Isolation and functional characterization of the kiss2 promoter in European sea bass

Resumen del trabajo presentado en la 26th Conference of European Comparative Endocrinologists (CECE 2012), celebrada en Zurich (Suiza) del 21 al 25 de agosto de 2012Kisspeptins, encoded by the Kiss1 gene, have emerged as key modulators of reproduction in mammals via stimulation of GnRH neurons. In contrast to mammals, some fish, including European sea bass, have two distinct genes encoding kisspeptins, kiss1 and kiss2. In vivo kisspeptin administration, in situ hybridization and immunohistochemistry studies in seabass and zebrafish, have shown that kiss2 could be the predominant regulator of gonadotropin synthesis while kiss1 function is still unclear. In this frame, the factors that directly regulate the expression of the kiss genes could help to understand their roles in fish. In mammals, several studies on the KiSS1 promoter have been accomplished. However, there is little information about which transcription factors are implicated in the regulation of the kiss gene promoters in fish. In this study, we have cloned and functionally characterized the putative promoter region of European sea bass kiss2. We isolated 3 Kb of the 5' flanking region of seabass kiss2, next, the putative promoter sequences of kiss2 from several fish were analyzed in silico for conserved regulatory elements using bioinformatic tools (Matinspector, TFDialign). This analysis predicted the binding of regulatory elements including CCAAT/enhancer-binding protein alpha (CEBP¿), Y-box binding transcription factor (YBX1) and complexes of TALE homeobox genes like PBX1_MEIS1. Different kiss2 promoter fragments were examined for their ability to direct luciferase expression in CHO and HEK 293 cells. Our data indicate that less than 1 kb promoter is needed to direct maximal luciferase expression in the mentioned cell lines. Further co-transfections of promoter fragments with the CCAAT/enhancer-binding protein alpha revealed that this factor downregulates kiss2 promoter activity in CHO and HEK 293 cells. Although further studies should be performed, these results suggest a prominent role of a proximal CCAAT binding sequence in the regulation of kiss2 expression in sea bass.Supported by MICINN (AGL2011-28890) and EU Project LIFECYCLE (FP7-222719-1