title: Biochemical characterization of diverse Stat5b-binding enhancers that mediate growth hormone-activated insulin-like growth factor-I gene transcription.
abstract: Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States of America.
authors.forename: Ben
authors.initials: B
authors.lastname: Varco-Merth
authors.forename: Kasim
authors.initials: K
authors.lastname: Mirza
authors.forename: Damir T
authors.initials: DT
authors.lastname: Alzhanov
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Chia
authors.forename: Peter
authors.initials: P
authors.lastname: Rotwein
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D056426:Genetic Loci; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases; D051379:Mice; D011485:Protein Binding; D051381:Rats; D050799:STAT5 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D050799:STAT5 Transcription Factor; C494097:Stat5b protein, rat; C548904:insulin-like growth factor-1, rat; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D008156:Luciferases