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Animals (Basel). 2018 Nov 7;8(11). pii: E200. doi: 10.3390/ani8110200.

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

Pan Z1,2, Wang X3, Di R4, Liu Q5, Hu W6, Cao X7, Guo X8, He X9, Lv S10, Li F11, Wang H12, Chu M13.

Author information

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. pzq170450077@163.com.
2
College of Agriculture and Forestry Science, Linyi University, Linyi 276000, China. pzq170450077@163.com.
3
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. xiangyu_wiggle@163.com.
4
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. dirangirl@163.com.
5
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. qiuyue1983921@163.com.
6
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. pinkyhoho@163.com.
7
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. caoxiaohan2007@hotmail.com.
8
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. guoxfnongda@163.com.
9
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. hexy2015x@163.com.
10
College of Agriculture and Forestry Science, Linyi University, Linyi 276000, China. lvshenjin@lyu.edu.cn.
11
College of Agriculture and Forestry Science, Linyi University, Linyi 276000, China. lifukuan@lyu.edu.cn.
12
College of Agriculture and Forestry Science, Linyi University, Linyi 276000, China. wanghui0512@lyu.edu.cn.
13
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. mxchu@263.net.

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; mRNA expression; methylation; regulatory mechanism; tissue-specific

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