Regulation and Function of SRF in Vascular Pathiobiology

SRF 在血管病理生物学中的调节和功能

• 批准号: 9764180
• 负责人: Joseph M Miano
• 金额: $ 53.39万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-05 至 2019-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764180
• 关键词: Acute; Aneurysm; Apoptosis; Arterial Injury; Atherosclerosis; Automobile Driving; Base Pairing; Binding; Binding Sites; Bioinformatics; Biology; Blood Vessels; CRISPR/Cas technology; Cells; ChIP-seq; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Complement; DNA Binding; Data; Development; Differentiated Gene; Differentiation and Growth; Disease; Disease model; Elements; Excision; Future; Gastrointestinal tract structure; Gender; Gene Abnormality; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Growth; Homeostasis; Human; Hyperplasia; IL6 gene; In Vitro; Inflammation; Inflammatory; Injury; Knowledge; Lesion; Lipids; LoxP-flanked allele; Luciferases; Mediating; Mediator of activation protein; Methods; Modeling; Mus; Pathogenicity; Pathologic; Phenotype; Regulation; Regulatory Element; Research; Role; Serum Response Factor; Single base substitution; Smooth Muscle Myocytes; Specificity; Tamoxifen; Testing; Text; Therapeutic; Time; Transcriptional Regulation; Untranslated RNA; Variant; Vascular Diseases; Vascular Smooth Muscle; Visceral; base; cell growth; cell type; cofactor; combat; experimental study; gain of function; gene function; genome editing; genomic data; in vivo; injured; insight; loss of function; macrophage; mouse model; novel; novel therapeutic intervention; novel therapeutics; overexpression; programs; promoter; response; tool; transcription factor; transcriptome sequencing; transdifferentiation; uptake

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.

现在有无可争议的证据表明血管平滑肌细胞（VSMC）对 血管疾病。 VSMC 的功能或功能障碍部分由关键转录活性驱动 因素（TF）。血清反应因子 (SRF)，一种在 VSMC 中大量表达的 TF，可结合大量的细胞因子 CArG 盒俗称 CArGome，协调许多不同的基因程序。 令人惊讶的是，人们对 Srf 转录的体内调节几乎一无所知，而 SRF在血管发育中的作用已广为人知，但尚无关于其在血管发育中直接作用的信息 疾病；鉴于最流行的 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（特别是长非编码 RNA，lncRNA）的完整补充不是 已知。我们一直是 SRF 研究领域的领先实验室，现在为这些主要科学提供新的见解 这些差距共同构成了本应用程序的基础。首先，我们提供CRISPR-Cas9基因组编辑 结果、生物信息学预测、ChIP-seq 和支持功能性转录因子结合的荧光素酶数据 控制Srf体内转录的位点（TFBS）。二、基因表达与Srf功能丧失（LOF） 研究支持 SRF 作为 VSMC 生长、炎症和新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 的分析限制在很窄的时间窗口内 由于胃肠道 (GI) 的竞争性致命表型，仅在他莫昔芬治疗后 10-14 天出现。如何- 我们最近生成并验证了一种新的 Cre 驱动小鼠，具有限制性 Cre 介导的切除 VSMC；在胃肠道的内脏 SMC 中观察到很少的活性，并且与 floxed Srf 小鼠的杂交显示 延长生存期提供了有史以来第一次有机会在急性和慢性疾病中询问 SRF 的功能 没有混淆表型的慢性血管疾病模型。最后，基因组研究暗示了一种新的 SRF依赖性VSMC炎症基因程序和CRISPR研究显示特定碱基替换 CArG 盒内的 SRF 依赖性基因表达无效体内。三大综合目标将受到严格考验 假设多个 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 TFBS 管理 Srf 的功能 在小鼠中使用 CRISPR 编辑进行转录。目标 2 将阐明与 Srf LOF 相关的表型和 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。 Aim 3 将进一步利用 Aim 2 中的小鼠进行综合 VSMC ChIP-seq 和 RNA- seq 研究阐明新的 SRF 靶基因，特别是控制 VSMC 的 lncRNA 类别 表型；关键 CArG 盒的 CRISPR 编辑计划作为研究基因功能的新范例。这些 研究将垂直推进我们对 SRF 调节和功能的了解，为新的研究铺平道路 对抗血管疾病的治疗方法，同时为进一步研究提出新的指导。