Structure and functional analysis of a Tilapia (Oreochromis mossambicus) growth hormone gene: Activation and repression by pituitary transcription factor Pit-1

A gene encoding the Tilapia mossambica (Oreochromis mossambicus) growth hor mone (tiGH) was isolated and sequenced. The gene spans 5.6 kb, including 3. 7 kb of 5' and 0.2 kb of 3' flanking sequences and a 1.7-kb transcription u nit comprised of six exons and five introns, The gene and the 5' flanking r egion contain several potential binding sites for Pit-1, a key transcriptio n activator of mammalian GH genes. One of these (-57/-42) is highly conserv ed in fish GH genes. It activates transcription in pituitary cells and bind s Pit-1. Transfection of luciferase reporter plasmids containing either the -3602/$19 tiGH sequence or one of its 5' deletion mutants (-2863/, -1292/ and -463/+19) resulted in strong activity in Pit-1-producing rat pituitary GC cells. A dose-dependent activation of the tiGH promoter was achieved in nonpituitary fish EPC and monkey COS cells cotransfected with a rat Pit-1 e xpression vector, demonstrating the crucial role played by Pit-1 as an acti vator of the tiGH gene. Fusion of the tiGH promoter with the beta-galactosi dase gene led to transient expression specifically in the nervous system of microinjected zebrafish embryos. The activity of the tiGH promoter in GC a nd EPC cells was strongly repressed by extending its 3' end from +19 to +40 , a sequence in which a Pit-1-binding site was identified using gel retarda tion assays. Point mutations of the site that suppressed Pit-1 binding in v itro restored full tiGH promoter activity. Thus, a Pit-1-binding site locat ed in the 5' untranslated region mediates Pit-1-dependent repression of the tiGH gene.