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 hormone (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 unit comprised of six exons and five introns. The gene and the 5' flanking region contain several potential binding sites for Pit-1, a key transcription activator of mammalian GH genes. One of these (-57/-42) is highly conserved in fish GH genes. It activates transcription in pituitary cells and binds 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 expression vector, demonstrating the crucial role played by Pit-1 as an activator of the tiGH gene. Fusion of the tiGH promoter with the beta-galactosidase gene led to transient expression specifically in the nervous system of microinjected zebrafish embryos. The activity of the tiGH promoter in GC and 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 retardation assays. Point mutations of the site that suppressed Pit-1 binding in vitro restored full tiGH promoter activity. Thus, a Pit-1-binding site located in the 5' untranslated region mediates Pit-1-dependent repression of the tiGH gene.
Sekkali
B
B
Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Institut de Chimie B6, Sart-Tilman, Belgium.
Brim
H
H
Muller
M
M
Argenton
F
F
Bortolussi
M
M
Colombo
L
L
Belayew
A
A
Martial
J A
JA
D020413:3' Untranslated Regions / Q000737:chemistry; D020121:5' Untranslated Regions / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D013006:Growth Hormone / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009420:Nervous System / Q000196:embryology / Q000378:metabolism; D017686:Oncorhynchus mykiss; D010911:Pituitary Neoplasms; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D017422:Sequence Analysis, DNA; D017210:Tilapia / Q000235:genetics*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D015027:Zebrafish / Q000196:embryology
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C494186:Pou1f1 protein, rat; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D013006:Growth Hormone