组蛋白去甲基酶KDM3A介导小鼠骨骼肌细胞中活性氧诱导的E2F1表达

Induction of E2F1 expression by ROS in murine skeletal muscle cells is mediated by the histone demethylase KDM3A

目的:研究在小鼠骨骼肌细胞凋亡过程中E2F1表达上调的表观遗传机制。方法:利用H2O2处理C2C12细胞,利用干扰RNA和瞬时转染相关因子,用实时定量PCR(real-time quantitative PCR,RT-qPCR)与Western blot检测E2F1基因表达水平,染色质免疫沉淀(chromatin immunoprecipitation,ChIP)检测蛋白质与DNA的结合。结果:E2F1表达水平上调,同时伴随E2F1基因启动子上H3K9甲基化水平的下降。进一步研究发现,活性氧促进H3K9去甲基酶KDM3A结合到E2F1启动子上促进E2F1转录。相反,使用干扰RNA敲减KDM3A显著减弱活性氧引起的E2F1表达上调。KDM3A干扰同时降低了E2F1启动子上乙酰化H3与H3K4甲基化水平并恢复了H3K9甲基化水平。结论:KDM3A可能通过改变组蛋白修饰参 与活性氧诱导的E2F1转录激活。

Objective:Accumulation of reactive oxygen species(ROS)in skeletal muscle cells promotes apoptosis,which represents a key mechanism underlying the loss of muscle mass during aging.Previously we have demonstrated that the transcription factor E2F1promotes the transcription of the pro-apoptotic gene PERP in skeletal muscle cells(C2C12).In the present stuy we investigated the epigenetic mechanism whereby E2F1 expression is stimulated by ROS in C2C12 cells.Methods:Real-time q PCR and Western blotting were used to examine gene expression levels.Chromatin immunoprecipitation was used to determine the interaction between protein and DNA.Results:We report that E2F1 levels were up-regulated in C2C12 exposed to H2O2.Up-regulation of E2F1 expression by H2O2treatment was accompanied by erasure of dimethyl H3K9 from the E2F1 promoter.Further study revealed that H2O2treatment promoted the binding of KDM3A,a histone demethylase,on the E2F1 promoter.In contrast,KDM3A depletion by si RNA significantly dampened the induction of E2F1 expression by H2O2treatment.Concomitantly,KDM3A knockdown decreased the levels of acetylated H3 and trimethylated H3K4 but restored the levels of dimethylated H3K9 on the E2F1 promoter.Conclusion:Our data suggest that KDM3A may participate in H2O2treatment-induced E2F1 trans-activation by modulating histone modifications on the E2F1 promoter.