Evolutionary and Functional Significance of 5-methylcytosine and 5-hydroxymethylcytosine in Mitochondrial DNA

智力优势：真核生物同时维持两个基因组，二倍体核基因组和线粒体基因组，后者由小的环状DNA组成，包裹在称为类核的蛋白质结构中，分布在线粒体网络中，每个细胞有数百到数千个拷贝。虽然线粒体负责高效的能量产生，但它的基因组只编码13种蛋白质，每一种蛋白质都与ATP产生所需的电子传递密切相关。线粒体功能所需的所有其他蛋白质都在核基因组上编码并输入线粒体。核基因组中胞嘧啶残基对5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）的表观遗传修饰对基因表达调控至关重要，至少在高等真核生物中，对正常发育和生存至关重要。直到最近，胞嘧啶甲基化在线粒体基因组中的作用还没有被发现。然而，对哺乳动物线粒体中负责胞嘧啶转化为5mC的DNA甲基转移酶1 （mtDNMT1）的鉴定表明，线粒体基因组中同时存在5mC和5hmC。此外，改变这种线粒体酶的水平会以基因特异性的方式影响线粒体转录。当前的项目遵循这些观察结果，试图揭示mtDNA胞嘧啶残基上产生这些修饰的机制，它们在线粒体功能中的作用，以及线粒体基因组表观遗传修饰的进化意义。该项目的成功完成可能会为目前对该细胞器内控制基因转录机制的理解提供范式转变。这项研究有望揭示线粒体胞嘧啶甲基化和羟甲基化的生物化学过程，以及该过程是否与细胞核中的操作有显著不同。线粒体DNA表观遗传修饰的保存将使我们能够理解这一过程是否为其运作的生物体提供了代谢优势。更广泛的影响：该项目将为本科生和研究生提供一个独特的机会，参与发现新的和令人兴奋的生物学知识的过程。该项目将为学生提供批判性思维，数据分析，团队合作，口头和书面报告研究成果的经验，并参与在实验室环境中解决问题的多学科方法。将本科生纳入该项目将为研究生和博士后提供指导经验，这对他们作为科学发现的积极参与者的发展至关重要。

Intellectual Merit: Eukaryotic organisms simultaneously maintain two genomes, the diploid nuclear genome and the mitochondrial genome, which consists of small circular DNA, packaged in protein-containing structures called nucleoids and distributed throughout the mitochondrial network, with hundreds to thousands of copies per cell. Although the mitochondrion is responsible for efficient energy generation, its genome encodes only 13 proteins, each intimately involved in electron transport that is required for ATP production. All other proteins required for mitochondrial function are encoded on the nuclear genome and imported into the mitochondria. Epigenetic modification of cytosine residues to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the nuclear genome is critical for regulation of gene expression and, at least in higher eukaryotes, is essential for normal development and survival. Until recently, a role for cytosine methylation in the mitochondrial genome had not been found. However, identification of a mitochondrial form of the mammalian enzyme responsible for conversion of cytosine to 5mC, DNA methyltransferase 1 (mtDNMT1) has revealed the presence of both 5mC and 5hmC in the mitochondrial genome. Moreover, changing the level of this mitochondrial enzyme impacts mitochondrial transcription in a gene-specific fashion. The current project follows on these observations by seeking to uncover the mechanisms generating these modifications on mtDNA cytosine residues, their role in mitochondrial function, and the evolutionary significance of epigenetic modification of the mitochondrial genome. Successful completion of this project is likely to provide a paradigm shift in the current understanding of mechanisms controlling gene transcription within this organelle. This research is expected to uncover the biochemistry involved in mitochondrial cytosine methylation and hydroxymethylation and whether the process differs significantly from that operating in the nucleus. The conservation of epigenetic modification of mitochondrial DNA will allow an understanding of whether this process affords a metabolic advantage to the organisms in which it operates. Broader Impacts: This project will offer a unique opportunity to both undergraduate and graduate students to participate in the process of discovery of new and exciting biological knowledge. The project will provide students with experience in critical thinking, data analysis, teamwork, oral and written presentation of research results, and participation in a multidisciplinary approach to problem solving in the laboratory environment. The inclusion of undergraduate students in the project will offer mentoring experience to graduate students and postdoctoral fellows, which is essential to their development as active participants in scientific discovery.