Impact of a Histone H3K9 Methyltransferase on Mitochondria - Project Summary The histone lysine methyltransferase SETDB1 trimethylates lysine 9 on histone 3 (H3K9me3), which leads to the formation of heterochromatin, silences gene expression, and impacts three-dimensional chromatin structure. However, SETDB1 has recently been shown to play important roles in the cytoplasm as well as the nucleus. Altered activity of SETDB1 has been associated with multiple rare diseases including Huntington’s Disease, Duchenne muscular dystrophy, acute myeloid leukemia, and mesothelioma. It is important to identify and characterize the full range of different activities for SETDB1 in order to better understand how this protein impacts the development of these diseases. MET-2 is the C. elegans homolog of human SETDB1. Our imaging of a functional GFP-tagged MET-2 fusion protein showed that MET-2::GFP localizes to the cytoplasm of C. elegans body-wall muscles. We found that knockout of met-2 or sequestration of MET-2 in the nucleus caused hypersensitivity to the acetylcholine receptor agonist levamisole, suggesting a cytoplasmic role in muscles. Based on the periodic localization of cytoplasmic MET-2::GFP and our previous work, which showed that reduced cellular ATP causes levamisole hypersensitivity, we hypothesized that MET-2 could impact the mitochondria. Using RFP-tagged Translocase of Outer Mitochondrial Membrane 20 (TOMM-20) to visualize muscle mitochondria, we discovered that loss of met-2 caused severe defects in mitochondria morphology. This is highly significant, as a connection between SETDB1 and mitochondria has not been previously reported. The proposed research will use unique genome-edited C. elegans strains, advanced imaging techniques, and a new sample processing method for quantitative proteomics to define the impact of MET-2 on mitochondria morphology, function, and protein abundance. We will generate new strains using CRISPR/Cas9 to determine if MET-2 affects mitochondria by 1) methylation dependent or independent activity and 2) nuclear or cytoplasmic function. We will determine the functional consequences of the fragmented mitochondria in the met-2 mutant by measuring ATP levels and oxygen consumption rate. Finally, we will perform a quantitative comparison of the wild-type and met-2 mutant transcriptomes and proteomes, with initial focus on mitochondrial proteins. Since mitochondrial dysfunction is a key factor in SETDB1- associated diseases, this basic science research could have significant impact on our understanding of how altered SETDB1 activity contributes to pathogenesis of multiple rare diseases.
