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.