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.