Transcriptional Control of Myocardin and the MYOCARDome

心肌素和 MYOCARDome 的转录控制

• 批准号: 10210425
• 负责人: Joseph M Miano
• 金额: $ 56.95万
• 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: 2019-11-21 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10210425
• 关键词: Aortic Aneurysm; Bacterial Artificial Chromosomes; Binding; Biological Assay; Blood Vessels; Blood flow; CRISPR/Cas technology; Cell Differentiation process; ChIP-seq; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cre driver; DNA; Data; Dependence; Development; Disease; Disease Progression; Disease model; Elements; Embryo; Engineering; Enhancers; Epitopes; Fistula; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genomics; Homeostasis; Human; Immunofluorescence Immunologic; Knock-out; Knowledge; Luciferases; Maintenance; Mediating; Messenger RNA; Methods; Modeling; Molecular; Mus; Mutation; Nucleic Acid Regulatory Sequences; Phenotype; Prevention; Process; Proteins; Quantitative Reverse Transcriptase PCR; Regulation; Regulatory Element; Reporting; Research Design; Serum Response Factor; Smooth Muscle Myocytes; Supporting Cell; System; Testing; Tissues; Transcriptional Regulation; Transgenic Mice; Triad Acrylic Resin; Untranslated RNA; Vascular Diseases; Vascular Smooth Muscle; Venous; chromosome conformation capture; cofactor; experimental study; gain of function; gene discovery; gene function; genetic approach; genome editing; genomic data; human disease; human model; in vivo; insight; loss of function; member; mouse model; myocardin; next generation; novel; overexpression; postnatal; prevent; programs; tool; transcriptome sequencing; transcriptomics; translational approach; translational model; virtual

Vascular smooth muscle cells (VSMCs) support nascent blood vessels during early development, but then acquire an advanced differentiated phenotype essential for contraction and blood flow regulation. The major effector of VSMC differentiation is the Serum Response Factor-Myocardin (SRF/MYOCD) transcriptional switch, which binds CArG boxes found in many VSMC-restricted genes. This switch is often compromised in disease states leading to VSMC de-differentiation. While levels of Myocardin are known to be reduced in disease, we know virtually nothing about its regulatory control in vivo. Moreover, the downstream targets of MYOCD (most notably, long noncoding RNAs) are not entirely known. We have generated a number of new mouse models and enabling genomic data that allow us to rapidly define the transcriptional control of Myocd in vivo and elucidate the function of novel SRF-dependent and SRF-independent MYOCD targets. We propose three aims that leverage mouse models with the revolutionary CRISPR-Cas9 genome editing system and state-of-the-art tools in genetics and genomics to test the hypothesis that SRF-dependent and SRF-independent transcription of Myocd and the downstream MYOCARDome function to maintain VSMC homeostasis. Aim 1 will utilize a new, biallelic-tagged Myocd mouse to interrogate candidate enhancers and regulatory elements defined through ChIP-seq, computational prediction, luciferase assay, or circular chromosome conformation capture (4C) assays. Two and three component CRISPR will inform those regulatory regions of critical importance for Myocardin expression. Aim 2 will utilize CRISPR-mediated loss-of-function mice and RNA-seq to begin deciphering the function of two novel genes discovered in screens for MYOCD-inducibility: an SRF-dependent long noncoding RNA gene (Mymsl) and an SRF-independent protein-coding gene (Kank1). Both genes are enriched in VSMC and appear to function in the maintenance of normal VSMC differentiation. ChIP-seq studies will ascertain and validate these MYOCD targets while disclosing the full MYOCARDome in VSMC using the dual epitope- tagged mice of Aim 1. Aim 3 will further characterize the critical regulatory elements (Aim 1) and novel MYOCD target genes (Aim 2) in models of vascular pathobiology (arterial-venous fistula and aortic aneurysm). In addition, we will make use of new loss- and gain-of-function Myocd mice to further advance our understanding of this critical cofactor and its downstream program in relevant models of human disease where the VSMC differentiation program is compromised. Completion of the aims will vertically advance our understanding of the regulatory processes undergirding Myocd expression and the function of novel MYOCD target genes under normal and disease conditions. Such knowledge will inform the next generation of experimental and clinical studies designed to maintain normal levels of Myocardin as a means of thwarting the pervasive de-differentiation of VSMC observed in human disease.

