Histone H3 O-GlcNAcylation in Endothelial Cells Promotes Diabetic Vascular Complications - Project Summary/Abstract Diabetes accelerates cardiovascular disease, in part, through hyperglycemia-induced O-linked β-N- acetylglucosamine (O-GlcNAc) modification on proteins in vascular endothelial cells (ECs). Recent advances identified O-GlcNAc modification on histones, which is actively involved in regulating chromatin accessibility and adjacent gene expression. But it remains elusive whether and how hyperglycemia-induced histone O- GlcNAc affect atheroprotecive gene expression and endothelial function, as well as its contribution to diabetes- accelerated atherosclerosis. We recently made the novel discovery that O-GlcNAc modification occurs at the histone H3T32 site (H3T32OG) in ECs under high-glucose. Notably, a loss-of-function mutation (H3T32A) abolished H3 O-GlcNAc while simultaneously increasing H3K27ac enrichment in the promoters of atheroprotective genes. To further investigate the atheroprone effects of H3T32OG, we generated a mouse model with inducible EC-specific replacement of H3T32T with H3T32A, effectively blocking H3 O-GlcNAc. These newly acquired results led to the overarching hypothesis that histone H3T32OG, as a novel histone code in ECs, mediates diabetes-induced EC dysfunction and atherosclerosis. Mechanistically, H3T32OG competes with H3K27ac, restricting chromatin accessibility and downregulating atheroprotective genes in ECs. To test this hypothesis, two specific aims are proposed: Aim 1: To investigate the effect of EC OGT and H3T32OG on diabetes-accelerated atherosclerosis. Using male and female iEC-OGT KO ApoE-/-, iEC-H3T32A ApoE-/-, and H3T32T ApoE-/- mice with low-dose STZ-induced diabetes, the study will compare atherogenesis, lipid deposition, macrophage infiltration, and plaque stability. Mechanistic insights will also be gained through single-nucleus RNA-seq from freshly isolated mouse aortas from these animals. Aim 2: To define the epigenetic features of diabetic condition-induced H3T32OG in ECs and its relation to EC function. Genome- wide H3 O-GlcNAc loci will be mapped via ChIP-seq and integrated with existing datasets. Then, in aortic ECs from H3T32A and H3T32T mice, the interaction between H3T32OG and H3K27ac, as well as histone accessibility and gene expression, will be evaluated in the promoter regions of key atheroprotective and atheroprone genes. Functional differences between these ECs, including nitric oxide bioavailability, monocyte adhesion, and permeability, will also be studied in vitro. In summary, this innovative research, supported by strong preliminary studies and well-established resources, aims to explore the novel histone code involved in diabetes-accelerated atherosclerosis (Aim 1) and endothelial cell dysfunction (Aim 2). The findings are expected to provide a deeper understanding of this epigenetic mechanism and challenge current views on histone codes in diabetic vascular complications.
