Unveiling the Atheroprone Role of Novel Mechanosensitive Histone Modification - Project Summary/Abstract The vascular endothelium, lining the inner surface of the arterial wall, responds dynamically to shear stress from blood flow. Atheroprone shear stress reduces atheroprotective gene expression and induces endothelial cell (EC) dysfunction, initiating atherosclerosis. Over the past decade, we and others have identified epigenetic and epitranscriptional regulations that are integral to shear stress-regulated gene expression. Although epigenetic regulation and histone post-translational modifications (PTMs) have been identified in ECs and other cells, the full extent of histone PTMs, as well as novel histone codes, remain to be fully understood. Recently, we discovered that O-GlcNAc occurs at the histone H3T32 site (H3T32OG) under atheroprone shear stress. A loss- of-function mutation (H3T32A) decreased H3 O-GlcNAc and increased H3K27ac in promoters of atheroprotective genes, suggesting systemic effects on chromatin accessibility and gene expression. To investigate the role of H3T32OG in atherosclerosis, we created a mouse model with a tissue-specific H3T32A mutation. These preliminary results led to the hypothesis that histone H3T32OG, as a novel histone code in ECs, mediates atheroprone shear stress-induced EC dysfunction and atherosclerosis. Mechanistically, H3T32OG competitively interacts with H3K27ac, ultimately restricting chromatin accessibility and downregulating atheroprotective genes in ECs. To test this hypothesis, we propose the following three specific aims: Aim 1: To elucidate the epigenetic features of H3T32OG in ECs under shear stress. Specifically, the shear stress-induced H3T32 O-GlcNAc modification site will be validated by mass spectrometry. Then, the enrichment of H3 O-GlcNAc will be identified by H3 O-GlcNAc ChIP-seq. Additionally, we will integrate H3 O-GlcNAc ChIP- seq data with existing datasets to understand the shear stress-governed epigenetic features of H3T32OG. Aim 2: To delineate the role of shear stress-regulated H3T32OG in EC function. Specifically, step flow channel- induced shear stress will be applied to mouse aortic ECs isolated from H3T32A mutation mice and their H3T32T littermates. Gene expression profiles and cell lineage mapping will be elucidated by single-cell RNA-seq. Furthermore, EC functional alterations between H3T32A and H3T32 will be evaluated in vivo and ex vivo. Aim 3: To investigate the effect of shear stress-regulated EC H3T32OG on atherosclerosis. We will evaluate the potential differences in atherogenesis in male and female iEC-OGT KO ApoE-/- mice vs. their wildtype littermates, as well as in iEC-H3T32A ApoE-/- and H3T32T ApoE-/- mice, by feeding them a high-fat diet or using a partial carotid artery ligation model. This research will enhance our understanding of epigenetic mechanisms in ECs and challenge current paradigms of histone modifications in health and disease.
