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

项目总结/摘要 本申请中包含的研究和培训计划旨在提高我的 在肺细胞和分子生物学的专业知识，并发展技能，追求一个独立的职业生涯在肺 research.通过这个KO 1项目，我希望提高我在项目设计，赠款写作，项目和 实验室管理层及时成功地过渡到独立调查员。我的近期目标是 获得肺基因调控转录机制分析的高级培训， 复杂的肺表型。 在我的导师和合作导师的支持下，这些领域的专家，我建议分析 Nkx 2 -1对肺上皮细胞增殖相关基因的调控及其对肺上皮细胞增殖的影响 Nkx 2 -1突变小鼠。Nkx 2 -1转录因子对肺分支和远端上皮细胞至关重要。 在发育过程中的细胞分化。在成人中，Nkx 2 -1通过细胞分化维持远端上皮表型。 特异性基因调控在肺肿瘤中，Nkx 2 -1是谱系存活癌基因和预后因子。 已经提出Nkx 2 -1在控制细胞增殖中的作用，但是可能介导细胞增殖的靶基因是不稳定的。 体内增殖是未知的。本项目的目标是评估Nkx 2 -1直接靶的作用 Nkx 2 -1在小鼠肺发育和疾病中调节细胞增殖的基因。 我们之前的ChIP芯片分析揭示了增殖相关基因的过度表达， Nkx 2 -1结合的miRNAs在肺发育早期的数量。这里我假设Nkx 2 -1 通过这些基因和miRNA调节肺部细胞增殖，并且它们有助于 当通过Nkx 2 -1功能的破坏改变时，异常肺表型的发病机制。 这一假设将通过以下方式进行检验：1）分析所选细胞的发育表达 增殖相关的Nkx 2 -1靶点，并评估Nkx 2 -1与 2）表征Nkx 2 -1调节的miRNA及其mRNA的表达 与体外细胞增殖相关，并将其表达与下游基因的表达相关联， 参与肺细胞增殖的Nkx 2 -1驱动的miRNA调控网络; 3）评估 这些Nkx 2 -1靶基因和miRNAs对Nkx 2 -1无效突变型肺表型的贡献， 其中分支形态发生和分化被破坏，并且在Nkx 2 -1磷酸化突变体中， 尽管有正常的分支但肺发育不全的肺。这些将提供深入了解 磷酸化、非磷酸化或缺失Nkx 2 -1在细胞增殖控制中的不同作用 体内的基因 波士顿大学医学院的肺中心因其 支持环境，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，正在进行的工作会议和学院 医学系提供的发展计划是我培训计划的重要组成部分。

PROJECT SUMMARY/ABSTRACT 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 for 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.