Transcriptional repression of the ovine follicle-stimulating hormone-beta gene by 17 beta-estradiol

Abstract

Direct transcriptional inhibition of the gene that encodes ovine FSH beta-subunit (oFSH beta) by 17 beta-estradiol (E2) has been previously demonstrated by our laboratory. To determine which cis-acting elements in the 5'-flanking region of this gene may be involved in E2 regulation, DNA constructs containing deletions of the 5'-end of the oFSH beta gene were fused to a luciferase reporter and tested in transient transfection assays. These oFSH beta-luciferase constructs and the human E2 receptor expression vector (HEO) were transfected into primary cultures of ovine pituitary cells and subsequently tested with E2. Expression of the largest oFSH beta-luciferase construct (-4741 to +759 of oFSH beta) was inhibited 50% by 20 nM E2. Repression was dependent upon cotransfection of estrogen receptor (HEO) and was E2 dose dependent, with an apparent ED50 similar to that of the positive control consensus estrogen-responsive element construct, ERETk-LUC (ED50 = 50 pM). Deletion studies indicated that sequences between- 105 and -84 bp are necessary for this repression. In addition, a synthetic nucleotide containing oFSH beta sequences from - 105 to -72 could direct E2-dependent repression of a heterologous thymidine kinase promoter that drives luciferase expression. Additional experiments showed that no tissue-specific elements were required for either basal expression or E2-directed transcriptional repression. Although there are no consensus DNA response elements for the estrogen receptor between -105 and +759 of the oFSH beta gene, cotransfection of a mutant E2 receptor lacking the DNA-binding domain (HE-11) failed to mediate E2-dependent inhibition. Gel retardation studies, using the oligonucleotide-containing oFSH beta sequences from -105 to -72, indicated no evidence of direct binding of the estrogen receptor to DNA from -105 to -72. The studies presented here indicate that transcriptional repression of the oFSH beta gene by E2 may be directed in vivo by 5'-flanking sequences between -105 and -72 of the oFSH beta gene. Furthermore, the data suggest that inhibition is mediated via E2 receptor-protein interactions with basal transcription factors that may bind to the -105/-72 DNA directly.