Control of Proliferation-related Genes and miRs by Nkx2-1 in Lung Development

描述（由申请人提供）：本申请中包含的研究和培训计划旨在增加我在肺细胞和分子生物学方面的专业知识，并培养我在肺研究领域独立发展的技能。通过这个KO1项目，我希望提高我在项目设计、拨款写作、项目和实验室管理方面的技能，以便及时成功过渡到独立研究者。我的近期目标是获得肺基因调控转录机制和复杂肺表型分析方面的高级培训。在我的导师和共同导师以及这些领域的专家的支持下，我提议分析Nkx2-1对增殖相关基因的调控及其对Nkx2-1突变小鼠肺上皮细胞增殖的影响。Nkx2-1转录因子在发育过程中对肺分支和远端上皮细胞分化至关重要。在成人中，Nkx2-1通过细胞特异性基因调控维持远端上皮表型。在肺肿瘤中，Nkx2-1是一个谱系存活癌基因和预后因素。Nkx2-1在控制细胞增殖中的作用已被提出，但可能介导体内增殖的靶基因尚不清楚。本项目的目的是评估Nkx2-1直接靶基因在小鼠肺部发育和疾病中调控Nkx2-1引起的细胞增殖中的作用。我们之前的ChIP-on-chip分析显示，在早期肺发育中，增殖相关基因和Nkx2-1结合的一些mirna过度表达。在这里，我假设Nkx2-1通过这些基因和mirna调节肺中的细胞增殖，并且当Nkx2-1功能被破坏时，它们有助于异常肺表型的发病机制。这一假设将通过以下方法得到验证：1)分析筛选到的与细胞增殖相关的Nkx2-1靶点的发育表达，并评估Nkx2-1在肺组织中与这些靶点的结合；2)在体外鉴定Nkx2-1调控的miRNA及其与细胞增殖相关的mrna的表达，并将其与下游基因的表达联系起来，建立Nkx2-1驱动的参与肺细胞增殖的miRNA调控网络；3)评估这些Nkx2-1靶基因和mirna对分支形态发生和分化被破坏的Nkx2-1零突变肺和尽管正常分支但肺发育不良的Nkx2-1磷酸化突变肺的表型的贡献。这将有助于深入了解磷酸化、未磷酸化或缺失的Nkx2-1在体内细胞增殖基因控制中的差异作用。波士顿大学医学院肺中心因其支持性环境而广受认可，帮助初级教师过渡到独立的职业生涯。每周与我的导师和共同导师的讨论，正在进行的会议和医学系提供的教师发展计划是我培训计划的关键组成部分。（摘要结束）

DESCRIPTION (provided by applicant): The research and training plans included in this application are designed to increase my expertise in lung cell and molecular biology, and to develop skills to pursue an independent career in lung research. With this KO1 project I expect to advance my skills in project design, grant writing, project and lab management to succeed in a timely transition to independent investigator. My immediate goals are to acquire advanced training in the analysis of transcriptional mechanisms of lung gene regulation, and of complex lung phenotypes. With the support of my mentor and co-mentors, experts in these fields, I propose to analyze Nkx2-1 regulation of proliferation-related genes and their effect on lung epithelial cell proliferation in Nkx2-1 mutant mice. The Nkx2-1 transcription factor is essential fr lung branching and distal epithelial cell differentiation during development. In the adult, Nkx2-1 maintains distal epithelial phenotypes by cell specific gene regulation. In lung tumors, Nkx2-1 is a lineage survival oncogene and a prognostic factor. A role of Nkx2-1 in control of cell proliferation has been suggested, but the target genes that may mediate proliferation in vivo are unknown. The goal of this project is to evaluate the role of Nkx2-1 direct target genes in the regulation of cell proliferation attributed to Nkx2-1 in mouse lung development and disease. Our previous ChIP-on-chip analyses revealed an overrepresentation of proliferation-related genes and a number of miRNAs bound by Nkx2-1 in early lung development. Here I hypothesize that Nkx2-1 regulates cell proliferation in the lung through these genes and miRNAs, and that they contribute to the pathogenesis of the abnormal lung phenotypes when altered by disruption of Nkx2-1 function. This hypothesis will be tested by: 1) Analyzing the developmental expression of selected cell proliferation-related Nkx2-1 targets previously identified in our screen, and evaluating Nkx2-1 binding to these targets in lung tissues, 2) Characterizing expression of Nkx2-1-regulated miRNAs and their mRNAs linked to cell proliferation in vitro and correlate their expression to that of downstream genes to establish an Nkx2-1 driven miRNA regulatory network involved in lung cell proliferation; 3) Evaluating the contribution of these Nkx2-1 target genes and miRNAs to the phenotype of Nkx2-1 null mutant lungs in which branching morphogenesis and differentiation are disrupted, and in Nkx2-1 phosphorylation mutant lungs in which the lung is hypoplastic in spite of normal branching. These will provide insights into the differential role of phosphorylated, unphosphorylated or absent Nkx2-1 in control of cell proliferation genes in vivo. The Pulmonary Center at Boston University School of Medicine is widely recognized for its supporting environment, helping Junior Faculty to transition to an independent career. The weekly discussions with my mentor and co-mentors, the work-in-progress meetings and the Faculty Development Program offered by the Department of Medicine are key components of my training plan. (End of Abstract)