The 3D architecture of the mitochondrial nucleoid and its role in organelle regulation

项目总结/摘要 线粒体基因组的结构及其在线粒体功能中的作用在很大程度上尚未探索。 线粒体DNA调控中的错误可能导致许多人类疾病， 任何年龄和任何器官，包括运动神经病、肾功能障碍、不孕症、心肌病，以及 神经变性一般来说，DNA的包装及其产生的高阶结构是至关重要的。 在真核生物和哺乳动物中参与基因表达、基因组复制和基因组分离的调节 和原核生物。众所周知，线粒体基因组是作为核蛋白复合体存在的 被称为类核，一个单克隆宿主平均有5个基因组拷贝， 每个细胞有100到10,000个拷贝。关于基因组是如何构建的， 细胞器，这种结构如何在细胞类型之间存在差异，以及这种结构如何在疾病中失调。 此外，尚不清楚单个核内的单个类核是否具有不同的结构并起作用。 不同的功能。 了解线粒体基因组结构的关键第一步是开发和优化 技术来表征类核的整体三维结构。我们建议 几个强大的，高分辨率的技术，并结合从每一个知识来开发 线粒体类核组织的模型。我们将优化Hi-C以映射物理交互 区域的线粒体基因组，ATAC-seq和NOMe-seq，以确定开放和可访问的区域， DNA和ChIP-nexus来开发主要类核结合蛋白的高分辨率图谱。创新实施 这些研究在几种不同的人类细胞系中进行，这些细胞系已被证明具有不同的能量， 不同的线粒体基因表达。此外，我们将调整该系统， 分化线粒体生物发生高度上调的成肌细胞。利用这些强大的 技术，我们将确定主要的转录因子和结构蛋白TFAM的作用， 建立和维持基因组结构。我们将使用已知的疾病突变体来了解 基因组结构在疾病中受到干扰。最后，我们认为类核之间可能存在异质性 在一个小圈子里。为此，我们将开发方法来破碎不同种群的类核 来描述它们的整体结构是如何不同的总之，这项研究将 推进我们对线粒体基因组结构的了解，使我们能够更好地了解线粒体如何 基因是如何被调控的以及基因组复制和拷贝数是如何被调控的。这些知识将 对于理解线粒体突变如何导致疾病是非常有价值的。

PROJECT SUMMARY / ABSTRACT The structure of the mitochondrial genome and its role in mitochondrial function are largely unexplored. Mistakes in the regulation of mitochondrial DNA can lead to a number of human diseases that can manifest at any age and in any organ including motor neuropathies, kidney dysfunction, infertility, cardiomyopathy, and neurodegeneration. Generally, the packaging of DNA and its resulting higher-order structure are critically involved in the regulation of gene expression, genome replication, and genome segregation in both eukaryotic and prokaryotic organisms. It is well known that the mitochondrial genome exists as a nucleoprotein complex called a nucleoid, and a single mitochondrion hosts, on average, 5 copies of the genome, resulting in between 100 and 10,000 copies per cell. There is a significant lack of knowledge on how the genome is structured in organello, how this structure differs between cell types, and how this structure is misregulated in disease. Further, it is not known if individual nucleoids within a single mitochondrion have different structures and serve different functions. A critical first step to understanding the structure of the mitochondrial genome is to develop and optimize techniques to characterize the overall three-dimensional architecture of the nucleoid. We propose adapting several powerful, high-resolution technologies, and combining the knowledge gained from each to develop models of the organization of the mitochondrial nucleoid. We will optimize Hi-C to map physically interacting regions of the mitochondrial genome, ATAC-seq and NOMe-seq to determine regions of open and accessible DNA, and ChIP-nexus to develop high-resolution maps of the major nucleoid binding proteins. We will carry these studies out in several distinct human cell lines, which have been shown to have different energy requirements and thus different mitochondrial gene expression. Further, we will adapt this system to differentiating myoblasts in which mitochondrial biogenesis is highly upregulated. Using these powerful techniques, we will determine the role of the major transcription factor and structural protein TFAM in establishing and maintaining genome structure. We will use known disease mutants to understand how the genome structure is disturbed in disease. Finally, we believe that heterogeneity may exist between nucleoids within a mitochondrion. To this end, we will develop methods to fractionate different populations of nucleoids from mitochondria to characterize how their overall architectures differ. In summary, this proposed research will advance our knowledge of mitochondrial genome structure, allowing us to better understand how mitochondrial genes are regulated and how genome replication and copy number are regulated. This knowledge will be invaluable in understanding how mitochondrial mutants lead to disease.