| Summary: | Growth differentiation factor 9 (<i>GDF9</i>) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine <i>GDF9</i> in different tissues by real-time PCR. <i>GDF9</i> exhibits significantly higher levels of expression (<i>p</i> < 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 <i>GDF9</i> 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 (<i>p</i> < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with <i>GDF9</i> mRNA expression (R = −0.75, <i>p =</i> 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of <i>GDF9</i>. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (<i>p</i> < 0.05) basal transcriptional activity of <i>GDF9</i> promoter in oocytes. These results imply that methylation of <i>GDF9</i> promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the <i>GDF9</i> promoter region in sheep, thereby regulating <i>GDF9</i> expression and resulting in a tissue-specific expression.
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