This proposal is concerned with alterations in lung gene expression in response to the physiological change in oxygen that occurs at birth. We have found that a gene, gamma-glutamyl transferase (GGT), which is expressed exclusively in the lung alveolar type 2 cell in late fetal and early postnatal life, is transcriptionally regulated by non-toxic concentrations of oxygen. This regulation takes the form of oxygen related use of alternative GGT promoters. The fetal lung utilizes at least three alternative GGT promoters, P1, P2 and P3. At birth, P2 is rapidly down- regulated, P1 is gradually down-regulated and P3 is up-regulated to become the sole promoter active in the late postnatal and adult lung. We plan to define to molecular mechanisms of this regulation by oxygen and to explore the biological consequences of this form of alternative promoter utilization. There studies will be carried out in a fetal lung epithelial cell line using deletion constructs, electrophoretic mobility shift assay, and Dnase 1 and methylation interference footprinting in order to define cis-acting elements in the GGT promoters and transcription factors responsible for the oxygen responses. We will study the biological consequences of GGT alternative promoter utilization by determining if the mRNAs derived from the different promoters are differentially translated and if the different GGT mRNAs provide a biologically-important mechanism for regulation of GGT over a broad range of oxygen environments. This will be studied with type 2 cell lines in vitro where GGT mRNA l and ll are expressed under the control of a viral promoter and cells are exposed to oxygen conditions where endogenous P1 and P2 are down-regulated and with transgenic mice in which each GGT mRNA is driven by the SP-C promoter to prevent the normal down-regulation of the mRNAs in the perinatal period. The effects of preventing P3 mRNA expression in postnatal lung will be tested by using newly-described methods to produce a targeted mutation in the GGT P3 promoter. The role of lung GGT will be also be studied in a recently identified mouse model of genetic GGT-deficiency. These studies will provide new insight into oxygen regulation of gene expression at birth.
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