PIT-1/GHF-1 BINDS TO TRH-SENSITIVE REGIONS OF THE RAT THYROTROPIN-BETA GENE

Three regions within the 5'-flanking region of the TSHbeta gene have A-T-rich sequences which have sequence similarity to binding sites for the pituitary-specific POU domain transcription factor Pit-1/GHF-1. These three regions have been termed TSH A (-274 to -258 bp), TSH B (-336 to -326 bp), and TSH C (-402 to -384 bp). TSH A and TSH C are able to confer 2-6-fold TRH stimulation to the heterologous viral thymidine kinase (tk) promoter in transient expression assays in GH3 pituitary cells; TSH C can confer a 3-10-fold increase in basal enhancer activity as well. TSH A, B, and C DNAs all bound Pit-1 from GH3 cell nuclear extracts, based on gel mobility shift analysis in which antibody against Pit-1 prevented the formation of specific DNA-GH3 nuclear protein complexes. TSH A and TSH C also each formed several additional DNA-nuclear protein complexes which were not observed with TSH B. Some of these complexes may contain Pit-1 as their formation was inhibited by the addition of Pit-1 antibody; other complexes, however, were not altered by antibody treatment. All three A-T-rich elements bound in vitro translated Pit-1, with calculated affinities of 360 (A), 125 (B), and 38 (C) nM, respectively. In order to test the ability of Pit-1 to transactivate the TSHbeta gene, luciferase reporter constructs containing either the homologous rTSHbeta gene promoter and 5'-flanking region or synthetic TSH gene elements fused to the heterologous HSV thymidine kinase (tk) promoter were transfected into 293 cells which lack Pit-1. Cotransfection of a Pit-1 expression vector with TSHbeta-luciferase constructs into 293 cells increased homologous TSHbeta promoter activity 3-10-fold, indicating that Pit-1 could transactivate the gene. Chimeric tk-TSH A-luciferase construct expression was not stimulated in Pit-1 cotransfection experiments; however, tk-TSH C-luciferase activity was stimulated up to 10-fold. These data suggest that Pit-1 may play a role in the basal and TRH-stimulated expression of the rat TSHbeta gene.