MED220/Thyroid Receptor-Associated Protein 220 Functions as a Transcriptional Coactivator with Pit-1 and GATA-2 on the Thyrotropin-β Promoter in Thyrotropes

Mediator (MED) 220/thyroid receptor-associated protein (TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSHβ transcripts, suggesting a specific function for TSHβ transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSHβ promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSHβ promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHβ promoter. Thus, the TSHβ gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.