinase (p38) pathway plays a fundamental role in conversion of myoblasts to differentiated myocytes. p38 phosphorylates specific transcription factors and chromatin-associated proteins promoting assembly of the myogenic transcriptome. Here, we demonstrate that p38 alpha and beta isoforms also control muscle-gene expression posttranscriptionally, by stabilizing critical myogenic transcripts. KSRP, an important factor for AU-rich element (ARE)-directed mRNA decay, undergoes p38-dependent phosphorylation during muscle differentiation. KSRP phosphorylated by p38 displays compromised binding to ARE-containing transcripts and fails to promote their rapid decay, although it retains the ability to interact with the mRNA degradation machinery. Overexpression of KSRP selectively impairs induction of ARE-containing early myogenic transcripts, without affecting p38-mediated transcriptional responses. Our results uncover an unanticipated role for KSRP in establishing a biochemical link between differentiation-activated p38 signaling and turnover of myogenic mRNAs.

Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/ SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/ SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38a kinase is the signal that promotes incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c-MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.