The role of the long non-coding RNA Falcor in early endoderm and lung development

长非编码RNA Falcor在早期内胚层和肺发育中的作用

基本信息

批准号：
9013538
负责人：
DANIEL, T (MD) Todd Swarr
金额：
$ 7.3万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-28 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9013538
关键词：
Address Adult Advisory Committees Amino Acids Asthma Beer Binding Bioinformatics Biology Board Certification CRISPR/Cas technology Cell Culture Techniques Cell Lineage Cells Cellular biology Chromatin Remodeling Factor Complex Congenital Abnormality Development Developmental Gene Developmental Process Disease Doctor of Philosophy Elements Embryo Embryonic Development Endoderm Epigenetic Process Epithelium Equilibrium Faculty Failure Fellowship Fellowship Program Flow Cytometry Funding Gene Expression Gene Expression Profiling Genes Genetic Genome Genomics Goals Histocompatibility Testing Histology Human Human Development Immunohistochemistry In Situ Hybridization In Vitro Injury Institutes Knock-out Knockout Mice Knowledge Label Laboratories Lead Life Liver Lung Lung diseases Malignant Neoplasms Malignant neoplasm of lung Medical Genetics Medicine Mentors Mentorship Molecular Molecular Biology Mus National Research Service Awards Neonatal Neonatology Pancreas Pathogenesis Pathway interactions Pattern Pennsylvania Phenotype Physicians Play Postdoctoral Fellow Primitive foregut structure Process Program Development Proteins Proteomics Pulmonary Fibrosis RNA RNA Binding Radioisotopes Recruitment Activity Regenerative Medicine Research Research Personnel Role Scientist Series Signal Pathway Signal Transduction Specific qualifier value Stable Isotope Labeling Structure Technical Expertise Technology Time To specify Tracheoesophageal Fistula Training Programs Transcription Coactivator Universities Untranslated RNA Work airway epithelium base career cell type chromatin immunoprecipitation dark matter epigenetic regulation forkhead protein genome editing graduate student human disease human embryonic stem cell human embryonic stem cell line improved in vivo insight interest knock-down lung development lung regeneration lung repair malformation member mouse development mouse model notochord novel perinatal medicine professor public health relevance pulmonary hypoplasia research study skills small hairpin RNA stem cell biology symposium tandem mass spectrometry transcription factor transcriptome

项目摘要

DESCRIPTION (provided by applicant): This proposal describes a five-year training program for development of a career as a physician-scientist in neonatology and medical genetics, with particular expertise in early endoderm and lung development. The candidate is completing a unique five-year fellowship program that will lead to board certification in both neonatal-perinata medicine and medical genetics. This fellowship included three years of intensive research funded by the Ruth L. Kirschstein National Research Service Award (T32). During this time, Dr. Swarr has become particularly interested in the role that the non-coding genome, and lncRNAs in particular, plays in directing normal development, and when disrupted, how these non-coding elements contribute to human disease. During the proposed study period, Dr. Swarr will continue to develop his knowledge of endoderm and lung biology, acquiring additional skills in mouse genetics, epigenetics, stem cell biology, and bioinformatics through a combination of intensive laboratory work, graduate-level courses, conferences, and one-on-one mentoring. This project will be carried out under the mentorship of Edward E. Morrisey, PhD, a recognized leader in the field of pulmonary biology. Dr. Morrisey is a Professor of both Medicine and Cell and Molecular Biology, as well as the Scientific Director of the Institute for Regenerative Medicine at the University of Pennsylvania. He has mentored numerous postdoctoral fellows and graduate students. An Advisory Committee, consisting of Drs. Michael Beers, Klaus Kaestner, Ken Zaret, and Rebecca Simmons, will assist Dr. Morrisey in mentoring Dr. Swarr to independence. Each of these faculty members is a highly regarded scientist in his or her own right, and all have successfully mentored many graduate students, post-doctoral fellows, and trainee physician- scientists. The proposed research focuses on the role that long non-coding RNAs (lncRNAs) play during early endoderm and lung development. Although a number of critical transcription factors and molecular pathways have been identified that direct fundamental aspects of lung development, the molecular mechanisms by which these "master regulatory" signals are carefully coordinated remains poorly defined. Long non-coding RNAs (lncRNAs) have recently been recognized to provide a crucial layer of control over proper gene expression, playing essential roles in both normal development and human disease. Dr. Swarr, with others in the Morrisey laboratory, have recently demonstrated that lncRNAs are spatially correlated with transcription factors and regulate lung development (Herriges MJ and Swarr DT, et al. Genes Dev. 2014 Jun 15;28(12):1363-79). In this same study, Dr. Swarr identified a lncRNA located 2kb downstream of Foxa2, herein referred to as Falcor (for Foxa2-Adjaent Long non-COding RNA). The pioneering transcription factor Foxa2 is known to play important roles in early endoderm specification and subsequent differentiation of the lung. The central hypothesis of this proposal is that Falcor plays a critical role in regulating the proper expression of Foxa2, and as a result is an essential orchestrator of endoderm and lung development. In Aim 1, CRISPR-Cas9 technology will be employed to study the role of Falcor in both mouse and human development. In Aim 1a, a Falcor knockout mouse model, which has already been generated, will be used to study the effects of loss of Falcor in vivo. In Aim 1b, the role of Falcr in human development will be studied by differentiating Falcor human embryonic stem (ES) cell knockout lines into definitive endoderm, and ultimately, mature airway epithelium. In Aim 2, the molecular mechanisms by which Falcor regulates the expression of Foxa2 will be defined. A comprehensive proteomics-based approach will be used to identify proteins interacting with Falcor, and the functional significance of these interactions will be interrogated using a series o shRNA knockdown experiments. The research outlined in this proposal will provide novel insights into the function of lncRNAs in early endoderm and lung development, with potential impact for a broad range of lung diseases, from congenital malformations to adult lung cancer. Moreover, by completing this work, I will acquire the necessary technical skills and scientific expertise to become a physician-scientist in neonatal-perinatal medicine and medical genetics with expertise in the mechanisms by which the non-coding genome and epigenetic processes direct normal development, and how disruptions of these developmental processes lead to congenital malformations and pulmonary disease.

