Abstract

The growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis regulates somatic growth during childhood and orchestrates tissue repair throughout the life span. Recently described inactivating mutations in Stat5b in humans with impaired growth have focused attention on this transcription factor as a key agent linking GH-stimulated signals to IGF-I gene expression, and several putative Stat5b sites have been identified in the IGF-I gene. Here, we define and characterize potential GH- and Stat5b-activated chromosomal enhancers that can regulate IGF-I gene transcription. Of 89 recognizable Stat5 sequences in 200 kb centering on the rat IGF-I gene, 22 resided within conserved regions and/or were identical among different species. Only 15 of these sites, organized into 7 distinct domains, were found to bind Stat5b by quantitative chromatin immunoprecipitation assays in liver chromatin of rats, but only after acute GH treatment. These sites could bind Stat5b in vitro, and individual domains could mediate GH- and Stat5b-stimulated IGF-I promoter activity in cultured cells. Further analyses revealed that four Stat5b domains possessed chromatin signatures of enhancers, including binding of co-activators p300 and Med1, and RNA polymerase II. These modifications preceded GH-stimulated recruitment of Stat5b, as did lysine 4 monomethylation of histone H3, which was enriched in 6/7 Stat5b-binding elements. In contrast, histone acetylation was induced by GH but was limited to Stat5b binding domains found within the IGF-I transcription unit. We conclude that GH stimulates recruitment of Stat5b to multiple dispersed regions within the igf1 locus, including several with properties consistent with long range transcriptional enhancers that collectively regulate GH-activated IGF-I gene transcription.

Highlights

  • Interferons ␣/␤ and ␥ [5, 6], and subsequent studies have both broadened the biological significance of this protein family as critical agents in multiple physiological and pathophysiological processes and have further defined their mechanisms of action at biochemical, molecular, and atomic levels of resolution [1,2,3,4]

  • Pituitary-derived growth hormone (GH)2 plays a pivotal role in regulating somatic growth during childhood and adolescence [12,13,14], primarily by stimulating the biosynthesis of insulin-like growth factor-I (IGF-I), a 70-residue secreted growth-promoting protein [15, 16] that is an important regulator of intermediary metabolism and tissue repair throughout the life span [14, 17, 18]

  • Genetic studies in mice and humans have established that the ognition sequences that are dispersed throughout the genome, transcription factor Stat5b is a central mediator in the GH-IGF- being found in introns and in intergenic DNA, as well as near

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Summary

EXPERIMENTAL PROCEDURES

Materials—The following reagents were purchased: recombinant rat GH, National Hormone and Pituitary Program, NIDDK, National Institutes of Health; fetal calf serum, Dulbecco’s modified Eagle’s medium, and phosphate-buffered saline, Mediatech-Cellgro (Herndon, VA); QuikChange site-directed mutagenesis kit, Stratagene (La Jolla, CA); SuperScript III reverse transcriptase system, protein A-Sepharose beads, SYBR Green platinum quantitative PCR mixture, and trypsin/EDTA solution, Invitrogen; whole genome amplification kit, Sigma; QIAquick PCR purification kit (Valencia, CA); Transit-LT1, Mirus (Madison, WI); protease inhibitor tablets, Roche Applied Sciences; okadaic acid, Alexis Biochemicals (San Diego); BCA protein assay kit, Pierce; and restriction enzymes, buffers, ligases, and polymerases, Roche Applied Sciences and Clontech. For gene expression studies and ChIP experiments, pH 8.1, and used as the template in quantitative PCRs. Primers up to four independent series of rats were used. 8 –10 are representative of three or Protein Immunoblotting—Immunoblotting was performed more independent quantitative PCR experiments from indeas described previously [30], using hepatic nuclear protein pendent immunoprecipitations. Extracts (5 ␮g) and the following dilutions of primary antibod- DNA-Protein Binding Studies—Electrophoretic gel mobility ies: anti-Stat5b-1:5000, anti-pStat Ϫ1:2000, and anti-cAMP- shift assays and antibody super-shift and DNA competition experiments were performed as described previously [30] with COS-7 (1–2 ␮g) or rat hepatic nuclear protein extracts (5 ␮g) and 5Ј-IR Dye700-labeled double-stranded oligonucleotides. Results were scanned and analyzed on a LiCoR Odyssey Infrared Imaging System, using software version 1.2

RESULTS
We used quantitative ChIP assays
DISCUSSION
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