GENE REGULATION AND FUNCTION - A
Tuesday, August 3, 2004
10:30 AM–12:30 PM
(371) IDENTIFICATION OF AN ACTIVIN RESPONSE ELEMENT ON THE PROMOTER OF OVINE FOLLICLE STIMULATING HORMONE BETA-SUBUNIT.
Su, Pei1, Miller, William 1, 1 North Carolina State University, Raleigh, North Carolina
ABSTRACT- Follicle stimulating hormone (FSH) is an alpha/beta heterodimer that orchestrates the development of ovarian follicles in mammals. Its overall production is controlled primarily by the transcription rate of its beta-subunit which depends largely on activin or activin-like factors found within the pituitary. The ovine FSH-beta promoter (4.7 kb) was linked to a luciferase reporter gene (oFSHLuc) and screened for activin response elements by transiently transfecting a series of oFSHLuc promoter mutants into L-betaT2 cells and testing for activin induction. Activin routinely induced wild-type oFSHLuc 5- to 10-fold, and only deletion of sequences from -175 to -151 totally destroyed this induction by activin. Selective deletion or mutation of 17 nucleotides within this region showed that only two nucleotides were absolutely necessary for activin induction (-166 bp and -165 bp). The importance of -166/-165 bps was confirmed in vivo using an oFSHLuc transgene mutated from -168 bp to -165 bp. Expression of this transgene was primarily found in the pituitaries of 5 founder lines, but expression was low, being < 5 % of that found for wild-type oFSHLuc in transgenic mice (Endocrinology 142: 2260-6, 2001). As expected, luciferase activity in pituitary cultures from these mice was less dependent on activin or activin-like factors made within the cultures as shown by the limited effect of follistatin (an inhibitor of activin action) on luciferase expression compared to its effect on expression of wild-type oFSHLuc (35 % and 74 % inhibition, respectively). Studies with a minimal thymidine kinase promoter (pT109Luc), showed that a region of the ovine FSH-beta promoter that included the -166/-165 nucleotides was sufficient to confer full activin responsiveness on pT109Luc in L-betaT2 cells. This region included sequences from -255 bp to -163 bp. Taken together, these data indicate that nucleotides -166/-165 are necessary for activin induction of oFSH beta and they and presumably several nucleotides surrounding them are also responsible for a significant amount of FSH expression in vivo. (Funded by NIH grant HD042459)
KEY WORDS: activin response element, follistatin, follicle stimulating hormone beta subunit promoter, transgenic mice