A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep
by
Zhangyuan Pan
1,2, 
Xiangyu Wang
1,
Ran Di
1,
Qiuyue Liu
1,
Wenping Hu
1,
Xiaohan Cao
1,
Xiaofei Guo
1,
Xiaoyun He
1,
Shengjin Lv
2,
Fukuan Li
2,
Hui Wang
2 and
Mingxing Chu
1,* 
1
Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2
College of Agriculture and Forestry Science, Linyi University, Linyi 276000, China
*
Author to whom correspondence should be addressed.
Animals 2018, 8(11), 200; https://doi.org/10.3390/ani8110200
Submission received: 31 July 2018
/
Revised: 21 October 2018
/
Accepted: 24 October 2018
/
Published: 7 November 2018
Simple Summary
Growth differentiation factor 9 (GDF9) is an important gene for ovine fertility. GDF9 is highly expressed in the ovary as opposed to other tissues, but the reason for this is unknown. Our study found this can be caused by the methylation level of the promoter CpG island mC-4 site. This finding contributes to the understanding of the regulatory mechanism of GDF9 gene in reproduction.
Abstract
Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 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 GDF9 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 (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.
Keywords:
GDF9; methylation; mRNA expression; tissue-specific; regulatory mechanism
