The long-term goals of this work are to study the functional roles of nuclear receptor co-activators in mediating signaling pathways critical to lung development and surfactant protein B (SP-B) gene expression in respiratory epithelial cells. Co-activators including p160 family members (SRC-1, ACTR and TIF2) and CBP/p300 possess histone acetyltransferase (HAT) activity and are known to play important roles in gene activation, cell differentiation and proliferation. The state of the art approaches of biochemical/molecular biology and conditional transgenic mouse models will be utilized.
The specific aims are: 1): Characterization of SP-B chromatin acetylation in H441 cells to test hypothesis that histones in the SP-B promoter/enhancer region are hyperacetylated during transcriptional activation. Chromatin cross-linking and chromatin immunoprecipitation (CHIP) assays will be performed; 2): Characterization of trans-interactions between nuclear receptor coactivators and TTF-1 to test hypothesis that nuclear receptor co-activators interact directly with TTF-1 to control target SP-B gene expression. Co-immunoprecipitation, Western blot, GST pulldown and mammalian two-hybrid system will be performed to identify the precise interacting domains of both nuclear receptor co-activators and TTF-1; 3): Characterization of TTF-1 acetylation by nuclear receptor co-activators to test hypothesis that nuclear receptor co-activators acetylate TTF-1 in lung epithelial cells. Specific nuclear receptor co-activators (e.g. SRC-1, ACTR, CBP/p300) acetylating TTF-1 will be identified. The precise acetylated lysine residues on TTF-1 will be identified by domain mapping and site-specific mutagenesis; 4): Characterization of dominant negative RAR in doxycycline-regulatable transgenic mice to test hypothesis that nuclear receptor co-activators are critical to lung development and recovery/remodeling of respiratory epithelial cells from emphysema.
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