High Mobility Group B Knockdown Decreases Heterochromatin Modifications
Authors:Rachel Lopez, Emma Monte, Thomas Vondriska
- Diana Azurdia, Assistant Director Undergraduate Research Center- Sciences, University of California, Los Angeles
- Thomas Vondriska, Associate Professor of Anesthesiology, Medicine, and Physiology, Departments of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine at UCLA
Chromatin is differentially packaged to create accessible and restricted regions, called eu- and heterochromatin, respectively. The chromatin structural protein High Mobility Group B (HMGB, a family with three isoforms) regulates transitions between these forms of chromatin through unknown mechanisms. We recently found that HMGB2 protein abundance increases with cardiac hypertrophy in the mouse, a condition that can lead to heart failure, a disease affecting >5 million people in the USA. In isolated cells, HMGB2 knockdown increases the heterochromatic post-translational modification (PTM) histone H3 lysine 9 trimethylation (H3K9me3) and decreases the euchromatic PTM H3K4me3; however, the effect HMGB levels have on other chromatin proteins remains unknown. To further characterize HMGB, we knocked down HMGB2 or HMGB3 in neonatal rat ventricular myocytes and mouse 3T3 fibroblasts and measured the abundance of euchromatic (H3K4me3, active RNA Polymerase II) or heterochromatic (H3K9me3, H3K27me3, Linker Histone H1, CTCF) proteins by Western blot. We hypothesize that the nucleus regulates chromatin accessibility through the combined actions of chromatin structural proteins (HMGB, CTCF and H1) and histone PTMs. Therefore, we predicted a decrease in heterochromatin proteins after knockdown of HMGB. HMGB2 and HMGB3 knockdown increased CTCF, potentially reflective of functional compensation. As predicted, HMGB2 knockdown increased H3K4me3 and decreased H3K9me3. HMGB3 knockdown increased RNA Polymerase II and decreased H3K9me3 and H3K27me3, as predicted; histone H1 remained unchanged. Thus, HMGB knockdown caused a shift from heterochromatin to euchromatin, implying HMGB normally preserves heterochromatin. The increase in CTCF and decrease in H3K9me3 following knockdown of either HMGB2 and HMGB3 suggests HMGBs have similar effects on heterochromatic PTMs. Future studies will explore the universality of HMGB actions by testing other cell types and will explore the changes in chromatin structure and gene expression that result from varied levels of HMGB protein in vivo.