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

线粒体疾病细胞系生物能拯救遗传修饰剂的表征

• 批准号: 9761543
• 负责人: Christopher F. Bennett
• 金额: $ 6.16万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2020-11-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761543
• 关键词: Address; Affect; Attenuated; Binding; Bioenergetics; Biogenesis; Brain; Bromodomain; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell Death; Cell Line; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Dana-Farber Cancer Institute; Data; Defect; Dependence; Diabetes Mellitus; Disease; Drug Targeting; Failure; Fibroblasts; Galactose; Gene Activation; Gene Expression; Gene Mutation; Genes; Genetic; Genomics; Glucose; Goals; Growth; Heterogeneity; Histones; Hour; Human; Human Pathology; Individual; Inherited; Institutes; Intervention; Kidney; Lead; Liver; Malignant Neoplasms; Measures; Metabolic; Metabolic Pathway; Methodology; Mitochondria; Mitochondrial Diseases; Mitochondrial Respiratory Chain Deficiencies; Modeling; Molecular; Mutation; Neurodegenerative Disorders; Nuclear; Nutrient; Oxidative Phosphorylation; Pathology; Pathway interactions; Patients; Phenotype; Population; Proteins; Proteomics; Publishing; Research; Research Proposals; Resources; Respiration; Respiratory Chain; Role; Skeletal Muscle; Stress; Symptoms; Technology; Testing; Therapeutic; Tissues; Transcription Coactivator; base; cell type; complex IV; design; drug development; effective therapy; gain of function; gene function; genome-wide; improved; innovation; insight; knock-down; loss of function; medical schools; metabolomics; mitochondrial DNA mutation; mitochondrial dysfunction; mutant; negative affect; novel; oxidation; prevent; promoter; proteomic signature; recruit; respiratory; response; therapeutic target; transcription factor

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.

项目摘要 线粒体功能障碍越来越多地欣赏，导致多种疾病，例如癌症， 糖尿病，神经退行性疾病和线粒体疾病。线粒体疾病由 线粒体基因的突变，导致高能组织的生物能缺陷导致组织 损害。目前，对于患有线粒体疾病的人没有有效的治疗选择 几乎没有承诺的药物开发目标。在这项研究建议中，我们旨在阐明遗传 拯救生物能学的机制定义了与人类细胞中线粒体突变有关的机制。我 提出提示为1）机械剖析如何抑制BRD4（在 我们最近的公正屏幕）营救线粒体生物能学，2）确定细胞效应（因素或 代谢途径）BRD4抑制的下游，3）确定挽救线粒体的其他基因 通过CRISPR基因编辑技术，呼吸链缺陷。我将首先确定PGC1α 在BRD4背景下更新线粒体呼吸基因表达的转录因子 通过基因敲低研究抑制。这将使我能够进一步测试线粒体基因的模型 BRD4启动子占用者使用至少一个PGC1α转录因子访问启动子的访问。 芯片分析。为了我的第二个目标，我将执行配对的线粒体保护麦克风和代谢组学 揭示BRD4抑制下游的生效器，为了我的第三个目标，我将扩展我们的基于CRISPR的效果 识别挽救与呼吸链相关的生物能缺陷的基因突变的平台 复杂的IV缺乏。这些研究利用资源在哈佛医学 学校，以及广泛的研究所，以洞悉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标。