描述（由适用提供）：该提案描述了一项为期五年的培训计划，以发展为新生儿和医学遗传学的身体科学家，在早期内胚层和肺部发展方面具有特别的专业知识。该候选人正在完成一项独特的五年奖学金计划，该计划将导致新生儿 - 植物医学和医学遗传学的董事会认证。该奖学金包括由露丝·柯希斯坦国家研究服务奖（T32）资助的三年的密集研究。在此期间，Swarr博士对非编码基因组和LNCRNA的作用特别感兴趣，在指导正常发育方面发挥了作用，而在破坏时，这些非编码元件如何促进人类疾病。在拟议的研究期间，Swarr博士将继续发展他对内胚层和肺部生物学的了解，通过强化实验室工作，研究生水平的课程，会议，会议和一对一的心理精神，获得了小鼠遗传学，表观遗传学，干细胞生物学和生物信息学的其他技能。该项目将在Edward E. Morrisey博士的心态下进行，该博士是肺部生物学领域的公认领导者。 Morrisey博士是医学和细胞和分子生物学的教授，也是宾夕法尼亚大学再生医学研究所的科学主任。他指导了许多博士后研究员和研究生。由Drs组成的咨询委员会。迈克尔·贝尔斯（Michael Beers），克劳斯·卡斯特纳（Klaus Kaestner），肯·扎雷（Ken Zaret）和丽贝卡·西蒙斯（Rebecca Simmons）将协助莫里西博士指导斯瓦尔博士至独立。这些教职员工中的每一个都是他或她自己的一位备受推崇的科学家，所有人都成功地指导了许多研究生，博士后研究员和实习生的身体科学家。拟议的研究重点是长期非编码RNA（LNCRNA）在早期内胚层和肺发育期间起的作用。尽管已经确定了许多关键的转录因子和分子途径，这是肺发育的直接基本方面，但这些“主调节”信号经过精心协调的分子机制仍然很差。长期的非编码RNA（LNCRNA）最近被认为是对适当基因表达的关键层，在正常发育和人类疾病中起着至关重要的作用。 Swarr博士与Morrisey实验室中的其他人最近证明，LNCRNA在空间上与转录因子相关并调节肺发育（Herriges MJ和Swarr DT等人，等等基因Dev。2014Jun 15; 28（12）：1363-79）。在同一项研究中，Swarr博士确定了一个位于FOXA2下游的2KB的lncRNA，此处称为Falcor（用于FOXA2-ADJAENT长期非编码RNA）。众所周知，开创性转录因子FOXA2在早期内胚层规范和随后的肺部分化中起重要作用。该提议的核心假设是，Falcor在调节FOXA2的适当表达中起着至关重要的作用，因此，是内胚层和肺发育的重要编排。在AIM 1中，将采用CRISPR-CAS9技术来研究Falcor在老鼠和人类发展中的作用。在AIM 1A中，已经生成的Falcor敲除小鼠模型将用于研究体内Falcor丢失的影响。在AIM 1B中，FALCR在人类发育中的作用将通过将Falcor人类胚胎（ES）细胞基因敲除线区分为确定的内胚层，最终将成熟的气道上皮来研究。在AIM 2中，将定义Falcor调节FOXA2表达的分子机制。一种全面的基于蛋白质组学的方法将用于识别与Falcor相互作用的蛋白质，这些相互作用的功能意义将使用系列O SHRNA敲低实验进行询问。该提案中概述的研究将为LNCRNA在早期内胚层和肺发育中的功能提供新的见解，对从先天性畸形到成人肺癌的广泛肺部疾病的潜在影响。此外，通过完成这项工作，我将获得必要的技术技能和科学专业知识，以成为新生儿 - 拘留医学和医学遗传学的物理科学家，并具有专业知识，这些机制是非编码基因组和表观遗传过程直接正常发展的机制，以及这些发育过程如何破坏这些发展过程，导致共生畸形畸形和纯种畸形疾病。