Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells. PacBio sequencing of exogenously methylated mitochondrial DNA from: HeLa cells; HeLa cells with perturbed TFAM levels, inhibited mitochondrial transcription, and disrupted complex III function; human skeletal myoblasts across three differentiation time points; U2OS cells; and in vitro generated mtDNA with varying levels of recombinant TFAM.

细胞代谢依赖于线粒体DNA（mtDNA）的调控与维持。每个细胞内存在数百至数千份mtDNA拷贝，但由于线粒体缺乏组蛋白及其他对核基因组压缩至关重要的机制，mtDNA如何组装为独立的线粒体类核仍未明确。本研究采用长读长单分子可及性图谱技术，以近单核苷酸分辨率对单条完整mtDNA分子的压缩状态进行检测。研究发现，与核基因组不同，人类mtDNA整体上呈现全或无式的全局压缩模式：绝大多数线粒体类核处于不可接近的失活状态。高度可及的线粒体类核同时结合转录与复制复合物，并选择性形成三链置换环结构。此外，本研究证实，主要的线粒体类核结合蛋白线粒体转录因子A（TFAM）可在体内外直接调控不可接近类核的比例，其作用机制符合成核-扩散模型，通过包被并压缩线粒体类核发挥功能。综上，本研究揭示了人类细胞内mtDNA组装与调控的核心架构。本研究对外源甲基化线粒体DNA开展PacBio测序，所涉及的样本包括：海拉（HeLa）细胞；TFAM水平扰动、线粒体转录受抑制且复合物III功能受损的HeLa细胞；三个分化时间点的人类骨骼肌成肌细胞；U2OS细胞；以及重组TFAM水平各异的体外合成mtDNA。