A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep.

Growth differentiation factor 9 () plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine in different tissues by real-time PCR. exhibits significantly higher levels of expression ( < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (-1453 to -1854) of promoter in ovary and heart was determined. In this region (-1987 to -1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased ( < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with mRNA expression (R = -0.75, 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of . The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced ( < 0.05) basal transcriptional activity of promoter in oocytes. These results imply that methylation of promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the promoter region in sheep, thereby regulating expression and resulting in a tissue-specific expression.