mtDNMT1介导线粒体表观遗传学调控在早衰中的作用及其干预机制

Mitochondrial DNA methylation and nucleoid package are the next-generation epigenetic biomarkers. Our pervious study demonstrated that the mRNA expression for mitochondria DNMT1 (mtDNMT1) decreased gradually with increased population doubling levels in human embryonic lung fibroblasts subjected to premature senescence and that the defective mtDNMT1 would up-regulate expression of mitochondrial transcriptional regulation factor for the first time, which implied that mtDNMT1 might mediate mitochondria DNA methylation in premature senescence. However, the role of mtDNA methylation mediated by mtDNMT1 in premature senescence is unclear. This proposed study will explore the epigenetic characteristics and change rules of mitochondria including mitochondrial DNA methylation, hydroxymethylation and the nucleoid package during premature senescence using specific mitochondria epigenetic methods, such as mitochondria immunoprecipitation combined sequencing (mtIP-Seq), bisulfite-PCR-pyrosequencing, 5mC mitochondria methylated DNA immunoprecipitation combined chip (mtMeDIP-chip), etc. This study is also intended to investigate the key events of mitochondrial DNA methylation mediated by mtDNMT1 and their potential role in premature senescence using the methyltransferase inhibitor, transcription activator-like effector nucleases (TALEN) and transcription activator-like effector activator (TALEA) techniques to intervene in the function of mtDNMT1. The project will not only explore the molecular mechanism underlying oxidative stress-induced premature senescence from the view point of mitochondria epigenetics, but will also help identify the early epigenetic biomarkers of mitochondrial damage, which can be exploited in the epigenetic targeted intervention of premature senescence and associated diseases.

线粒体DNA甲基化和类核组装是新一代表观遗传学标志，申请者前期研究发现早衰中人胚肺成纤维细胞线粒体DNA甲基转移酶mtDNMT1表达随增龄降低，其缺陷导致线粒体转录调控因子上调，提示mtDNMT1可能介导早衰中mtDNA甲基化，但其介导mtDNA甲基化在早衰中作用未明。本项目拟应用线粒体免疫沉淀测序（mtIP-Seq）、亚硫酸氢盐修饰焦磷酸测序及5mC甲基化免疫沉淀芯片（mtMeDIP-chip）等线粒体表观遗传学技术分析早衰发生中线粒体DNA甲基化、羟甲基化及类核组装等其表观遗传学修饰的变化特征和规律；应用甲基化酶抑制剂并结合TALEN和TALEA技术干预mtDNMT1表达，探讨其介导mtDNA甲基化关键事件及在早衰中的作用。本项目不仅从线粒体表观遗传学这一崭新的视角探讨氧化应激诱导早衰的分子机制，而且为寻找线粒体早期表观遗传生物学标志及对早衰等相关疾患表观遗传靶向干预提供重要理论依据。

本课题为验证mtDNMT1介导早衰中线粒体表观遗传学调控机制及其干预措施，运用人胚肺成纤维细胞复制性衰老模型、中等剂量过氧化氢诱导早衰模型及CRISPR/Cas9技术敲除mtDNMT1细胞株，应用激光共聚焦、化学发光、酶联免疫及Q-PCR，检测细胞衰老中线粒体生物学性状变化，发现线粒体围绕细胞核呈弥散分布，复制性衰老线粒体数量、mtDNA拷贝数下降，8-OHdG含量升高；早衰线粒体数量和8-OHdG含量降低，ATP浓度和mtDNA拷贝数升高，表明复制性衰老与早衰线粒体生物学功能有差异并逐渐衰退，过氧化氢有早衰初期线粒体毒性。运用mtMeDIP-Seq及mtIP-Q-PCR，筛选细胞衰老中差异甲基化的线粒体基因，检测mt5mc对其表达调控，发现两种衰老中ND5、ND6高甲基化，COX1是早衰特有，两种衰老存在线粒体基因差异甲基化谱，捕获线粒体相关mRNA、LncRNA及Small ncRNA差异甲基化区域，较全面揭示细胞衰老线粒体表观遗传特征。荧光检测及DAC干预检测mtDNMTs酶活性、mtDNA甲基化相关酶表达，发现mtDNMTs活性和mtDNMT1表达随衰老而升高，为衰老相关线粒体生物学标志，mtDNMT1敲除促进早衰，TET1-3、MBD2在复制性衰老及早衰差异表达。Q-PCR、WB及亚硫酸氢盐修饰BSP检测线粒体相关基因表达及甲基化状态，即核编码TFAM、TFB2M、MTERF、POLRMT及PGC1ɑ、NRF1，线粒体编码D-loop、MT-TF、MT-RNR1及ND1、ND6和Cytb，发现复制性衰老及早衰中差异表达，TFB2M及ND6受启动子区甲基化调控，证实核-线粒体存在表观遗传交叉串话。在2、4及10月龄快速老化小鼠SAMP8和抗快速老化小鼠SAMR1，电镜下早衰小鼠肺组织线粒体增多肿胀、嵴消失，甲基化相关酶和线粒体功能基因表达谱差异，调控动物早衰，且体内外早衰线粒体表观遗传调控存在差异。经MeRIP-Seq发现细胞衰老中m6A RNA甲基化位点增多，复制性衰老与早衰新的RNA差异甲基化与其mRNA表达密切关联，是衰老RNA甲基化新特征。本研究为体内外早衰mtDNA表观遗传调控提供新资料，是其表观遗传调控网络的重要补充，mtDNMT1可作为早衰线粒体表观遗传生物学标志及潜在干预靶点，并为合理评价氧化应激与年龄相关疾患的线粒体毒性提供科学依据。