In order to address this issue, we sought to identify MyoD-binding partners that could play a role in facilitating MyoD access to the chromatin. Our work identified BAF60c as a MyoD binding partner. BAF60c is a subunit of a chromatin remodeling complex, called SWI/SNF, that uses the energy of ATP to dissociate DNA-histone contacts, therefore, we considered it to be an excellent candidate for helping MyoD access the chromatin of silent loci. Indeed and importantly, BAF60c co-localizes with MyoD at the promoter of an early myogenic differentiation marker gene (myogenin) prior to the activation of transcription without other SWI/SNF subunits such as Ini1 or Brg1 (Fig. 1A).⁷ Furthermore, genome wide ChIP (ChIP on ChIP) showed that RNAi-mediated downregulation of BAF60c expression prevents the recruitment of MyoD to most of its target genes impairing their transcription. In turn, the absence of MyoD prevents BAF60c binding to the chromatin of myogenin. This evidence supports a model of reciprocal cooperation, whereby BAF60c and MyoD facilitate each other’s binding to muscle specific loci.

Rather than a direct repressor role for the MyoD-BAF60c interaction at the chromatin of silent myogenic genes, we suggest that the concomitant presence of BAF60c and MyoD poises these regions for a rapid transcriptional activation when the differentiation signals arrive. Therefore, we propose that the MyoD-BAF60c complex provides an epigenetic memory of poised genes during myoblast proliferation. Supporting this idea, downregulation of BAF60c by RNAi does not activate myogenin transcription at any time in undifferentiated, proliferating myoblasts. Indeed, the absence of BAF60c prevents MyoD binding and histone H3 acetylation, while it does not decrease the presence of typical repressor histone modifications such as H3K9me3 and H3K27me3 at the myogenin locus prior to the activation of transcription. In agreement with the idea that MyoD-BAF60c could induce a poised or intermediate chromatin state, recent data by ChIP-seq¹¹ shows that MyoD binding to the chromatin is associated with histone H4 acetylation even at silent loci, which suggests that MyoD-BAF60c chromatin binding co-occurs with a more relaxed or open chromatin, an intermediate/ready-to-go state that may be easier and faster to remodel as soon as the differentiation signals arrive.

It is well known that p38 MAPK plays a fundamental role in the transition of myoblast to differentiated myocytes, as p38 activity is essential for myogenesis.^19-21 We had previously reported that SWI/SNF recruitment to myogenic regulatory regions was dependent on p38 activity, and that p38 kinases α and β could phoshorylate a SWI/SNF subunit with a predicted size of one of the three BAF60 proteins in vitro.² However, the identity of the specific BAF60 variant targeted by p38 α/β-mediated phosphorylation, and the in vivo role of this event remained unanswered. To address these questions, we analyzed the BAF60 family of SWI/SNF subunits and only BAF60c contained a suitable phospho-acceptor site for p38 α/β kinases. Notably, this phosphorylation target motif appears conserved in human, rat and mouse BAF60c sequences and was not present in BAF60a or BAF60b. This site was a Threonine Proline-directed residue in position 229. Mutation of this site to an Alanine impaired the incorporation of the phosphorylation in an in vitro kinase assay, confirming that it was the acceptor site for p38-direct phoshorylation. Furthermore, in vivo experiments confirmed this Threonine as the “sensor” of p38 activity since overexpression of a wild-type BAF60c induced an earlier and strong myogenic differentiation (formation of large multinucleated myotubes) while the mutant induced a weaker and delayed response. At the chromatin level, the BAF60c phosphorylation-deficient mutant facilitates MyoD binding in the same manner as the BAF60c wild-type does, however, the mutant BAF60c is unable to recruit an active SWI/SNF complex to the myogenin promoter and thus myogenin transcription is impaired. In agreement with these results, our previous work² showed that p38 α/β kinase inhibition by soluble compounds (SB203580) prevented BAF60c phosphorylation and consequent Brg1 recruitment to the myogenic loci. In this scenario, histone acetylation and the recruitment of factors such as MyoD and acetyltransferases (p300, PCAF) were unaltered, revealing the specificity of p38-mediated phosphorylation in mediating recruitment of Brg1-based SWI/SNF complex to MyoD-target genes. All these data underscore BAF60c-phosphorylation as a specific event that directs SWI/SNF recruitment to the chromatin subjected to active remodeling during cellular differentiation. Other SWI/SNF subunits may be modified post-translationally by other enzymes. Future proteomic studies will help to reveal the nature of these modifications and their mediators.

