Coordinated regulation of vascular smooth muscle phenotype by p300, CBP, and TET2

成熟的血管平滑肌细胞(SMC)保留了可逆性去分化和显著改变的能力 它们的表型对环境线索的反应。这种可塑性允许血管修复和生长，但也 导致心血管病变，包括动脉粥样硬化、内膜增生、动脉瘤、移植 动脉硬化症等。对SMC表型转换的表观遗传控制知之甚少。我们有 最近发现TET2是一种新的SMC表型的主要表观遗传调节因子，促进SMC 分化，并在病变血管中下调。TET2促进关键转录因子的表达 SMC分化的驱动因素包括myocardin和SRF，同时抑制KLF4的表达， 一种转录因子，与SMC的去分化和干细胞的多能性有关。除了它的 已知的产生表观遗传标记5-羟甲基胞嘧啶(5hmC)的功能，我们已经确定 TET2还影响组蛋白甲基化，表明TET2参与了大鼠的整体染色质重塑。 SMC。其他研究表明，造血细胞中的TET2和HDACs之间存在关联。我们的新预赛 数据表明，TET2和组蛋白乙酰转移酶(HATS)之间存在物理和功能上的联系。帽子 乙酰化增强子区域的组蛋白以促进细胞类型特异性基因的表达。我们已经做出了 令人惊讶的是，经常被认为可以互换的帽子P300和CBP有相反的情况 在调控SMC基因表达中的作用。我们发现p300是诱导培养的SMC分化所必需的。 而CBP是去分化所必需的。值得注意的是，这些帽子在收缩时也相反地影响5hmC SMC中的启动子，我们检测到p300和TET2之间存在依赖分化的关联。我们 假设p300和CBP在收缩和合成表型特异性调控不同的增强子 基因，是影响SMC表型转换的关键因素。我们进一步建议TET2/p300 相互作用可能协调调节染色质的构象。这项提案的总体目标是确定 P300、CBP和Tet协同调节SMC表型可塑性的机制。我们会 利用最先进的分子生物学方法、动物模型和先进的基因组学技术 解决核心假设，即p300和CBP通过以下方式调节相反的基因表达程序 乙酰化不同的顺式调控元件，HATS和TET2协同工作，在 SMC表型调控。目标1将确定p300和CBP在SMC表型中的机制作用 调制。目的2确定p300和CBP在体内血管损伤反应中的相反作用。 AIM 3将在体外通过缺失方法确定p300、CBP和TET2是如何调节增强子的 在活体内。总的来说，这些研究将导致对SMC表型调节的新理解，SMC表型调节具有 有可能产生新的心血管疾病预防和治疗战略。

Mature vascular smooth muscle cells (SMC) retain the ability to reversibly dedifferentiate and dramatically alter their phenotype in response to environmental cues. This plasticity allows for vascular repair and growth, but also contributes to cardiovascular pathologies including atherosclerosis, intimal hyperplasia, aneurysm, transplant arteriosclerosis, and others. Little is known about the epigenetic control of SMC phenotypic switching. We have recently identified TET2 as a novel master epigenetic regulator of SMC phenotype that promotes SMC differentiation and is downregulated in diseased vessels. TET2 promotes expression of key transcriptional drivers of SMC differentiation including myocardin and SRF while simultaneously inhibiting expression of KLF4, a transcription factor associated with dedifferentiation in SMC and pluripotency in stem cells. In addition to its known function of generating the epigenetic mark 5 hydroxymethylcytosine (5hmC), we have determined that TET2 also influences histone methylation, indicating that TET2 is involved in global chromatin remodeling in SMC. Others have shown association between TET2 and HDACs in hematopoietic cells. Our new preliminary data indicate a physical and functional association between TET2 and histone acetyltransferases (HATs). HATs acetylate histones in enhancer regions to promote cell type-specific gene expression. We have made the surprising observation that the HATs p300 and CBP, often considered to be interchangeable, have opposing roles in regulating SMC gene expression. We find that p300 is required to induce SMC differentiation in culture, while CBP is required for de-differentiation. Notably, these HATs also oppositely influence 5hmC at contractile promoters in SMC, and we detect a differentiation-dependent association between p300 and TET2. We hypothesize that p300 and CBP regulate distinct enhancers at contractile- and synthetic phenotype-specific genes, respectively, and are critical factors in SMC phenotypic switching. We further propose that TET2/p300 interactions may coordinately regulate chromatin conformation. The overall goal of this proposal is to identify the mechanisms by which p300, CBP, and TET coordinately regulate SMC phenotypic plasticity. We will employ state-of-the art molecular biology approaches, animal models, and advanced genomics techniques to address the central hypothesis that p300 and CBP regulate opposing programs of gene expression by acetylating distinct cis regulatory elements, and that HATs and TET2 work in concert to remodel chromatin during SMC phenotype modulation. Aim 1 will determine the mechanistic roles of p300 and CBP in SMC phenotypic modulation. Aim 2 will aim to define the opposing roles of p300 and CBP on vascular injury response in vivo. Aim 3 will determine how enhancers are regulated by p300, CBP, and TET2 using deletion approaches in vitro and in vivo. Collectively, these studies will lead to new understanding of SMC phenotypic modulation, which has potential for generating new preventive and therapeutic strategies for cardiovascular diseases.