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: 10060485
• 负责人: Joseph M Miano
• 金额: $ 52.63万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-11-22 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10060485
• 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; Cre driver; 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.

现在有不可争议的证据表明，血管平滑肌细胞（VSMC）实质上有助于 血管疾病。 VSMC的功能或功能障碍部分由关键转录的活动驱动 因素（TF）。血清反应因子（SRF），在VSMC中大量表达的TF，结合了大的干部 Carg Boxes通俗地称为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 血管发育中的SRF已熟悉，没有关于其直接作用在血管中的信息 疾病；鉴于最受欢迎的SMC CRE驱动程序的限制，无法进行基于性别的研究 （MYH11）。此外，SRF靶基因的完整补体（尤其是不编码的RNA，LNCRNA）不是 已知。我们一直是SRF研究的领先实验室，现在为这些主要科学提供新的见解 总体上，差距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转录因子结合 - 在体内控制SRF转录的ING位点（TFB）。其次，基因表达和SRF功能丧失（LOF） 研究支持SRF作为VSMC生长，炎症和新内膜形成的早期介体 急性血管侮辱。重要的是，现有的CRE驱动小鼠将SRF LOF的分析限制为狭窄的时间窗口 由于胃肠道（GI）的竞争，致命的表型，培养氨昔芬后仅10-14天。如何- 曾经，我们最近生成并验证了一种新的CRE驱动器鼠标，并具有受限的CRE介导的切除 到VSMC；在胃肠道的内脏SMC中观察到很少的活性，并且带有Floxed SRF小鼠的十字架显示 扩展生存提供了有史以来第一个质疑SRF功能的机会 血管疾病的慢性模型，没有混淆表型。最后，基因组研究暗示了一个新的 依赖SRF的VSMC炎症基因程序和CRISPR研究表明了特定的基础替代 在carg盒中，体内无效SRF依赖性基因表达。三个综合目标将严格测试 多个TFB控制SRF表达以直接依赖Carg依赖性稳态或 血管壁中的致病基因程序。 AIM 1将评估管理SRF的新TFB的功能 使用鼠标中的CRISPR编辑转录。 AIM 2将阐明与SRF LOF相关的表型 使用一种新型的CRE驱动小鼠的急性和慢性血管疾病模型的功能 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芯片seq和RNA- SEQ研究以控制VSMC的新型SRF靶基因，特别是LNCRNA类别 表型； CRISPR编辑的关键CARG盒子计划作为研究基因功能的新范式。这些 研究将垂直提高我们对SRF监管和功能的了解，从而铺平了通往新的道路 治疗方法打击血管疾病，同时推进新指令进行进一步研究。