Wang Qingwei, Ozer Hatice Gulcin, Wang Bowen, Zhang Mengxue, Urabe Go, Huang Yitao, Kent K Craig, Guo Lian-Wang
Department of Surgery, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
Mol Ther Methods Clin Dev. 2021 Feb 27;21:54-66. doi: 10.1016/j.omtm.2021.02.021. eCollection 2021 Jun 11.
Bromodomain protein BRD4 reads histone acetylation (H3K27ac), an epigenomic mark of transcription enhancers. CCAAT enhancer binding protein delta (CEBPD) is a transcription factor typically studied in metabolism. While both are potent effectors and potential therapeutic targets, their relationship was previously unknown. Here we investigated their interplay in vascular smooth muscle cell (SMC) inflammation. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed H3K27ac/BRD4 enrichment at in injured rat carotid arteries. While genomic deletion of BRD4-associated enhancer in SMCs decreased transcripts, BRD4 gene silencing also diminished mRNA and protein, indicative of a BRD4 control over CEBPD expression. Bromodomain-1, but not bromodomain-2, accounted for this BRD4 function. Moreover, endogenous BRD4 protein co-immunoprecipitated with CEBPD, and both proteins co-immunoprecipitated the promoter and enhancer DNA fragments. These co-immunoprecipitations (coIPs) were all abolished by the BRD4-bromodomain blocker JQ1, suggesting a BRD4/CEBPD /promoter/enhancer complex. While BRD4 and CEBPD were both upregulated upon tumor necrosis factor alpha (TNF-α) stimulation of SMC inflammation (increased interleukin [IL]-1b, IL-6, and MCP-1), they mediated this stimulation via preferentially elevated expression of platelet-derived growth factor receptor alpha (PDGFRα, versus PDGFRβ), as indicated by loss- and gain-of-function experiments. Taken together, our study unravels a hierarchical yet collaborative BRD4/CEBPD relationship, a previously unrecognized mechanism that prompts SMC inflammation and may underlie other pathophysiological processes as well.
溴结构域蛋白BRD4识别组蛋白乙酰化（H3K27ac），这是转录增强子的一种表观基因组标记。CCAAT增强子结合蛋白δ（CEBPD）是一种通常在代谢研究中涉及的转录因子。虽然二者都是强效效应分子和潜在治疗靶点，但它们之间的关系此前并不清楚。在此，我们研究了它们在血管平滑肌细胞（SMC）炎症中的相互作用。染色质免疫沉淀结合高通量测序（ChIP-seq）显示，在损伤的大鼠颈动脉中，H3K27ac/BRD4在[具体位置未给出]处富集。虽然SMC中BRD4相关增强子的基因缺失降低了[具体基因未给出]转录本，但BRD4基因沉默也减少了[具体基因未给出]的mRNA和蛋白质，表明BRD4对CEBPD表达具有调控作用。溴结构域1而非溴结构域2负责BRD4的这一功能。此外，内源性BRD4蛋白与CEBPD发生共免疫沉淀，且二者都与[具体基因未给出]启动子和增强子DNA片段发生共免疫沉淀。这些共免疫沉淀（coIP）均被BRD4溴结构域阻断剂JQ1消除，提示存在BRD4/CEBPD/启动子/增强子复合物。虽然在肿瘤坏死因子α（TNF-α）刺激SMC炎症（白细胞介素[IL]-1β、IL-6和单核细胞趋化蛋白-1增加）时，BRD4和CEBPD均上调，但功能缺失和功能获得实验表明，它们通过优先提高血小板衍生生长因子受体α（PDGFRα，相对于PDGFRβ）的表达来介导这种刺激。综上所述，我们的研究揭示了一种分层但协作的BRD4/CEBPD关系，这是一种此前未被认识的机制，它引发SMC炎症，也可能是其他病理生理过程的基础。
