Notch signaling in cardiomyocyte transcriptome signatures

心肌细胞转录组特征中的 Notch 信号传导

• 批准号: 9755477
• 负责人: Jared Churko
• 金额: $ 23.43万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-03 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755477
• 关键词: Adult; Advisory Committees; Affect; Arrhythmia; Arrhythmogenic Right Ventricular Dysplasia; Binding; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiac development; Cardiology; Cardiovascular Diseases; Cardiovascular system; Career Mobility; Cells; ChIP-seq; Communities; Contracts; DNA Binding; Data; Derivation procedure; Development; Disease; Disease model; Drug Screening; Electrophysiology (science); Engineering; Event; Exhibits; Family member; Foundations; Funding; Future; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic Transcription; Goals; Heart; Heart Atrium; High-Throughput Nucleotide Sequencing; Human; Human Engineering; Immunofluorescence Immunologic; Institutes; Knock-out; Knowledge; Lead; Ligands; Localized Disease; Mass Spectrum Analysis; Medicine; Mentors; Mentorship; Molecular; Monitor; Mus; Myocardium; NOTCH1 gene; NOTCH3 gene; Pathway interactions; Pattern; Pharmaceutical Preparations; Phase; Play; Population; Positioning Attribute; Program Development; Proteomics; Reaction; Research Personnel; Research Project Grants; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Structure; Technology; Testing; Therapeutic; Therapeutic Human Experimentation; Tissues; Transcription Initiation Site; Universities; Ventricular; Western Blotting; Work; base; career; career development; differential expression; drug discovery; experience; genetic signature; improved; induced pluripotent stem cell; insight; knock-down; meetings; member; notch protein; null mutation; professor; programs; regenerative; regenerative therapy; skills; small molecule; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; whole genome

 DESCRIPTION (provided by applicant): PROJECT SUMMARY This proposal describes a five-year career development program to prepare Dr. Churko for a career as an independent investigator. This program builds on Dr. Churko's background as a cell and molecular biologist by providing him with expertise in gene editing and sequencing technology to advance our understanding of notch signaling within the heart. Dr. Churko's mentor is Dr. Joseph Wu, a Professor of Medicine/Cardiology and Director of the Stanford Cardiovascular Institute at Stanford University. Dr. Wu is an excellent mentor with extensive experience in hiPSC derivation, differentiation into cardiomyocytes (hiPSC-CMs), and using hiPSC-CMs to study cardiovascular development and disease. The K99 phase will consist of structured mentorship by the primary mentor, complementary meetings with the advisory committee, formal coursework, a provocative research project, and a program of career transition. The K99 phase of Dr. Churko's studies will focus on supporting Dr. Churko's RNA-seq data and further develop tools (CRISPR-Cas9 notch knock- out lines) to comprehensively study notch signaling at a whole genome level. In Dr. Churko's preliminary data, he has observed a striking enrichment in notch-specific genes and transcription factors only found within the adult human ventricles. This finding is also supported in mouse and chick studies which demonstrated that knocking out members of the notch pathway cause atrial genes to be expressed within the ventricles. With the current hiPSC technology to study human cardiomyocytes in culture, combined with advancements in high-throughput sequencing technology, Dr. Churko is in a unique position to uncover unknown regulatory and signaling events which define the gene signatures of the human ventricles. In AIM1 of Dr. Churko's proposal, he will develop a PCR panel of genes which are specifically expressed in the different chambers. This panel will be a valuable contribution to the cardiac community because it will allow researchers to define and assess pathways which regulate the development and identify of each heart chamber. Dr. Churko will use this panel to assess and verify which small molecules and ligands promote the ventricular gene signature, and, in using CRISPR-Cas9 technology outlined in AIM2, which notch family members are necessary for this signature. Dr. Churko will then verify these markers as being cardiac specific by immunofluorescence and quantify these differences within the heart by mass spectroscopy. Further, in AIM1, Dr. Churko will be the first to study notch-specific signaling in cardiomyocytes at a single-cell level (using single-cell Western blotting). Finally, in AIM3, Dr. Churko will assess the function and transcription changes which occur from notch signaling in cardiomyocytes. He will perform RNA-seq on hiPSC-CMs with activating notch signaling and identify DNA binding patterns and motifs specific to notch signaling (NOTCH1-ICD, HEY2). A unique goal in this aim is to integrate both RNA-seq data with ChIP-seq data to experimentally verify which transcription factor binding events lead to changes within cardiomyocyte gene signatures. Collectively, the proposed work will provide a comprehensive picture of notch signaling within human cardiomyocytes. In addition, this work will provide the foundation for future studies on engineering hiPSC-CMs into expressing ventricular and atrial gene signatures, which will be carried out by Dr. Churko as an independent investigator.

