Mediator (MED) is a multi-subunit complex that plays a central role in transcription initiation by integrating regulatory signals from gene-specific transcriptional activators to RNA polymerase II. Genetic studies have identified mutations in specific MED subunits to be associated with human cardiac diseases. Specifically, deletions in MED15 are common in patients with DiGeorge/velocardiofacial syndrome, which typically includes congenital heart defects. A missense mutation in MED30 causes cardiomyopathy in mice. Emerging evidence suggests a critical role for ?specialized? MED subunits in the regulation of temporal- and/or spatial-specific gene expression. However, little is known as to the subunit composition of MED in cardiomyocytes (CMs) at distinct developmental stages, and no studies have examined enrichment of MED complexes harboring individual ?specialized? MED subunits at distinct genomic loci, as well as the specific transcription factors (TFs) they directly interact with to anchor them to functionally critical enhancer/promoter elements during heart development, or during the progression of cardiac disease. We found that transcript levels of MED subunits fluctuate within CMs during development. To gain further understanding into MED subunit specific roles in CMs, I generated novel mouse models with cardiac-specific knockout (cKO) of selected MED subunits. MED15, MED25, and MED30 cKO mice showed distinct phenotypes. MED15 cKO mice died immediately after birth, with cardiac defects observed from E12.5, whereas MED30 cKO mice died at E10-10.5 with dilated hearts. Conversely, MED25 cKO mice survived to adulthood and displayed no cardiac defects, demonstrating that not all MED subunits are indispensable for cardiac function. Accordingly, my hypothesis is that MED15 and MED30 subunits exhibit unique functional activities that shape key events in CM transcriptional regulation, morphogenesis, and heart function.
My Specific Aims are:
Aim 1. (K99) To elucidate stage-specific and locus- specific roles of MED15 or MED30 subunits in developing CMs by analyzing cardiac phenotypes of MED15 and MED30 cKO mice, identifying unique transcriptional regulatory elements enriched for MED15 or MED30 during early heart development, and identifying TFs that specifically interact with MED15 or MED30;
and Aim 2. (R00) To understand the pathophysiological functions of MED15 and MED30 subunits during the progression of adult heart disease, including DCM and pathological hypertrophy, by utilizing inducible MED15 and MED30 CM-specific knockout (icKO) mouse models. I have a strong background and training record in mouse genetics and molecular cardiology. My training in the K99 phase will consist of structured mentorship by my primary mentor and complementary co-mentors/consultants, formal coursework, additional training in experimental skills (RNAseq, ChIP-seq and bioinformatics) and a program of career transition. This career plan and research project will ensure my successful transition to independent research, to fulfill my ultimate career goal of understanding the development, progression, and molecular basis of human cardiovascular diseases.
Genetic studies have identified mutations in specific subunits of Mediator (MED), which plays a central role in transcription initiation, associated with congenital heart disease and heart failure. However, little is known as to the specific roles of MED subunits in developing cardiomyocytes, or adult cardiac function, and how specialized MED subunits regulate temporal- and/or spatial-specific gene expression in cardiomyocytes. Proposed studies are aimed at understanding the role of cardiac disease-related MED15 and MED30 in cardiomyocytes at the molecular, cellular, and physiological levels, and gaining insight into mechanisms by which cardiac specialized MEDs regulate the cardiac transcriptional program during heart development, or during the progression of cardiac disease.