FOLLICLE-STIMULATING-HORMONE RECEPTOR GENE PROMOTER ACTIVITY

The promoter region of the;rat follicle-stimulating-hormone receptor ( FSHR) was characterized to understand better the transcriptional regul ation of this receptor, and in particular its cell-specific expression , Several constructs containing up to 5 kb of DNA 5' to the translatio nal start site were used to direct the synthesis of the chloramphenico l acetyltransferase (CAT) gene in transiently transfected rat primary Sertoli cells and several different cell lines. As little as 286 bp (- 30 to -316 bp) of proximal promoter sequence was sufficient for transc ription in rat primary Sertoli cells, a mouse Sertoli cell line (MSC 1 ) and a Leydig cell line (MA-10). Some of the promoter constructs were active at a low but detectable level in NIH3T3 cells whereas none of the four constructs were transcriptionally active in Cos-7 cells. Tran sfected primary Sertoli cells responded to the addition of serum to th e medium by increasing CAT activity two- to threefold. Examination of the methylation state of the FSHR promoter using methylation-sensitive enzymes and Southern blot analysis revealed that the proximal 5' flan king region of the FSHR gene was differentially methylated depending o n whether FSHR mRNA was being actively expressed. Electrophoretic mobi lity shift assays revealed a specific banding pattern of retarded prot ein-DNA complexes that varied greatly in intensity using nuclear extra cts isolated from rat primary Sertoli, MSC 1, NIH3T3 and Cos-7 cells. At least one of these bands was specific to the primary Sertoli cells and MSC 1 cells and was responsive to the presence of serum, 5 kb of F SHR 5' flanking sequence directed gonad-specific expression of the E. coli B-galactosidase reporter mRNA in two transgenic lines.