Abstract 10740: Epigenetic Basis for the Heterogeneity of VCAM1 Expression in Cytokine Activated Vascular Endothelium
We hypothesized that epigenetic mechanisms play a key role in regulating VCAM1 gene expression in vascular endothelial cells (EC) in response to cytokine stimulation. Epigenetics is broadly defined as chromatin-based mechanisms important in the regulation of gene expression that do not involve changes in the DNA sequence per se. These chromatin modifications involve histone density and post-translational modifications, DNA methylation and RNA-based mechanisms. Using ChIP, we found that histone H3 & H4 acetylation, histone eviction & RNA Pol II loading at the VCAM1 promoter in human umbilical vein EC (HUVEC) are concurrent with VCAM1 mRNA induction. Remarkably, cell surface expression varied markedly in a homogeneous population between ECs. Basal expression of p65 and TNF-induced nuclear translocation did not explain this heterogeneity. Remarkably, we found marked heterogeneity in DNA methylation at the VCAM1 promoter in HUVECs with MeDIP or sodium bisulfite genomic sequencing. Using cell sorting, we found that the VCAM1 promoter from the 5% highest and lowest VCAM1 protein expressing cells show sparse and dense DNA methylation, respectively. VCAM1 mRNA and heterogeneous nuclear RNA levels were increased, 12 & 20-fold respectively, in high expressing cells, relative to low expressing cells (n=3, p≤0.05). Functional activity of luciferase promoter-reporter constructs do not differ between the high and low expressing populations. MicroRNA-126, which targets the VCAM1 3’-UTR, was increased 3-fold in VCAM1-high EC cells (n=3, p≤0.05). Mechanistically, ChIP indicated that RNA Pol II was selectively engaged on hypomethylated VCAM1 promoter regions. When we established single cell derived-clones of HUVEC we observed heterogeneity in VCAM1 promoter methylation and VCAM1 mRNA inducibility both between clones and compared to the founder HUVEC population. We take these data to indicate that heterogeneous expression of VCAM1 is mediated, in part, by epigenetic mechanisms. It is of great interest that seemingly homogeneous populations of EC can exhibit profound differences in gene expression based upon distinct promoter epigenetic states. The relative contribution of epigenetic processes to EC heterogeneity warrants further study.
- © 2013 by American Heart Association, Inc.