Characterization of genetic modifiers of bioenergetic rescue in mitochondrial disease cell lines

项目摘要 越来越多的人认识到线粒体功能障碍会导致几种疾病，如癌症， 糖尿病、神经变性疾病和线粒体疾病。线粒体疾病是由 线粒体基因突变导致高能组织中的生物能量缺陷，导致组织 损害目前，对于患有线粒体疾病的个体没有有效的治疗选择 也没有什么有希望的药物开发目标。在这项研究中，我们的目标是阐明遗传 这些机制可以挽救与人类细胞中线粒体突变相关的生物能量学缺陷。我 提出1）机械地剖析BRD4（在哺乳动物中鉴定的含溴结构域蛋白）的抑制如何 我们最近的无偏筛选）拯救线粒体生物能量学，2）确定细胞效应物（因子或 代谢途径）下游的BRD4抑制，和3）确定额外的基因，拯救线粒体 通过CRISPR基因编辑技术治疗呼吸链缺陷我将首先确定PGC1α 在BRD 4背景下上调线粒体呼吸基因表达的转录因子 通过基因敲除研究抑制。这将使我能够进一步测试我们的模型， 至少一种PGC1α转录因子的启动子接近被BRD4启动子占用抑制， ChIP分析。对于我的第二个目标，我将进行配对线粒体蛋白质组学和代谢组学， 发现BRD4抑制的下游效应器，对于我的第三个目标，我将扩展我们基于CRISPR的 平台，以确定基因突变，拯救与呼吸链相关的生物能量学缺陷 复合物IV缺乏症。这些研究利用了哈佛医学院达纳法伯癌症研究所的资源 学校和布罗德研究所提供的见解，细胞如何科普线粒体缺陷， 确定线粒体疾病的潜在治疗靶点。

PROJECT SUMMARY There is a growing appreciation of mitochondrial dysfunction contributing to several diseases such as cancer, diabetes, neurodegenerative disease, and mitochondrial diseases. Mitochondrial diseases result from mutations in mitochondrial genes that cause bioenergetic defects in high-energy tissues leading to tissue damage. Currently, there are no effective treatment options for individuals afflicted with mitochondrial diseases and few promising targets for drug development. In this research proposal, we aim to elucidate genetic mechanisms that rescue the bioenergetics deficits associated with mitochondrial mutations in human cells. I propose to 1) mechanistically dissect how inhibition of BRD4 (a bromodomain-containing protein identified in our recent unbiased screens) rescues mitochondrial bioenergetics, 2) determine cellular effectors (factors or metabolic pathways) downstream of BRD4 inhibition, and 3) identify additional genes that rescue mitochondrial respiratory chain deficiencies through CRISPR gene-editing technology. I will first determine the PGC1α transcription factor that upregulates expression of mitochondrial respiratory genes in the context of BRD4 inhibition through gene knockdown studies. This will allow me to further test our model that mitochondrial gene promoter access by at least one PGC1α transcription factor is inhibited by BRD4 promoter occupancy using ChIP analyses. For my second aim, I will perform paired mitochondrial proteomics and metabolomics to uncover the effectors downstream of BRD4 inhibition and for my third aim, I will expand our CRISPR-based platform to identify gene mutations that rescue the bioenergetics defects associated with respiratory chain complex IV deficiency. These studies with utilize resources at Dana-Farber Cancer Institute, Harvard Medical School, and the Broad Institute to provide insights into how cells cope with mitochondrial deficiencies and identify potential therapeutic targets for mitochondrial diseases.