Project Summary Mitochondrial diseases comprise a heterogeneous group of genetic inherited disorders resulting from mutations in mitochondrial or nuclear DNA that cause failures in mitochondrial energetic and metabolic function. As a consequence of this mitochondrial failure, high energy demanding tissues such as brain, skeletal muscle, liver, kidney, endocrine and respiratory systems are severely affected. Current available therapies remain supportive but an effective cure is still missing. Therefore, there is an urgent medical need to identify new therapeutic targets to treat mitochondrial diseases. Identification of specific targets and drugs that increase and rescue mitochondrial bioenergetics through different complexes of the electron transport chain can be of therapeutic value to treat mitochondrial diseases. An example is activation of the transcriptional coactivator PGC-1α, a major component of mitochondrial biogenesis, that rescue bioenergetic defects caused by mutations or mouse models of mitochondrial diseases and ameliorates clinical symptoms. Using a chemical and genome-wide CRISPR editing screens in trans-mitochondrial cybrids cell carrying a mutation in a mitochondrial encoded complex I subunit, we have identified Brd4 (Bromodomain protein 4) as potential target to treat mitochondrial diseases. Bromodomain inhibition or loss-of-Brd4 enhances oxidative phosphorylation activity and rescues the bioenergetic defects caused by genetic inhibition of mitochondrial complex I and promotes cell survival under high energetic demands. However, the precise mechanisms of how bromodomain inhibition controls mitochondrial bioenergetics and the effects on mitochondrial disease in in vivo models are unknown. The major goal of this grant application is to identify and analyze the molecular mechanisms whereby bromodomain inhibition activates mitochondrial energetics function and whether it rescues mitochondrial disease symptoms in in vivo mouse models. The research strategy is focused on three central aims: 1) Molecular and functional analysis of how Brd4 controls mitochondrial gene expression programs in trans-mitochondrial cybrid cells. (Specific Aim 1), 2) Cellular, metabolic and bioenergetic analysis mediated by bromodomain inhibition in cybrid cell lines and mitochondrial disease patient derived fibroblasts (Specific Aim 2) and, 3) In vivo and Ex vivo metabolic, energetic and survival analysis by bromodomain inhibitors in mitochondrial disease mouse models (Specific Aim 3). The outcomes from these studies will identify novel molecular mechanisms and regulatory components by which bromodomain inhibition and loss of Brd4 rescue bioenergetic defects caused by mitochondrial electron transfer chain defects. Since mitochondrial bioenergetic failure is a hallmark of mitochondrial diseases, our studies may translate into potential therapies.