MDFIC alteration of the GR transcriptome in COS-1 cells

Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules and are widely used in the clinic. The cellular response to glucocorticoids is remarkably diverse; however, the molecular factors that govern tissue specificity are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast 2 hybrid assay using as bait the hinge region of GR. The MyoD family inhibitor domain-containing (MDFIC) protein was identified as a binding partner for GR. Knockdown of MDFIC in A549 cells alters the GR transcriptome. To further investigate the functional effect of MDFIC on the GR transcriptome, we performed a genome-wide microarray in COS-1 cells that were transfected with GR alone or GR and MDFIC. The transfected cells were treated with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. Overexpression of MDFIC was found to expand the GR transcriptome. COS-1 cells were transfected with GR alone or GR and MDFIC. The day after transfection, medium was replaced with new medium supplemented with 10% charcoal-stripped FBS. Forty-eight hours post-transfection, cells were stimulated with either vehicle (control) or 100nM Dex for 6 hours. Total RNA was harvested for microarray analysis using the RNeasy Mini Kit and RNase-Free DNase Kit (Qiagen) from 3 biological replicates from each group.