In our work, we have not only described that SWI/SNF is recruited to myogenic loci in response to the differentiation-activated p38 pathway, but importantly, we have identified the SWI/SNF subunit being directly phosphorylated by p38 kinase (BAF60c), and determined the functional impact of this phosphorylation on myogenic differentiation. While BAF60c phosphorylation is crucial for initiating the chromatin remodeling that leads to transcriptional activation, there are still some questions regarding the nature of this recruitment. For instance, genome-wide studies are needed to determine the possible interactions of SWI/SNF subunits with enhancer regions and their possible role in looping and long-distance interactions with promoters, as shown with the CIITA promoter where various combinations of BAF155, BAF170, Ini1 and Brg1 have been detected at different enhancer elements and therefore could mediate these looping interactions.^10,22 Recent work by ChIP-seq analysis has revealed that MyoD binds Eboxes present in enhancer regions of the myogenin gene located at -6.5 Kb and -4.5 Kb.¹¹ Furthermore, epigenomic data shows that RNApol II is present in some of these enhancer regions.²³ Therefore, it would not be surprising to find other SWI/SNF subunits bound in distal areas of the myogenin gene that are recruited to the promoter region following p38-mediated phosphorylation of BAF60c. Integrating genome-wide data from ChIP, nucleosome positioning and chromosome capture conformation techniques at several time points of differentiation could establish if there are pre-assembled SWI/SNF subunits at different myogenin regulatory regions (or other genomic loci) that come together to form a complete active SWI/SNF complex in response to p38 signaling (Fig. 2). This model could explain the rapid recruitment of an active SWI/SNF complex following p38 activation and the need to place subunits of the same complex distributed in genomic regulatory regions that come together on activation signals.

Knowledge of transcriptional regulation has advanced dramatically in the last decade; the identification of the histone code and the integration of genome-wide and proteomic techniques have been crucial for revealing new revolutionary mechanisms of transcriptional regulation. Nevertheless, many aspects of the transcriptional process remain unexplained. In the myogenic transcription some exciting questions remain to be addressed. How is chromatin architecture integrating regulatory factor binding and tethering to the chromatin? If loops are bringing together enhancer and promoter regions, who are the mediators and which are the genes or genomic domains being regulated in this fashion? What are the specific kinetics of regulatory factor binding that modulate transcriptional regulation? Does chromosomal 3D structure vary between proliferating and differentiating myoblasts?

Regarding BAF60c functions, it would be important to identify new BAF60c chromatin target sites; to monitor if BAF60c binds MyoD-independent loci; whether BAF60c chromatin binding is accompanied by other SWI/SNF subunits and its significance/impact for chromatin structure and regulation.

Furthermore, it is crucial to understand the role that different combinations of preassembled SWI/SNF components may play in regulating chromatin architecture and remodeling.

Immunoprecipitation assays followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) could be used to identify differentiation-stage specific BAF60c-complexes. This approach could also identify post-translational modifications in BAF60c and in other SWI/SNF subunits that can play an important role in transcriptional activation/repression. In addition, the identification of BAF60c-interacting enzymes that promote these modifications could also reveal new regulatory pathways that may be involved in myogenic transcription.

Finally, it would be very interesting to explore in detail how the nucleosomes are remodeled along the myogenin promoter and how nucleosome location genome-wide differs in precursor vs. differentiated cells. Do BAF60c-differential complexes have an impact on DNA accessibility and how?

Forcales SV, Albini S, Giordani L, Malecova B, Cignolo L, Chernov A, et al. Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex EMBO J 2012 31 301 16 doi: 10.1038/emboj.2011.391.