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. Th ese three regions have been termed TSH A (-274 to -258 bp), TSH B (-33 6 to -326 bp), and TSH C (-402 to -384 bp). TSH A and TSH C are able t o confer 2-6-fold TRH stimulation to the heterologous viral thymidine kinase (tk) promoter in transient expression assays in GH3 pituitary c ells; 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 e xtracts, based on gel mobility shift analysis in which antibody agains t Pit-1 prevented the formation of specific DNA-GH3 nuclear protein co mplexes. TSH A and TSH C also each formed several additional DNA-nucle ar protein complexes which were not observed with TSH B. Some of these complexes may contain Pit-1 as their formation was inhibited by the a ddition of Pit-1 antibody; other complexes, however, were not altered by antibody treatment. All three A-T-rich elements bound in vitro tran slated 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 transact ivate the TSHbeta gene, luciferase reporter constructs containing eith er the homologous rTSHbeta gene promoter and 5'-flanking region or syn thetic TSH gene elements fused to the heterologous HSV thymidine kinas e (tk) promoter were transfected into 293 cells which lack Pit-1. Cotr ansfection of a Pit-1 expression vector with TSHbeta-luciferase constr ucts into 293 cells increased homologous TSHbeta promoter activity 3-1 0-fold, indicating that Pit-1 could transactivate the gene. Chimeric t k-TSH A-luciferase construct expression was not stimulated in Pit-1 co transfection experiments; however, tk-TSH C-luciferase activity was st imulated 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.