Regulation and Function of SRF in Vascular Pathiobiology

现在有无可争议的证据表明，血管平滑肌细胞（VSMCs）实质性地促进了 血管疾病VSMCs的功能或功能障碍部分是由关键转录活性驱动的， 因子（TF）。血清反应因子（SRF）是一种在血管平滑肌细胞中大量表达的TF， CArG盒俗称CArGome，协调了许多不同的基因程序。 令人惊讶的是，关于Srf转录的体内调节几乎一无所知，而Srf的功能是已知的。 SRF在血管发育中的作用已被很好地理解，但关于其在血管发育中的直接作用还没有信息。 疾病;鉴于最受欢迎的SMC Cre驱动程序的限制，不可能进行基于性别的研究 （Myh11）。此外，SRF靶基因（特别是长链非编码RNA，lncRNA）的完整互补序列并不 知道的我们一直是SRF研究的领先实验室，现在为这些主要的科学研究提供新的见解。 这些差距共同构成了本申请的基础。首先，我们提供CRISPR-Cas9基因组编辑 结果，生物信息学预测，ChIP-seq和荧光素酶数据支持功能性转录因子结合- 在体内控制Srf转录的转录位点（TFBS）。第二，基因表达和Srf功能丧失（LOF） 研究支持SRF作为VSMC生长、炎症和新生内膜形成的早期介质， 急性血管损伤重要的是，现有的Cre驱动小鼠将Srf L0 F的分析限制在10分钟的窄时间窗。 由于胃肠道（GI）的竞争性、致命表型，他莫昔芬给药后仅10-14天。怎么-- 最近，我们已经产生并验证了一种新的Cre驱动小鼠，其具有限制性Cre介导的切除 在胃肠道的内脏SMC中观察到很少的活性，并且与floxed Srf小鼠的交叉显示 延长生存期，首次提供了在急性和慢性炎症中询问SRF功能的机会。 无混淆表型的血管疾病慢性模型。最后，基因组研究暗示了一种新的 SRF依赖的VSMC炎症基因程序和CRISPR研究显示了特定的碱基取代 在CArG盒内使SRF依赖性基因在体内表达无效。三个综合目标将严格测试 多个TFBS控制Srf表达以指导CArG依赖性稳态或 血管壁中的致病基因程序。目标1将评估新的TFBS管理Srf的功能 在小鼠中使用CRISPR编辑进行转录。目的2将阐明与Srf LOF相关的表型， 使用新型Cre驱动小鼠在急性和慢性血管疾病模型中获得功能， 无与伦比的VSMC特异性。目的3将进一步利用目的2中的小鼠用于整合VSMC ChIP-seq和RNA-聚合酶链反应。 seq研究阐明新的SRF靶基因，特别是一类lncRNA，在控制VSMC 关键CArG盒的CRISPR编辑计划作为研究基因功能的新范式。这些 研究将垂直推进我们对SRF调节和功能的认识，为新的研究铺平道路。 治疗方法，以打击血管疾病，同时推进新的指示，为进一步的研究。

There is now incontrovertible evidence that vascular smooth muscle cells (VSMCs) contribute substantively to vascular diseases. The function or dysfunction of VSMCs is driven, in part, by the activity of key transcription factors (TF). Serum response factor (SRF), an abundantly expressed TF in VSMCs that binds a large cadre of CArG boxes colloquially known as the CArGome, orchestrates a number of disparate gene programs. Surprisingly, almost nothing is known about the in vivo regulation of Srf transcription and, while the function of SRF in vascular development is well understood, there is no information about its direct role in vascular diseases; and gender-based studies are not possible given the limitation of the most popular SMC Cre driver (Myh11). Moreover, the full complement of SRF target genes (notably long noncoding RNAs, lncRNAs) is not known. We have been a leading lab in SRF research and now offer fresh insights into these major scientific gaps that, collectively, form the basis of this application. First, we provide CRISPR-Cas9 genome editing results, bioinformatic predictions, ChIP-seq, and luciferase data supporting functional transcription factor bind- ing sites (TFBS) controlling Srf transcription in vivo. Second, gene expression and Srf loss-of-function (LOF) studies support SRF as an early mediator of VSMC growth, inflammation, and neointimal formation following acute vascular insult. Importantly, existing Cre driver mice limit analysis of Srf LOF to a narrow time window of only 10-14 days post-tamoxifen due to a competing, lethal phenotype of the gastrointestinal (GI) tract. How- ever, we have recently generated and validated a new Cre driver mouse with restricted Cre-mediated excision to VSMCs; little activity is observed in visceral SMCs of the GI tract, and a cross with floxed Srf mice shows extended survival providing the first ever opportunity to interrogate the function of SRF in both acute and chronic models of vascular disease without confounding phenotypes. Finally, genomic studies implicate a new SRF-dependent VSMC inflammatory gene program and CRISPR studies show a specific base substitution within the CArG box nullifies SRF-dependent gene expression in vivo. Three integrated aims will rigorously test the hypothesis that multiple TFBS control Srf expression to direct CArG-dependent homeostatic or pathogenic gene programs in the vessel wall. Aim 1 will evaluate the function of new TFBS governing Srf transcription using CRISPR editing in the mouse. Aim 2 will elucidate phenotypes associated with Srf LOF and gain-of-function in acute and chronic models of vascular disease using a novel Cre driver mouse for unparalleled VSMC specificity. Aim 3 will further utilize mice in Aim 2 for integrative VSMC ChIP-seq and RNA- seq studies to elucidate novel SRF target genes, particularly the class of lncRNAs, in the control of VSMC phenotypes; CRISPR editing of key CArG boxes are planned as a new paradigm to study gene function. These studies will vertically advance our knowledge of SRF regulation and function, paving the way towards new therapeutic approaches to combat vascular diseases while advancing new directives for further research.