 描述(由申请人提供)：项目摘要本提案描述了一项为期五年的职业发展计划，为丘尔科博士作为一名独立研究员的职业生涯做准备。该项目以丘尔科博士作为细胞和分子生物学家的背景为基础，向他提供基因编辑和测序技术方面的专业知识，以促进我们对心脏内Notch信号的理解。丘尔科博士的导师是约瑟夫·吴博士，他是医学/心脏病学教授，斯坦福大学斯坦福心血管研究所所长。吴博士是一位优秀的导师，在HiPSC的衍生、分化为心肌细胞(HiPSC-CMS)以及使用HiPSC-CMS研究心血管发育和疾病方面拥有丰富的经验。K99阶段将包括主要导师的有组织的指导、与咨询委员会的补充会议、正式的课程作业、具有挑衅性的研究项目和职业过渡计划。Churko博士研究的K99阶段将侧重于支持Churko博士的RNA-SEQ数据，并进一步开发工具(CRISPR-Cas9缺口敲除系)，在整个基因组水平上全面研究缺口信号。在丘尔科博士的初步数据中，他观察到仅在成人脑室发现的缺口特异基因和转录因子显著丰富。这一发现在小鼠和小鸡的研究中也得到了支持，这些研究表明，敲除Noch通路的成员会导致心房基因在脑室内表达。随着目前HiPSC技术在培养中研究人类心肌细胞，再加上高通量测序技术的进步，Churko博士处于独特的地位，可以发现定义人类心室基因特征的未知调控和信号事件。在丘尔科博士提议的AIM1中，他将开发一组在不同空间中特异表达的基因的聚合酶链式反应(PCR)。这个小组将是对心脏界的宝贵贡献，因为它将允许研究人员定义和评估调节每个心腔发育和识别的途径。丘尔科博士将利用这个小组来评估和验证哪些小分子和配体促进了心脏基因的签名，以及在使用AIM2中概述的CRISPR-Cas9技术时，哪些缺口是这个签名所必需的。然后，丘尔科博士将通过免疫荧光验证这些标志物是心脏特异的，并通过质谱学对心脏内的这些差异进行量化。此外，在AIM1中，丘尔科博士将是第一个在单细胞水平上研究心肌细胞缺口特异性信号的人(使用单细胞Western blotting)。最后，在AIM3中，Churko博士将评估Notch信号在心肌细胞中发生的功能和转录变化。他将对HiPSC-CMS进行RNA-SEQ，激活Noch信号，并确定Noch信号的DNA结合模式和基序(NOTCH1-ICD，HEY2)。这一目标的一个独特目标是将RNA-SEQ数据与CHIP-SEQ数据相结合，以实验验证哪些转录因子结合事件会导致心肌细胞基因签名的变化。总的来说，这项拟议的工作将提供人类心肌细胞内Notch信号的全面图景。此外，这项工作将为未来工程HiPSC-CMS表达心室和心房基因签名的研究奠定基础，这项研究将由Churko博士作为独立研究员进行。