The MUC5AC gene, expressed by goblet cells in the conducting airway epithelium, encodes the protein backbone of MUC5AC mucin, which is heavily O-glycosylated and gel-forming. Mucin hypersecretion is associated with inflammatory airway diseases such as asthma. A current treatment for asthma is inhaled glucocorticoids, including dexamethasone (Dex), which binds to the glucocorticoid receptor (GR) located in the cytoplasm of most cells. Previous studies from our lab have shown that Dex transcriptionally represses MUC5AC gene expression by activating nuclear translocation of GR and binding to two GR cis-sites in the MUC5AC promoter. We hypothesize that GR binding to the functional GRE 3 and GRE 5 cis-sites recruits co-repressors, such as histone deacetylases (HDAC), to form a complex to repress expression of the MUC5AC gene. Immunofluorescence imaging was used to analyze localization of GR and HDAC2 in primary differentiated human bronchial epithelial (HBE) cells stimulated with 1μM Dex for 0.5, 1, 6, 18 and 24 h. These data showed nuclear colocalization of GR and HDAC2 at 0.5, 1 and 6 h but not at 18 or 24 h. To determine the proteins within this complex we utilized a DNA pull-down assay. Following exposure to 100 nM Dex, nuclear extracts from A549 cells were incubated first with biotinylated DNA probes specific for the two previously identified functional GRE cis-sites on the MUC5AC promoter region, then with streptavidin agarose resin. The eluted protein complexes were electrophoresed and analyzed by Western blot and Mass Spectrometry. These data indicated that following Dex exposure, a protein complex containing GR, HDAC2, HDAC1 and other co-repressor proteins including YY1, binds to the active GRE cis-sites to transcriptionally repress the MUC5AC gene expression.
|Advisor:||Rose, Mary C.|
|Commitee:||Elliott, Mark S.|
|School:||The George Washington University|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- District of Columbia|
|Source:||MAI 47/06M, Masters Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Asthma, Co-repressor, Dexamethasone, Gene regulation, Muc5ac|
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