ZEB2 Shapes the Epigenetic Landscape of Atherosclerosis

Abstract

Background: Smooth muscle cells (SMC) transition into a number of different phenotypes during atherosclerosis, including those that resemble fibroblasts and chondrocytes, and make up the majority of cells in the atherosclerotic plaque. To better understand the epigenetic and transcriptional mechanisms that mediate these cell state changes, and how they relate to risk for coronary artery disease (CAD), we have investigated the causality and function of transcription factors (TFs) at genome wide associated loci. Methods: We employed CRISPR-Cas 9 genome and epigenome editing to identify the causal gene and cell(s) for a complex CAD GWAS signal at 2q22.3. Subsequently, single-cell epigenetic and transcriptomic profiling in murine models and human coronary artery smooth muscle cells were employed to understand the cellular and molecular mechanism by which this CAD risk gene exerts its function. Results: CRISPR-Cas 9 genome and epigenome editing showed that the complex CAD genetic signals within a genomic region at 2q22.3 lie within smooth muscle long-distance enhancers for ZEB2 , a TF extensively studied in the context of epithelial mesenchymal transition (EMT) in development and cancer. ZEB2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and TGFβ signaling, thus altering the epigenetic trajectory of SMC transitions. SMC specific loss of ZEB2 resulted in an inability of transitioning SMCs to turn off contractile programing and take on a fibroblast-like phenotype, but accelerated the formation of chondromyocytes, mirroring features of high-risk atherosclerotic plaques in human coronary arteries. Conclusions: These studies identify ZEB2 as a new CAD GWAS gene that affects features of plaque vulnerability through direct effects on the epigenome, providing a new thereapeutic approach to target vascular disease.

The epigenomic landscape of single vascular cells reflects developmental origin and identifies disease risk loci

“…12,38 SMC specific deletion of Tcf21 and Zeb2 in the mouse revealed a significant effect on transcriptional regulation, epigenetic landscape, and plaque characteristic, further defining their roles as causal CAD genes. 22,31 Another notable observation in our study is that differential chromatin accessibility and gene expression profiles between vascular beds highlights specific genes with known roles in mediating vascular disease. For example, expression of the gene Ccn3 is higher in the ascending aorta smooth muscle (Figure 2I).…”

“…A limitation of this study is that we did not evaluate the epigenomic profiles in a disease state across vascular beds. We anticipate that there are dynamic changes in chromatin accessibility and gene expression programs in disease, as has been previously observed, 22,23 and future studies will be aimed at understanding how these dynamic chromatin accessibility changes occur across vascular beds. Similarly, recent work has demonstrated changes in chromatin accessibility in vascular tissue with aging.…”

“…Tissues were collected and dissociated for single cell capture as previously described. 22, 30, 31 Briefly, vascular tissue was washed three times in PBS, tissues were then placed into an enzymatic dissociation cocktail (2 U ml−1 Liberase (5401127001; Sigma–Aldrich) and 2 U ml−1 elastase (LS002279; Worthington) in Hank’s Balanced Salt Solution (HBSS)), and minced. After incubation at 37 °C for 1 h, the cell suspension was strained and then pelleted by centrifugation at 500g for 5 min.…”

“…Given the interrelationship between RNA expression and global chromatin accessibility, these datasets where then integrated together as previously described. 22,32 Integrated scRNAseq and scATACseq datasets underwent additional non-linear dimensional reduction and UMAP visualization (Figure 1D). The integrated UMAP representing both transcriptomic and chromatin accessibility visually reveals significant differences between cell types by vascular bed (Figure 1D).…”

“…18 This context is important when considering that heritability of complex disease is determined by common genetic variation which largely mediates disease risk through modifying genomic enhancer function within regions of open chromatin. 19 The recent advancement of single cell global chromatin accessibility profiling through Assay for Transposase Accessible Chromatin sequencing (scATACseq) in combination with single cell transcriptomic analysis (scRNAseq) has further defined that genetic risk of cardiac 20,21 and vascular disease [22][23][24] is driven in part through modification of cell type specific enhancers regulating expression of disease modifying genes in a cell type specific manner.…”