Abstract
Cell differentiation status is defined by the gene expression profile, which is coordinately controlled by epigenetic mechanisms. Cell type-specific DNA methylation patterns are established by chromatin modifiers including de novo DNA methyltransferases, such as Dnmt3a and Dnmt3b. Since the discovery of the myogenic master gene MyoD, myogenic differentiation has been utilized as a model system to study tissue differentiation. Although knowledge about myogenic gene networks is accumulating, there is only a limited understanding of how DNA methylation controls the myogenic gene program. With an aim to elucidate the role of DNA methylation in muscle development and regeneration, we investigate the consequences of mutating Dnmt3a in muscle precursor cells in mice. Pax3 promoter-driven Dnmt3a-conditional knockout (cKO) mice exhibit decreased organ mass in the skeletal muscles, and attenuated regeneration after cardiotoxin-induced muscle injury. In addition, Dnmt3a-null satellite cells (SCs) exhibit a striking loss of proliferation in culture. Transcriptome analysis reveals dysregulated expression of p57Kip2, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CDKIs), in the Dnmt3a-KO SCs. Moreover, RNAi-mediated depletion of p57Kip2 replenishes the proliferation activity of the SCs, thus establishing a role for the Dnmt3a-p57Kip2 axis in the regulation of SC proliferation. Consistent with these findings, Dnmt3a-cKO muscles exhibit fewer Pax7+ SCs, which show increased expression of p57Kip2 protein. Thus, Dnmt3a is found to maintain muscle homeostasis by epigenetically regulating the proliferation of SCs through p57Kip2.
Highlights
Myogenic differentiation program has been extensively studied as a model of tissue differentiation since the discovery of MyoD [1]
We investigated the role of Dnmt3a in muscle satellite cells by analyzing Dnmt3a-conditional knockout mice in which Dnmt3a loci are deleted utilizing Cre-recombinase driven by Pax7 or Paired box 3 (Pax3) promoters that are activated in the muscle precursor lineage
The Dnmt3acKO mice exhibited significantly smaller body sizes than WT littermates at 8- to 12-week old (Fig 1D), they were born at normal Mendelian ratios, and were viable
Summary
Myogenic differentiation program has been extensively studied as a model of tissue differentiation since the discovery of MyoD [1]. Gene expression is regulated by both genetic and epigenetic mechanisms. DNA methylation is an epigenetic modification, which usually occurs at CpG sites [5]; the cytosine residues at CpG sites are methylated to 5-methyl-cytosine. This DNA methylation is mediated by a group of DNA methyltransferases (Dnmt) [6]. Accumulating evidence suggests that DNA methylation by Dnmt proteins in the promoter regions is associated with gene silencing, linking DNA methylation to gene suppression [6,10]. Recent studies have clarified the roles of DNA methylation in gene bodies and intergenic regions in enhancing gene expression [11,12,13,14]
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