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.
Correct spatial and temporal patterns of gene expression during lung development are critical for the formation of a functional lung ready for the first breath at birth. In adults, patterns of gene expression also have to be maintained to preserve a healthy lung. One key regulator of the patterns of gene expression in the lung is the transcription factor Nkx2-1. This gene was suggested to regulate proliferation, growth and differentiation of lung cells. This project will address the regulation of cell proliferation related genes by Nkx2-1. The analysis of these regulatory mechanisms in normal and mutant lungs showing abnormal lung formation will be the base for development of further approaches to control altered cell behaviors in lung diseases.