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

项目摘要/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还没有被探索。 线粒体DNA调控中的错误可能导致许多人类疾病，这些疾病可能在 任何年龄和任何器官，包括运动神经病、肾功能障碍、不孕不育、心肌病和 神经退行性变。一般来说，DNA的包装及其产生的高阶结构是至关重要的 参与调控真核生物的基因表达、基因组复制和基因组分离 和原核生物。众所周知，线粒体基因组以核蛋白复合体的形式存在。 被称为类核，单个线粒体平均拥有5个基因组副本，导致在 每个细胞100和10,000个拷贝。关于基因组是如何构成的，目前还缺乏相关知识 这种结构在不同的细胞类型之间有何不同，以及这种结构在疾病中是如何被错误调控的。 此外，还不知道单个线粒体内的单个类核是否具有不同的结构和服务 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 描述类核整体三维结构的技术。我们建议改编 几种强大的高分辨率技术，并结合从每一种技术中获得的知识来开发 线粒体类核的组织模型。我们将优化Hi-C以映射物理交互 线粒体基因组、ATAC-seq和Nome-seq区域，以确定开放和可访问的区域 DNA和芯片连接，以开发主要类核结合蛋白的高分辨率地图。我们会带着 这些研究是在几个不同的人类细胞系中进行的，这些细胞系被证明具有不同的能量 要求，因此不同的线粒体基因表达。此外，我们将使这一系统适应 分化成肌细胞，其中线粒体生物合成高度上调。使用这些强大的 技术，我们将确定主要转录因子和结构蛋白TFAM在 建立和维护基因组结构。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，我们认为类核子之间可能存在不均一性。 在线粒体内。为此，我们将开发方法来分离不同的类核群体 从线粒体来描述它们的整体结构有何不同。总之，这项拟议的研究将 提高我们对线粒体基因组结构的认识，使我们能够更好地理解线粒体是如何 基因被调控，基因组复制和拷贝数如何被调控。这一知识将是 在理解线粒体突变如何导致疾病方面有非常宝贵的价值。

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.