Functional characterization of the follicle-stimulating hormone receptor core promoter: applying a comparative approach among primates

Abstract

Follicle-stimulating hormone (FSH) is essential for primate reproduction, acting via the FSH-receptor (FSHR) which is expressed in Sertoli and granulosa cells only. Despite its highly specific expression pattern, knowledge of the FSHR promoter and transcriptional regulation is still very limited. To gain insights into the regulatory elements controlling FSHR expression we characterized the core promoter activity of all important primate lineages including human. We isolated DNA fragments covering nucleotides -1 to -257 relative to the translational start site of the human, chimpanzee (Pan troglodytes), bonobo (Pan paniscus), cynomolgus monkey (Macaca fascicularis), marmoset (Callithrix jacchus) and lemur (Microcebus murinus) FSHR gene. The DNAs were cloned into the pGL3 vector to drive the expression of the luciferase reporter in transiently transfected COS7 and SK11 (mouse Sertoli) cell lines. Finally relative luciferase activity (RLA) was determined. Promoter activities varied significantly between species. Compared to the human FSHR promoter the chimpanzee displayed a 3.7-fold higher RLA, while for the bonobo only a 0.5 RLA was detected. The other primates displayed promoter activities similar to the human. Comparison of the human, chimpanzee and bonobo nucleotide sequences revealed only very few mismatches. Subsequent in-vitro mutagenesis of the human FSHR core promoter introducing one selected chimpanzee-specific alteration caused a significant 5-fold increase in RLA. Introducing the human nucleotide into the chimpanzee promoter decreased promoter activity to the bonobo wildtype level. Sequence analysis identified a binding site for ETS transcription factors to be involved, hitherto unknown for the FSHR promoter. Although FSHR promoters show very high degrees of sequences homology among primates, single nucleotide changes may have significant impact on FSHR promoter activities. Thus comparative functional studies using closely related species could yield important insights on different regulatory promoter elements within the same gene.