血管平滑肌细胞(VSMCs)在早期发育过程中支持新生血管，但随后 获得对收缩和血流调节必不可少的高级分化表型。少校 VSMC分化的效应因子是血清反应因子-Myocardin(SRF/MYOCD)转录 开关，它结合了许多VSMC限制性基因中发现的Carg盒。此交换机经常受到威胁 在疾病状态下导致VSMC去分化。而已知的Myocardin水平在 疾病，我们几乎对它在体内的调控一无所知。此外，该公司的下游目标 MYOCD(最值得注意的是长的非编码RNA)并不完全为人所知。我们已经产生了许多新的 小鼠模型和使我们能够快速定义MYOCD转录控制的基因组数据 并阐明新的SRF依赖和非SRF依赖的MYOCD靶点的功能。我们 提出三个目标，利用革命性的CRISPR-Cas9基因组编辑来利用小鼠模型 遗传学和基因组学中的系统和最先进的工具来测试SRF依赖的假设 和SRF不依赖的MYOCD转录和下游MYOCARDome功能 维持VSMC内环境平衡。目标1将利用一种新的双等位基因标记的MYOCD小鼠来询问 通过CHIP-SEQ定义的候选增强子和调节元件、计算预测 荧光素酶检测，或环形染色体构象捕获(4C)检测。两组分和三组分 CRISPR将向这些调控区域提供对Myocardin表达至关重要的信息。目标2将 利用CRISPR介导的功能丧失小鼠和RNA-SEQ开始破译两个新的 在MYOCD诱导性筛查中发现的基因：依赖SRF的长非编码RNA基因 (Myms1)和不依赖SRF的蛋白编码基因(Kank1)。这两个基因都富含VSMC和 似乎在维持正常的VSMC分化中起作用。CHIP-SEQ研究将确定 验证这些MYOCD靶点，同时披露VSMC中使用双重表位的完整MYOCARDome- 目标1的标记小鼠。目标3将进一步描述关键调控元件(目标1)和新的 血管病理生物学模型(动-静脉瘘和主动脉)中的MYOCD靶基因(AIM 2) 动脉瘤)。此外，我们还将利用新的功能丧失和功能获得的MYOCD小鼠来进一步推进 我们对这一关键辅因子及其下游程序在相关人类疾病模型中的理解 VSMC分化计划受到损害。这些目标的完成将垂直推进我们的 对支持MYOCD表达的调控过程和NORTH功能的理解 MYOCD在正常和疾病条件下的靶基因。这样的知识将会通知下一代 的实验和临床研究，旨在维持正常水平的肌钙蛋白作为一种手段 抑制在人类疾病中观察到的VSMC的普遍去分化。

项目成果

A New "Lnc" to Brake Inflammation.

抑制炎症的新“Lnc”。

• DOI: 10.1161/atvbaha.123.319444
• 发表时间: 2023
• 期刊: Arteriosclerosis, thrombosis, and vascular biology
• 作者: Long,Xiaochun;Miano,JosephM;Zhou,Jiliang
• 通讯作者: Zhou,Jiliang

Generating a CRISPR knockout mouse through a strong premature termination codon: a cautionary tale.

• DOI: 10.7555/jbr.34.20200106
• 发表时间: 2020-12-25
• 期刊: Journal of biomedical research
• 影响因子: 2.3
• 作者: Lyu QR;Yao P;Miano JM
• 通讯作者: Miano JM

Of mice and human-specific long noncoding RNAs.

• DOI: 10.1007/s00335-022-09943-2
• 发表时间: 2022-06
• 期刊: Mammalian genome : official journal of the International Mammalian Genome Society
• 作者: Ghanam AR;Bryant WB;Miano JM
• 通讯作者: Miano JM

CRISPR links to long noncoding RNA function in mice: A practical approach.

• DOI: 10.1016/j.vph.2019.02.004
• 发表时间: 2019-03
• 期刊: Vascular pharmacology
• 影响因子: 4
• 作者: J. Miano;Xiaochun Long;Qing R. Lyu

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

• DOI: 10.1186/s13059-021-02304-3
• 发表时间: 2021-03-16
• 期刊: Genome biology
• 影响因子: 12.3
• 作者: Gao P;Lyu Q;Ghanam AR;Lazzarotto CR;Newby GA;Zhang W;Choi M;Slivano OJ;Holden K;Walker JA 2nd;Kadina AP;Munroe RJ;Abratte CM;Schimenti JC;Liu DR;Tsai SQ;Long X;Miano JM
• 通讯作者: Miano JM