Dnmt3a Regulates Proliferation of Muscle Satellite Cells via p57Kip2

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.

细胞分化状态由基因表达谱决定，而该过程由表观遗传机制协同调控。细胞类型特异性DNA甲基化模式由染色质修饰因子建立，包括从头DNA甲基转移酶（de novo DNA methyltransferase），例如Dnmt3a与Dnmt3b。自肌源性主基因MyoD被发现以来，肌源性分化便被用作研究组织分化的经典模型系统。尽管目前关于肌源性基因调控网络的研究成果不断积累，但学界对于DNA甲基化如何调控肌源性基因程序的认知仍十分有限。为阐明DNA甲基化在肌肉发育与再生过程中的作用，本研究针对小鼠肌肉前体细胞中Dnmt3a的突变效应展开探究。由Pax3启动子驱动的Dnmt3a条件性敲除（conditional knockout, cKO）小鼠表现出骨骼肌器官质量下降，且在心脏毒素（cardiotoxin）诱导的肌肉损伤后再生能力减弱。此外，Dnmt3a敲除的卫星细胞（satellite cells, SCs）在体外培养中表现出显著的增殖能力丧失。转录组分析显示，在Dnmt3a敲除的卫星细胞中，细胞周期蛋白依赖性激酶抑制剂（cyclin-dependent kinase inhibitors, CDKIs）Cip/Kip家族成员p57Kip2的表达出现失调。此外，通过RNA干扰（RNA interference, RNAi）介导的p57Kip2敲降可恢复卫星细胞的增殖活性，由此证实Dnmt3a-p57Kip2信号轴在调控卫星细胞增殖过程中发挥重要作用。与上述研究结果一致，Dnmt3a条件性敲除小鼠的骨骼肌组织中Pax7阳性卫星细胞数量减少，且这些细胞内p57Kip2蛋白的表达水平升高。综上，本研究证实Dnmt3a可通过表观遗传调控p57Kip2的表达，进而维持卫星细胞的增殖活性，最终维持肌肉稳态。