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Keywords: Skeletal muscle differentiation, methylation, acetylation, transcription, epigenetics and chromatin modifying enzymes
Issue Date: 10-Jan-2013
Abstract: Skeletal myogenesis is a tightly regulated process involving a series of steps which begins with the initial commitment of multi-potent mesodermal progenitors towards the myogenic lineage. Once committed, they proliferate as undifferentiated mononucleated myoblasts followed by irreversible withdrawal from cell cycle. Concomitantly, they differentiate and fuse into multinucleated myotubes. The terminal step of differentiation culminates with the maturation of differentiated myotubes into muscle fibers. This process of skeletal muscle development and differentiation is orchestrated by the combinatorial activity of two transcription factors families that include Myogenic Regulatory Factors (MRFs) and the Myocyte Enhancer Factor-2 (MEF2). The expression and activities of MRFs and MEF2 are tightly controlled by both positive and negative regulatory factors. In addition there are several chromatin modifiers and remodelling complexes that regulate MRF and MEF2 activities and thereby regulating skeletal muscle differentiation. I have analyzed the role of one such chromatin modifier G9a, which mediates H3 lysine-9 di-methylation (H3K9me2) chromatin marks that are associated with transcriptional repression. G9a is expressed at high levels in undifferentiated myoblasts and is down-regulated upon differentiation. Over-expression of G9a in myoblasts retards skeletal muscle differentiation in a methyltransferase activity-dependent manner. RNAi-mediated knockdown of G9a in muscle precursor cells results in enhanced differentiation. This is concomitant with early induction of differentiation genes myogenin and Troponin T. G9a-dependent impairment of differentiation is correlated with H3K9me2 marks on muscle-specific promoters. In addition, G9a interacts with and methylates MyoD, a MRF that is central to differentiation of skeletal muscle cells. G9a methylates MyoD at a single lysine (K) residue 104, suppressing its transcriptional activity. This modification blocks the ability of MyoD to activate its downstream target genes such as myogenin, resulting in impairment of differentiation. Interestingly, K104 in MyoD is also a site for acetylation by a histone acetyltransferase P/CAF. Consistent with this, G9a disrupts P/CAF-mediated MyoD acetylation which is critical for its transactivation function and for execution of the myogenic differentiation program. RNAi mediated reduction of G9a results in altered kinetics of MyoD acetylation, suggesting a possibility of cross-talk between G9a and P/CAF in controlling MyoD transcriptional activity and myogenesis. In addition, pharmacological inhibition of P/CAF activity enabled us to identify gene networks broadening the spectrum of its regulatory function in skeletal muscle differentiation. These findings provide insights into the mechanisms by which chromatin modifiers regulate MyoD activity via direct post-translational modifications to control skeletal muscle differentiation.
Appears in Collections:Ph.D Theses (Open)

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