Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription.
A key agent in the anabolic actions of growth hormone (GH) is insulin-like growth factor-I (IGF-I), a 70-amino acid secreted protein with direct effects on somatic growth and tissue maintenance and repair. GH rapidly and potently stimulates IGF-I gene transcription by mechanisms independent of new protein synthesis, and recent studies have linked the transcription factor Stat5b to a regulatory network connecting the activated GH receptor on the cell membrane to the IGF-I gene in the nucleus. Here we analyze two distinct conserved GH response elements in the rat IGF-I locus that contain paired Stat5b sites. Each response element binds Stat5b in vivo in a GH-dependent way, as assessed by chromatin immunoprecipitation assays, and consists of one high affinity and one lower affinity Stat5b site, as determined by both qualitative and quantitative protein-DNA binding studies. In biochemical reconstitution experiments, both response elements are able to mediate GH-stimulated and Stat5b-dependent transcription when fused to a reporter gene containing either the major IGF-I promoter or a minimal neutral promoter, although the paired Stat5b sites located in the second IGF-I intron were more than twice as effective as the response element that mapped approximately 73 kb 5' to the IGF-I exon 1. Taken together, our results define the initial molecular architecture of a complicated GH-regulated transcriptional pathway, and suggest that apparently redundant hormone response elements provide a mechanism for amplifying GH action at a physiologically important target gene.
Chia
Dennis J
DJ
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239, USA.
Ono
Mitsuru
M
Woelfle
Joachim
J
Schlesinger-Massart
Mylynda
M
Jiang
Honglin
H
Rotwein
Peter
P
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D017930:Genes, Reporter; D013006:Growth Hormone / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008297:Male; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D012313:RNA; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D004247:DNA