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（VSMC）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至关重要的高级分化表型。主要 VSMC分化的效应因子是血清反应因子-心肌细胞素（SRF/MYOCD）转录 开关，其结合在许多VSMC限制性基因中发现的CArG盒。这个开关经常被破坏 导致VSMC去分化的疾病状态。虽然已知心肌素的水平会降低， 疾病，我们几乎不知道它在体内的调节控制。此外，下游目标 MYOCD（最值得注意的是，长非编码RNA）并不完全已知。我们已经产生了一些新的 小鼠模型和使基因组数据，使我们能够快速确定Myocd的转录控制 并阐明新的SRF依赖性和SRF非依赖性MYOCD靶点的功能。我们 提出三个目标，利用具有革命性CRISPR-Cas9基因组编辑的小鼠模型 系统和国家的最先进的工具，在遗传学和基因组学，以测试的假设， Myocd和下游MYOCARDome的SRF非依赖性转录， 维持VSMC稳态。Aim 1将利用一只新的双等位基因标记的Myocd小鼠来进行询问 通过ChIP-seq定义的候选增强子和调控元件，计算预测， 荧光素酶测定或环状染色体构象捕获（4C）测定。二组分和三组分 CRISPR将告知那些对Myocardin表达至关重要的调控区域。目标2将 利用CRISPR介导的功能丧失小鼠和RNA-seq，开始破译两种新的 MYOCD诱导基因筛选中发现的基因：一种SRF依赖性长非编码RNA基因 （Mymsl）和SRF非依赖性蛋白质编码基因（Kankl）。这两种基因在VSMC中富集， 似乎在维持正常VSMC分化中起作用。ChIP-seq研究将确定和 验证这些MYOCD靶点，同时使用双表位公开VSMC中的完整MYOCARDome- Aim 1的标记小鼠。目的3将进一步表征关键调控元件（目的1）和新的 血管病理学模型（动静脉瘘和主动脉瘤）中MYOCD靶基因（Aim 2） 动脉瘤）。此外，我们将利用新的功能丧失和功能获得的Myocd小鼠进一步推进 我们对这一关键辅因子及其在人类疾病相关模型中的下游程序的理解 VSMC的差异化计划受到影响。目标的完成将纵向推进我们的 了解Myocd表达的调控过程和新的Myocd基因的功能， 正常和疾病条件下的MYOCD靶基因。这样的知识将告知下一代 旨在维持正常水平的Myocardin作为一种手段， 阻止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