Notch signaling in cardiomyocyte transcriptome signatures

 描述（由申请人提供）：项目概要本提案描述了一个为期五年的职业发展计划，以准备博士Churko作为一个独立的调查员的职业生涯。该计划建立在Churko博士作为细胞和分子生物学家的背景之上，为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。Churko博士的导师是Joseph Wu博士，他是斯坦福大学医学/心脏病学教授和斯坦福大学心血管研究所主任。吴博士是一位优秀的导师，在hiPSC衍生、分化为心肌细胞（hiPSC-CM）以及使用hiPSC-CM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结构化指导，与咨询委员会的补充会议，正式的课程，挑衅性的研究项目和职业过渡计划。Churko博士研究的K99阶段将专注于支持Churko博士的RNA-seq数据，并进一步开发工具（CRISPR-Cas9 notch knock-out lines），以在全基因组水平上全面研究notch信号。在Churko博士的初步数据中，他观察到只有在成年人心室中才能发现的缺口特异性基因和转录因子的惊人富集。这一发现也得到了小鼠和小鸡研究的支持，这些研究表明，敲除notch通路的成员会导致心房基因在心室内表达。利用目前的hiPSC技术研究培养中的人类心肌细胞，结合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传导事件。在Churko博士的AIM 1中，他将开发一个在不同腔室中特异性表达的基因PCR面板。该小组将对心脏社区做出有价值的贡献，因为它将使研究人员能够定义和评估调节每个心腔发育和识别的途径。Churko博士将使用这个小组来评估和验证哪些小分子和配体促进心室基因签名，以及在使用AIM 2中概述的CRISPR-Cas9技术时，哪些notch家族成员是这个签名所必需的。然后，Churko博士将通过免疫荧光验证这些标记物是心脏特异性的，并通过质谱法量化心脏内的这些差异。此外，在AIM 1中，Churko博士将是第一个在单细胞水平上研究心肌细胞中Notch特异性信号传导的人（使用单细胞Western印迹法）。最后，在AIM 3中，Churko博士将评估心肌细胞中notch信号的功能和转录变化。他将对具有激活notch信号的hiPSC-CM进行RNA-seq，并鉴定notch信号特异性的DNA结合模式和基序（NOTCH 1-ICD，HEY 2）。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导致心肌细胞基因特征的变化。总的来说，拟议的工作将提供一个全面的图片缺口信号在人类心肌细胞。此外，这项工作将为未来研究hiPSC-CM表达心室和心房基因特征提供基础，这将由Churko博士作为独立研究者进行。

 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.