Muscle satellite cells are responsible for the postnatal growth and repair capacity of adult skeletal muscle. The transcription factor Pax7 is expressed in satellite cells and is required for satellite cell survival and function through satellite cell development. We have shown that Pax7 activates target genes such as Myf5 through recruitment of the Ash2l/Wdr5/MLL2 histone methyltransferase complex. In addition, we found that 10% of satellite cells have never expressed Myf5 (based on Myf5-Cre lineage marking), and this subset of cells (termed satellite stem cells) are capable of repopulating the satellite cell niche. Using mass spectrometry, we identified Carm1 as a Pax7 binding protein. Carm1 directly methylates Pax7 on N-terminal arginines. Mutation of these sites results in a markedly reduced ability of Pax7 to upregulate Myf5 transcription, but does not affect DNA binding. Importantly, our experiments indicate that Carm1 methylation of Pax7 is necessary for Pax7 to bind MLL2. We therefore hypothesize that Carm1 positively controls transcriptional-activation by Pax7 through regulating the ability of Pax7 to bind MLL2 (and recruitment of the Wdr5/Ash2L/MLL2 histone methyltransferase), to activate Pax7-target genes. In this application, we propose a research program to molecularly define the role of Carm1 in regulating Pax7 function. To investigate the hypothesis that Pax7 mediates entry into the myogenic program through epigenetic mechanisms, we will undertake a comparative analysis of Pax7, Carm1 and Ash2L binding sites between satellite stem and myogenic cells, as well as map the distribution of modified histones as a function of gene expression. The enzymatic activity of Carm1 is regulated by phosphorylation at serine 229. Therefore, we will express an activated mutant version of Carm1 in satellite stem cells to ask whether this is sufficient to induce Myf5 transcription. To investigate at which levels of the satellite cell developmental program Carm1 is regulating Pax7 function, we will investigate the modulation of the Pax7-MLL2 interaction in quiescent satellite stem cells and myogenic cells, in activated satellite cells, during cell-cycle progression of proliferating myoblasts, and during terminal differentiation. We will undertake a multiplex in vitro kinase screen using recombinant Carm1 protein as substrate to identify the kinase(s) that are capable of phosphorylating Carm1. We will perform mass spectrometric analysis to identify additional regulators of Carm1. Lastly, we will employ siRNA knock-downs and conditional loss-of-function analysis in mice to characterize the biological significance of the Carm1-mediated regulation of Pax7 function. These studies will provide important new information about the molecular regulation of satellite cell function. Potentially, such insights will lead to novel therapeutic interventions for muscle- wasting diseases such as muscular dystrophy and sarcopenia.
Understanding how regulatory genes control the growth and repair of skeletal muscle, in particular the formation, activation, proliferation, and terminal differentiation of myogenic stem cells, is highly relevant to understanding the regenerative processes that occur in patients with muscle wasting diseases such as muscular dystrophy or age-related sarcopenia. We believe that our proposed studies will provide novel insights into the biology of muscle regeneration. Potentially, such insights will lead to new modalities of therapeutic intervention.
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