Growth Hormone Regulation of Insulin-Like Growth Factor-I Gene Expression May Be Mediated by Multiple Distal Signal Transducer and Activator of Transcription 5 Binding Sites

Abstract

The transcription factor signal transducer and activator of transcription (STAT)-5 mediates GH stimulation of IGF-I gene expression in the liver. Previous studies suggested that STAT5 might exert this effect by binding to an IGF-I intron 2 region and a distal 5'-flanking region each containing two STAT5 binding sites. Here we report the identification of three additional chromosomal regions containing a total of five putative STAT5 binding sites that may mediate GH-induced STAT5 activation of IGF-I gene expression in the mouse liver. By comparing an 170-kb mouse genomic DNA containing the IGF-I gene with the corresponding human sequence, we identified 19 putative STAT5 binding sites that bear the consensus sequence of STAT5 binding site and are conserved across the two species. Chromatin immunoprecipitation assays indicated that five chromosomal regions containing a total of nine of the 19 putative STAT5 binding sites were bound by STAT5 in the mouse liver in response to GH administration and that these bindings preceded or coincided with GH-increased IGF-I gene transcription. Two of the five chromosomal regions correspond to those previously identified in other species, and the three new chromosomal regions that contain a total of five putative STAT5 binding sites are IGF-I intron 3 regions located at least 26 kb from the transcription start site. Gel-shift assays confirmed the binding of the five new putative STAT5 binding sites as well as three of the four previously identified STAT5 binding sites to GH-activated STAT5 from the mouse liver. Cotransfection analyses indicated that, although each of the five chromosomal regions was able to mediate STAT5 activation of reporter gene expression, together they mediated greater STAT5 activation of reporter gene expression in response to GH. Overall, these results suggest that GH-induced STAT5 activation of IGF-I gene expression in the mouse liver might be collectively mediated by at least eight STAT5 binding sites located in distal intronic and 5'-flanking regions of the IGF-I gene.

The hormonal action of IGF1 in postnatal mouse growth

“…10) was unsuitable for our purposes because expression of Igf1 in the liver of Igf1 nullizygous mice would be unregulated, making uncertain the physiological significance of the results. The mechanisms controlling mammalian Igf1 gene transcription are complex, as they involve, for example, two promoters and also dispersed regulatory elements that serve GH signaling acting through the Stat5b pathway (11).…”

Dispersed Chromosomal Stat5b-binding Elements Mediate Growth Hormone-activated Insulin-like Growth Factor-I Gene Transcription

“…In further support of this hypothesis, two distinct GH-inducible Stat5b binding domains have been mapped to chromatin in human IGF1 and rat Igf1 loci (28 -30), and both appear able to act as GH-and Stat5b-dependent regulators of IGF-I promoter function in cultured cells (28,30). As results of more recent experiments have suggested the potential existence of multiple Stat5b-binding elements in the IGF-I gene (31,32), we sought to identify and characterize putative GH-regulated and Stat5b-dependent chromosomal enhancers responsible for GH-activated IGF-I gene transcription. Our results show that GH acutely stimulates recruitment of Stat5b to a least seven distinct chromosomal domains found throughout the Igf1 locus coincident with induction of IGF-I gene transcription and that each can function in vitro to augment IGF-I promoter activity.…”

Identifying growth hormone-regulated enhancers in the Igf1 locus

“…Analogous regions have been identified near the mouse Igf1 gene (17,26) and to a far more limited extent near human IGF-I (55). The most remarkable feature of these chromosomal regions, perhaps besides their dynamic plasticity in chromatin, is that there are so many of them.…”