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

项目总结 越来越多的人认识到线粒体功能障碍是导致癌症等多种疾病的原因， 糖尿病、神经退行性疾病和线粒体疾病。线粒体疾病由以下原因引起 导致高能组织生物能缺陷的线粒体基因突变导致组织 损坏。目前，对于患有线粒体疾病的个体，还没有有效的治疗选择。 而且几乎没有什么有希望的药物开发目标。在这个研究方案中，我们的目标是阐明基因 挽救与人类细胞线粒体突变相关的生物能量学缺陷的机制。我 建议1)机械地剖析BRD4(一种含有溴域的蛋白质，在 我们最近的无偏见筛选)挽救了线粒体生物能量学，2)确定了细胞效应器(因素或 代谢途径)BRD4抑制的下游，以及3)识别拯救线粒体的其他基因 通过CRISPR基因编辑技术获得呼吸链缺陷。我将首先确定Pgc1α 在BRD4背景下上调线粒体呼吸基因表达的转录因子 通过基因敲除研究进行抑制。这将使我能够进一步测试我们的模型，线粒体基因 至少一个Pgc1α转录因子对启动子的访问被BRD4启动子占据所抑制 芯片分析。对于我的第二个目标，我将执行配对的线粒体蛋白质组学和代谢组学 发现BRD4抑制下游的效应器，为了我的第三个目标，我将扩展我们基于CRISPR的 识别修复呼吸链相关生物能量缺陷的基因突变的平台 复杂性静脉输液缺乏症。这些研究利用了哈佛大学医学院Dana-Farber癌症研究所的资源 学校和布罗德研究所提供了对细胞如何应对线粒体缺陷和 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.