IMPORTANCE OF THE MOST PROXIMAL GC BOX FOR ACTIVITY OF THE PROMOTER OF HUMAN THYROID-HORMONE RECEPTOR BETA-1

We wish to localize the sequences required for transcriptional express ion of the thyroid hormone receptor beta 1 (TR beta 1). Constitutive a ctivity of the promoter of human thyroid hormone receptor beta 1 was a ssessed by transient transfection of deletion constructs attached to l uciferase as reporter, into P19, GH3, HepG2, H19-7, and COS1 cells. A 40-base pair fragment of the beta 1 promoter including the TATA box in duced minimal luciferase activity, which was considered basal activity . The activities of various lengths of the beta 1 promoter were estima ted relative to the minimal promoter in five cell lines. The region be tween -130 and -40 was crucial for constitutive activity in all cell l ines. Further deletion analysis in HepG2 cells showed that two regions mainly augmented the transcriptional activity of the minimal 40 base pair fragment. One region located at -115 to -93, which is highly GO-r ich, included the most proximal of five putative GC boxes present in t he whole 1325-base pair promoter. A second region contributing to expr ession of TR beta 1 in HepG2 cells is at -70 to -40. Mutation of the m ost proximal GC box strongly suppressed transactivity of the whole pro moter in P19 and HepG2 cells. In contrast, mutations in the other GC b oxes did not suppress transactivation in P19 cells and slightly suppre ss activation in HepG2 cells. In Schneider cells, which do not express Sp1, transactivity of the region distal to -40 is positively regulate d by cotransfection with a vector expressing Spl. The region -438 to - 70, in which two putative GC boxes including the most proximal GC box are present, was not positively regulated by Spl. Mutation analysis de monstrated that coexpression of Spl did not augment transactivation th rough the most proximal GC box in Schneider cells. Gel mobility shift assays show that four complexes are formed on the most proximal GC box in the presence of HepG2 nuclear extracts. These data indicate that t he region including the most proximal GC box is crucial for the expres sion of TR beta 1 and that Sp1 acid other factors may control basal ex pression level of TR beta 1 through interacting with the most proximal GC box in mammalian cells.