Regulatory regions in the promoter and first intron of Sparus aurata growth hormone gene: Repression of gene activity by a polymorphic minisatellite.

Transcriptional activity of the gilthead sea bream (Sparus aurata) growth hormone (saGH) gene promoter has been investigated using transient transfection assays in GH3 cells. Analysis of two fragments (1.7 kb and a 5'-deleted 0.9 kb) of saGH gene promoter directed luciferase reporter gene activity, indicating transcriptional activity of both fragments in vitro. The shorter fragment, containing five potential binding sites for the pituitary-specific transcription factor GHF-1/Pit-1, two cAMP-response elements and two glucocorticoid-response elements conferred higher reporter gene activity than the longer fragment. This result suggests presence of an inhibitory region upstream of the saGH promoter. Transient transfection assays were also used to investigate the effect of the polymorphic minisatellite saGHFIM found in the first intron of saGH gene on gene expression in four cell lines: fish pituitary (RTP-2) and non-pituitary (RTH-149) and mammalian pituitary (GH3) and non-pituitary (HEK293T). Luciferase activity was repressed by saGHFIM containing a high number of tandem repeats compared to a Control construct and to a construct with a smaller number of tandem repeats. Moreover, this repression was dependent on the orientation of the repeats relative to the viral promoter. These in vitro results imply that long GH introns might influence GH gene expression in vivo.