PIT-1 AND GATA-2 INTERACT AND FUNCTIONALLY COOPERATE TO ACTIVATE THE THYROTROPIN BETA-SUBUNIT PROMOTER

The molecular determinants governing cell-specific expression of the t hyrotropin (TSH) beta-subunit gene in pituitary thyrotropes are not we ll understood, The P1 region of the mouse TSH beta promoter (-133 to - 88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern a nd Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors, In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuc lear extracts and GATA-2 expressed in COS cells, The complex demonstra ted binding specificity to the P1 region DNA probe and could be disrup ted by a GATA-2 antibody, When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interact ion was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 ce lls synergistically stimulated mouse TSH beta promoter activity 8.5-fo ld, while each factor alone had a minimal effect, Mutations that abrog ated this functional stimulatory effect mapped to the P1 region. Final ly, we show that GATA-2 directly interacts with Pit-1 in solution, In summary, these data demonstrate functional synergy and physical intera ction between homeobox and zinc finger factors and provide insights in to the transcriptional mechanisms of thyrotrope-specific gene expressi on.