Transcriptional Control of Myocardin and the MYOCARDome

心肌素和 MYOCARDome 的转录控制

基本信息

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
项目类别：
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

项目摘要

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的血管，但随后支持获取用于收缩和血流调节必不可少的高级分化表型。专业 VSMC分化的效应因子是血清反应因子 - 肌心蛋白（SRF/MYOCD）转录开关，它结合了许多VSMC限制基因中发现的carg盒子。此开关经常被妥协在疾病状态下，导致VSMC脱不同。众所周知，心肌素水平降低疾病，我们几乎对其在体内的监管控制一无所知。此外，下游目标 MyOCD（最值得注意的是，长期不编码的RNA）并不完全知道。我们已经生成了许多新的鼠标模型并启用基因组数据，使我们能够快速定义MyOCD的转录控制体内并阐明了新型SRF依赖性和独立于SRF的MYOCD靶标的功能。我们提出三个目标，以革命性的CRISPR-CAS9基因组编辑来利用鼠标模型遗传学和基因组学的系统和最先进的工具，以检验SRF依赖性的假设 MyOCD和下游心肌功能的独立于SRF的转录维持VSMC稳态。 AIM 1将利用一种新的，双重标记的MyOCD鼠标来询问候选增强子和调节元素通过chip-seq，计算预测，荧光素酶测定或圆形染色体构象捕获（4C）测定。两个组件 CRISPR将为那些对心肌表达的监管区域提供至关重要的重要性。 AIM 2意志利用CRISPR介导的功能丧失小鼠和RNA-seq开始解读两个新颖的功能在筛选中发现的基因具有肌动物诱导性：SRF依赖性长无编码RNA基因（MYMSL）和独立于SRF的蛋白质编码基因（KANK1）。这两个基因都富含VSMC和在维持正常VSMC分化的维持中似乎起作用。 Chip-seq研究将确定和验证这些MyOCD靶标，同时使用双表位 - AIM 1的标记小鼠AIM 3将进一步描述关键的调节元素（AIM 1）和新颖在血管病理学模型（动脉 - 病毒瘘和主动脉）模型中的MyOCD靶基因（AIM 2）动脉瘤）。此外，我们将利用新的损失和功能获得的MYOCD小鼠来进一步进步我们对这种关键辅助因子及其在人类疾病相关模型中的下游计划的理解 VSMC分化程序被妥协的地方。目的的完成将垂直促进我们的了解少于MyOCD表达的调节过程和新颖的功能在正常和疾病条件下，MyOCD靶基因。这样的知识将告知下一代实验和临床研究旨在维持正常水平的心肌素作为一种手段挫败在人类疾病中观察到的VSMC的普遍脱不同。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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
影响因子：
0
作者：
Ghanam AR;Bryant WB;Miano JM
通讯作者：
Miano JM

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

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