The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [A549_RNA-seq]

How individual genes are regulated from a mitochondrial polycistronic transcript to have variable expression remains an enigma. Here, through bisulfite sequencing and strand-specific mapping, we show mitochondrial genomes in human and other animals were strongly biased to light (L)-strand non-CpG methylation with conserved peak loci preferentially located at gene-gene boundaries, which was also independently validated by MeDIP and FspEI digestion. Such mtDNA methylation patterns are conserved across different species and developmental stages, but display dynamic local or global changes during development and aging. Knockout of DNMT3A alone perturbed mtDNA regional methylation patterns, but not global levels, and altered mitochondrial gene expression, copy number, and oxygen respiration. Overexpression of DNMT3A strongly increased mtDNA methylation and strand bias. Overall, methylation at gene body and boundaries was negatively associated with mitochondrial transcript abundance and also polycistronic transcript processing. Furthermore, HPLC-MS confirmed the methylation signals on mitochondria DNA. Together, these data provide high-resolution mtDNA methylation maps that revealed a strand specific non-CpG methylation, its dynamic regulation and its impact on the polycistronic mitochondrial transcript processing.

线粒体多顺反子转录本如何调控单个基因以实现差异化表达，这一问题至今仍是未解之谜。本研究通过亚硫酸氢盐测序（bisulfite sequencing）与链特异性图谱分析（strand-specific mapping），证实人类及其他动物的线粒体基因组显著偏向轻链（L链）非CpG甲基化，且保守的甲基化峰值位点优先分布于基因-基因边界处；该结果亦通过甲基化DNA免疫沉淀（methylated DNA immunoprecipitation, MeDIP）与FspEI酶切实验得到了独立验证。此类线粒体DNA（mitochondrial DNA, mtDNA）甲基化模式在不同物种与发育阶段中均保守存在，但在发育及衰老过程中会呈现局部或整体的动态变化。单独敲除DNA甲基转移酶3A（DNMT3A）会扰乱线粒体DNA的区域甲基化模式，但未改变整体甲基化水平，同时还会改变线粒体基因表达、拷贝数与氧呼吸能力。过表达DNMT3A则可显著提升线粒体DNA的甲基化水平与链偏好性。总体而言，基因本体及其边界处的甲基化与线粒体转录本丰度以及多顺反子转录本加工过程呈负相关。此外，高效液相色谱-质谱联用法（High Performance Liquid Chromatography-Mass Spectrometry, HPLC-MS）验证了线粒体DNA上存在甲基化信号。综上，本研究数据提供了高分辨率的线粒体DNA甲基化图谱，揭示了链特异性非CpG甲基化现象、其动态调控机制以及对线粒体多顺反子转录本加工过程的影响。