MED14 and MED1 differentially regulate target-specific gene activation by the glucocorticoid receptor

Abstract

The Mediator subunits MED14 and MED1 have been implicated in transcriptional regulation by the glucocorticoid receptor (GR) by acting through its activation functions 1 and 2. To understand the contribution of these Mediator subunits to GR gene-specific regulation, we reduced the levels of MED14 and MED1 using small interfering RNAs in U2OS-hGR osteosarcoma cells and examined the mRNA induction by dexamethasone of four primary GR target genes, interferon regulatory factor 8 (IRF8), ladinin 1, IGF-binding protein 1 (IGFBP1), and glucocorticoid-inducible leucine zipper (GILZ). We found that the GR target genes differed in their requirements for MED1 and MED14. GR-dependent mRNA expression of ladinin 1 and IRF8 required both MED1 and MED14, whereas induction of IGFBP1 mRNA by the receptor was dependent upon MED14, but not MED1. In contrast, GILZ induction by GR was largely independent of MED1 and MED14, but required the p160 cofactor transcriptional intermediary factor 2. Interestingly, we observed higher GR occupancy at GILZ than at the IGFBP1 or IRF8 glucocorticoid response element (GREs). In contrast, recruitment of MED14 compared with GR at IGFBP1 and IRF8 was higher than that observed at GILZ. At GILZ, GR and RNA polymerase II were recruited to both the GRE and the promoter, whereas at IGFBP1, RNA polymerase II occupied the promoter, but not the GRE. Thus, MED14 and MED1 are used by GR in a gene-specific manner, and the requirement for the Mediator at GILZ may be bypassed by increased GR and RNA polymerase II occupancy at the GREs. Our findings suggest that modulation of the Mediator subunit activities would provide a mechanism for promoter selectivity by GR.