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Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep

Li, Zhuanjian, Guo, Ruoting, Gu, Zhenzhen, Wang, Xiangnan, Wang, Yongcai, Xu, Huifen, Wang, Chunxiu, Liu, Xiaojun
Gene 2019 v.699 pp. 1-7
binding sites, bioinformatics, bioluminescence assay, breeding season, enzymes, gene expression, genes, gonadotropin-releasing hormone, hypothalamus, kisspeptin, neurons, promoter regions, secretion, sheep, transcription factors
Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of −1912 bp to −1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (−1786 to −1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (−1786 to −1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.