甲基转移酶Dot1L通过调控启动子甲基化水平促进骨骼肌成肌分化

Methyltransferase Dot1L promotes myogenic differentiation of skeletal muscle by regulating promoter methylation level

目的明确甲基转移酶Dot1L是否参与成肌分化调控过程,并初步探索其调控成肌分化过程的分子机制。方法应用小鼠体外成肌分化模型C2C12细胞系,诱导C2C12细胞在0、1、3、5 d发生成肌分化,使用Dot1L特异性抑制剂抑制其甲基转移酶活性,并应用免疫荧光实验观察Dot1L对成肌分化过程的影响;检测成肌分化标志分子肌球蛋白重链(myosin heavy chain,MHC)和肌细胞调节因子(Myogenin)的蛋白质水平和转录水平及启动子区域的组蛋白甲基化水平;使用RNA-Seq基因组范围内检测抑制剂处理与否发生变化的基因阵列;并用CHIP-qPCR实验验证H3K79me2结合位点。结果Dot1L组蛋白底物H3K79的甲基化水平随成肌分化进程逐渐升高(P<0.01);抑制Dot1L酶活性后,成肌分化过程明显受阻;免疫荧光实验显示C2C12细胞多核肌管形成显著下降(P<0.01),MHC和Myogenin的蛋白质水平及转录水平均显著降低(P<0.01);启动子区域组蛋白H3K4甲基化水平降低(P<0.01);RNA-Seq显示受Dot1L调控的基因阵列主要集中在骨骼肌的成肌分化和肌肉的发育;经CHIP-qPCR验证参与成肌分化的部分基因存在H3K79me2结合位点。结论甲基转移酶Dot1L通过直接调控一群成肌分化相关基因的转录水平参与调控骨骼肌的成肌分化过程。

Objective To confirm whether methyltransferase Dot1 L is involved in the regulation of myogenic differentiation,and explore its molecular mechanism in the regulation process.Methods Myoblast differentiation model of mouse C2 C12 cell line was used to induce myogenic differentiation for 0,1,3 and 5 d.The specific inhibitor of Dot1 L was used to inhibit the activity of methyltransferase,and the impacts of Dot1 L on the process of myoblast differentiation was observed by immunofluorescence assay.The protein and transcription levels of myosin heavy chain(MHC)and myogenin,markers of myogenic differentiation,were subsequently detected,so was the histone methylation level of the promoter region.Moreover,RNA-Seq analysis was adopted to determine the gene arrays affected by inhibitor treatment in the genome,and chromatin immunoprecipitation coupled with quantitative PCR(CHIP-qPCR)was performed to verify the binding site of H3 K79 me2.Results The methylation level of H3 K79,Dot1 L histone substrate,was gradually increased with the process of myogenic differentiation(P<0.01).With the inhibition of Dot1 L activity,the process of myogenic differentiation was obviously suppressed,the formation of multinucleated myotubes in C2 C12 cells was significantly decreased(P<0.01),the protein and transcription levels of MHC and Myogenin were remarkably reduced(P<0.01),and the histone H3 K4 methylation level in the promoter region was also declined(P<0.01).RNA-Seq analysis indicated that the gene arrays regulated by Dot1 L mainly focused on the myogenic differentiation and muscle development of skeletal muscle.Finally,CHIP-qPCR verified that some genes involved in myogenic differentiation have H3 K79 me2 binding sites.Conclusion Methyltransferase Dot1 L participates in the regulation of the myogenic differentiation process of skeletal muscle by directly affecting the transcription level of myogenic differentiation related genes.