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

 描述（由申请人提供）：本提案描述了一个为期五年的培训计划，用于发展医学和医学遗传学领域的医生-科学家职业生涯，特别是在早期内胚层和肺发育方面的专业知识。候选人正在完成一个独特的五年奖学金计划，这将导致董事会认证在这两个围产期医学和医学遗传学。该奖学金包括由Ruth L. Kirschstein国家研究服务奖（T32）。在此期间，Swarr博士对非编码基因组，特别是lncRNA在指导正常发育中的作用，以及当这些非编码元件被破坏时，这些非编码元件如何导致人类疾病特别感兴趣。Swarr将继续发展他的内胚层和肺生物学知识，通过密集的实验室工作，研究生课程，会议和一对一指导的组合，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额外技能。该项目将在爱德华·E·Morrisey博士是肺生物学领域公认的领导者。Morrisey博士是医学和细胞与分子生物学教授，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。一个由迈克尔·比尔斯博士、克劳斯·凯斯特纳博士、肯·扎雷特博士和丽贝卡·西蒙斯博士组成的咨询委员会将协助莫里西博士指导斯瓦尔博士独立。这些教师中的每一个都是一个高度重视的科学家在他或她自己的权利，并都成功地指导了许多研究生，博士后研究员，和实习医生-科学家。这项研究的重点是长链非编码RNA（lncRNA）在早期内胚层和肺发育过程中的作用。尽管已经鉴定了一些关键的转录因子和分子途径，其指导肺发育的基本方面，但是这些“主调节”信号被仔细协调的分子机制仍然不清楚。长链非编码RNA（lncRNA）最近被认为是控制基因表达的关键层，在正常发育和人类疾病中起着重要作用。Swarr博士和Morrisey实验室的其他人最近证明lncRNA与转录因子在空间上相关并调节肺发育（Herriges MJ和Swarr DT等人，Genes Dev. 2014年6月15日;28（12）：1363-79）。在同一项研究中，Swarr博士鉴定了位于Foxa 2下游2kb的lncRNA，本文称为Falcor（Foxa 2-Adjaent Long non-Coding RNA）。转录因子Foxa 2在肺的早期内胚层特化和随后的分化中起重要作用。该提议的中心假设是Falcor在调节Foxa 2的适当表达中起关键作用， 因此是内胚层和肺发育的重要协调者。在目标1中，CRISPR-Cas9技术将用于研究Falcor在小鼠和人类发育中的作用。在目标1a中，已经产生的Falcor基因敲除小鼠模型将用于研究体内Falcor缺失的影响。在目标1b中，Falcr在人类发育中的作用将通过将Falcor人胚胎干（ES）细胞敲除系分化为定形内胚层并最终分化为成熟的气道上皮来研究。在目标2中，Falcor调节Foxa 2表达的分子机制将被定义。一个全面的蛋白质组学为基础的方法将被用来确定蛋白质与Falcor相互作用，这些相互作用的功能意义将被询问使用一系列的shRNA敲除实验。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具有潜在影响。此外，通过完成这项工作，我将获得必要的技术技能和科学知识，成为围产期医学和医学遗传学的医生科学家，具有非编码基因组和表观遗传过程指导正常发育的机制的专业知识，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.