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

• 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
• 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

 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（由申请人提供）：该提案描述了一个为期五年的培训计划，旨在发展新生儿学和医学遗传学方面的医师科学家的职业生涯，特别是早期内胚层和肺部发育方面的专业知识。该候选人正在完成一项独特的为期五年的奖学金计划，该计划将获得新生儿围产期医学和医学遗传学的委员会认证。该奖学金包括由 Ruth L. Kirschstein 国家研究服务奖 (T32) 资助的三年深入研究。在此期间，Swarr 博士对非编码基因组（尤其是 lncRNA）在指导正常发育中所发挥的作用，以及当这些非编码元件被破坏时如何导致人类疾病特别感兴趣。在拟议的研究期间，Swarr 博士将继续发展他的内胚层和肺生物学知识，通过密集的实验室工作、研究生水平课程、会议和一对一指导的结合，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额外技能。该项目将在肺生物学领域公认的领导者爱德华·E·莫里西博士的指导下进行。 Morrisey 博士是宾夕法尼亚大学医学、细胞和分子生物学教授，也是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。咨询委员会，由博士组成。迈克尔·比尔斯 (Michael Beers)、克劳斯·凯斯特纳 (Klaus Kaestner)、肯·扎雷特 (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走向独立。这些教员中的每一位都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、博士后研究员和见习医师科学家。拟议的研究重点是长非编码 RNA (lncRNA) 在早期内胚层和肺发育过程中发挥的作用。尽管已经确定了许多指导肺发育基本方面的关键转录因子和分子途径，但这些“主调节”信号仔细协调的分子机制仍然不清楚。长链非编码 RNA (lncRNA) 最近被认为可以为正确的基因表达提供关键的控制层，在正常发育和人类疾病中发挥重要作用。 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 Jun 15;28(12):1363-79）。在同一项研究中，Swarr 博士鉴定了位于 Foxa2 下游 2kb 处的 lncRNA，本文称为 Falcor（Foxa2-Adjaent Long non-COding RNA）。众所周知，开创性的转录因子 Foxa2 在早期内胚层规范和随后的肺分化中发挥着重要作用。该提案的中心假设是 Falcor 在调节 Foxa2 的正确表达方面发挥着关键作用， 因此，它是内胚层和肺部发育的重要协调者。在目标 1 中，将采用 CRISPR-Cas9 技术来研究 Falcor 在小鼠和人类发育中的作用。在目标 1a 中，已经生成的 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，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气道上皮来研究 Falcr 在人类发育中的作用。在目标 2 中，将定义 Falcor 调节 Foxa2 表达的分子机制。将使用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，并且将使用一系列 shRNA 敲低实验来探讨这些相互作用的功能意义。该提案中概述的研究将为lncRNA在早期内胚层和肺部发育中的功能提供新的见解，并对从先天畸形到成人肺癌等多种肺部疾病产生潜在影响。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，成为新生儿围产期医学和医学遗传学领域的医师科学家，在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破坏如何导致先天性畸形和肺部疾病方面具有专业知识。