Ozer, G. | bioRxiv Science

bioRxiv2020

Partnership between epigenetic reader BRD4 and transcription factor CEBPD

Vascular smooth muscle cell (SMC) state/phenotype transitions underlie neointimal hyperplasia (IH) predisposing to cardiovascular diseases. Bromodomain protein BRD4 is a histone acetylation reader and enhancer mark that co-activates transcription elongation. CCAAT enhancer binding protein delta (CEBPD) is a transcription factor typically studied in adipogenesis and immune cell differentiation. Here we investigated the association between BRD4 and CEBPD in SMC state transition. Chromatin immunoprecipitation sequencing (ChIPseq) showed enrichment of BRD4 and histone acetylation (H3K27ac) at Cebpd and enhancer in rat carotid arteries undergoing IH. In vitro, BRD4 silencing with siRNA reduced SMC expression of CEBPD. Bromodomain-1 but not bromodoamin-2 accounted for this BRD4 function. Endogenous BRD4 co-IPed with CEBPD; Cebpd promoter and enhancer DNA fragments co-IPed with CEBPD or endogenous BRD4 (ChIP-qPCR). These co-IPs were abolished by the BRD4 bromodomain blocker JQ1. TNF upregulated both BRD4 and CEBPD. Silencing CEBPD averted TNF-induced inflammatory SMC state transition (heightened IL-1{beta}, IL6, and MCP-1 mRNA levels), so did JQ1. CEBPD overexpression increased PDGFR preferentially over PDGFR{beta}; so did TNF, and JQ1 abolished TNFs effect. Our data reveal a BRD4/CEBPD partnership that promotes CEBPDs own transcription and inflammatory SMC state transition, thus shedding new light on epigenetic reader and transcription factor cooperative actions in SMC pathobiology.

cell biology↗

bioRxiv2020

Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

Atherosclerosis is commonly treated with angioplasty which, however, evokes neointimal hyperplasia (IH) and recurrent stenotic diseases. Epigenomic investigation was lacking on post-angioplasty IH. The histone acetylation reader BRD4 and H3K27me3 writer EZH2 are potent epigenetic factors; their relationship is little understood. Through genome-wide survey in the rat angioplasty model, we studied BRD4 and EZH2 functional regulations involved in IH. We performed chromatin immunoprecipitation sequencing (ChIPseq) using rat carotid arteries. While H3K27me3 ChIPseq signal prevalently intensified in balloon-injured (vs uninjured) arteries, BRD4 and H3K27ac became more enriched at Ezh2. Indeed, BRD4-siRNA or CRISPR-deletion of BRD4-associated enhancer abated the smooth muscle cell (SMC) expression of EZH2, and SMC-specific BRD4 knockout in BRD4fl/fl; Myh11CreERT2 mice reduced both H3K27me3 and IH in wire-injured femoral arteries. In accordance, post-angioplasty IH was exacerbated and mitigated, respectively, by lentiviral expression and pharmacological inhibition of EZH2/1; EZH2 (or EZH1) loss- and gain-of-function respectively attenuated and aggravated pro-IH SMC proliferative behaviors. Furthermore, while H3K27me3 ChIPseq signal magnified at P57 and ebbed at Ccnd1 and Uhrf1 after injury, silencing either EZH2 or EZH1 in SMCs up-regulated P57 and down-regulated Ccnd1 and Uhrf1. In summary, our results reveal an upsurge of EZH2/H3K27me3 after angioplasty, BRD4s control over EZH2 expression, and non-redundant EZH2/1 functions. As such, this study unravels angioplasty-induced loci-specific H3K27me3/ac redistribution in the epigenomic landscape rationalizing BRD4/EZH2-governed pro-IH regulations.

pathology↗