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

 描述(由申请者提供)：这份建议书描述了一项为期五年的培训计划，旨在发展成为新生儿学和医学遗传学领域的内科科学家，特别是早期内胚层和肺发育方面的专业知识。这位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围产期医学和医学遗传学方面的董事会认证。这项奖学金包括由Ruth L.Kirschstein国家研究服务奖(T32)资助的三年密集研究。在这段时间里，斯沃尔博士对非编码基因组，尤其是lncRNAs在指导正常发育中所起的作用，以及当这些非编码元件被破坏时如何导致人类疾病产生了特别的兴趣。在拟议的研究期间，Swarr博士将继续发展他的内胚层和肺部生物学知识，通过密集的实验室工作、研究生水平的课程、会议和一对一指导相结合，获得小鼠遗传学、表观遗传学、干细胞生物学和生物信息学方面的其他技能。该项目将在爱德华·E·莫里西博士的指导下进行，他是肺部生物学领域公认的领导者。莫里西博士是医学和细胞分子生物学的教授，也是宾夕法尼亚大学再生医学研究所的科学主任。他曾指导过许多博士后研究员和研究生。一个由Michael Beers博士、Klaus Kaestner博士、Ken Zaret博士和Rebecca Simmons博士组成的咨询委员会将协助Morrisey博士指导Swarr博士走向独立。这些教职员工本身就是一位备受尊敬的科学家，他们都成功地指导了许多研究生、博士后研究员和实习医生兼科学家。这项拟议的研究集中在长非编码RNA(LncRNAs)在早期内胚层和肺发育中所起的作用。虽然已经确定了一些关键的转录因子和分子通路，这些转录因子和分子通路直接影响肺发育的基本方面，但这些“主要调节”信号被仔细协调的分子机制仍然不清楚。长非编码RNA(LncRNAs)最近被认为是控制基因表达的关键一层，在正常发育和人类疾病中都发挥着至关重要的作用。Swarr博士和Morrisey实验室的其他人最近证明了lncRNAs与转录因子在空间上相关，并调节肺发育(Herriges MJ和Swarr DT等人)。吉尼斯·德夫(Gene Dev.)2014年6月15日；28(12)：1363-79)。在同一项研究中，Swarr博士发现了一个位于Foxa2下游2kb的lncRNA，这里称为Falcor(Foxa2-Adjaent长非编码RNA)。先驱性转录因子Foxa2在早期内胚层特化和随后的肺分化中发挥重要作用。这一提议的中心假设是，Falcor在调节Foxa2的正确表达方面发挥了关键作用， 因此，它是内胚层和肺发育的重要协调者。在目标1中，将使用CRISPR-Cas9技术来研究Falcor在小鼠和人类发育中的作用。在Aim 1a中，已经建立的Falcor基因敲除小鼠模型将用于研究体内Falcor基因缺失的影响。在目标1b中，将通过将Falcor人类胚胎干细胞(ES)敲除株分化为最终的内胚层，并最终分化为成熟的呼吸道上皮，来研究Falcr在人类发育中的作用。在目标2中，将定义Falcor调控Foxa2表达的分子机制。将使用一种基于蛋白质组学的综合方法来鉴定与Falcor相互作用的蛋白质，并将通过一系列shRNA敲除实验来询问这些相互作用的功能意义。这项提案中概述的研究将为lncRNAs在早期内胚层和肺发育中的功能提供新的见解，并可能对从先天性畸形到成人肺癌等广泛的肺部疾病产生影响。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长，成为一名新生儿围产期医学和医学遗传学方面的内科科学家，擅长非编码基因组和表观遗传过程指导正常发育的机制，以及这些发育过程的中断如何导致先天性畸形和肺部疾病。

 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.