Regulation and Function of SRF in Vascular Pathiobiology

现在有无可辩驳的证据表明，血管平滑肌细胞(VSMCs)对 血管疾病。VSMCs的功能或功能障碍在一定程度上是由关键转录活动驱动的 因子(Tf)。血清反应因子(SRF)，一种在VSMC中大量表达的转铁蛋白，结合了大量的 Carg Box通俗地称为CArGome，它编排了许多不同的基因程序。 令人惊讶的是，对SRF转录的体内调控几乎一无所知，而SRF的功能 SRF在血管发育中的作用已被熟知，但没有关于其在血管发育中的直接作用的信息 疾病；由于最受欢迎的SMC CRE驱动程序的限制，基于性别的研究是不可能的 (MYH11)此外，SRF靶基因(特别是长非编码RNAs，lncRNAs)的完整补充并不是 为人所知。我们一直是SRF研究的领先实验室，现在为这些重大科学研究提供新的见解 这些差距共同构成了这项申请的基础。首先，我们提供CRISPR-Cas9基因组编辑 结果，生物信息学预测、芯片序列和荧光素酶数据支持功能转录因子结合- 体内调控SRF转录的ING位点(TFBS)。第二，基因表达与SRF功能丧失(LOF) 研究支持SRF是VSMC生长、炎症和血管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 LOF的分析限制在 仅在他莫昔芬服药后10-14天，由于胃肠道(GI)的竞争致死表型。怎么- 曾经，我们最近产生并验证了一种新的具有限制性Cre介导的切除的Cre驱动小鼠 对VSMC，在胃肠道内脏SMC中观察到很少的活动，与SRF小鼠杂交显示 延长生存期提供了第一次机会来询问SRF在急性和慢性疾病中的功能 没有混淆表型的慢性血管疾病模型。最后，基因组研究揭示了一种新的 SRF依赖的VSMC炎症基因程序和CRISPR研究显示特定的碱基替换 在Carg框内，在体内使依赖SRF的基因表达无效。三个综合目标将受到严格考验 假设多个TFBS控制SRF的表达以引导Carg依赖的内稳态或 血管壁上的致病基因程序。目标1将评估新的TFBS管理SRF的功能 在鼠标中使用CRISPR编辑进行转录。AIM 2将阐明与SRF LOF和 在急性和慢性血管疾病模型中使用新型CRE驱动小鼠的功能获得 无与伦比的VSMC专一性。AIM 3将进一步利用AIM 2中的小鼠来整合VSMC芯片-seq和rna- SEQ研究阐明新的SRF靶基因，特别是控制VSMC的一类lncRNAs 表型；关键CAG框的CRISPR编辑被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调节和功能的了解，为新的 与血管疾病作斗争的治疗方法，同时为进一步研究提出新的指导方针。

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.