Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression

Ying Wang,Honglin Jiang

doi:10.1074/jbc.m412808200

Abstract

Growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression through signal transducer and activator of transcription 5b (STAT5b) and STAT5a. The objective of this study was to identify the cis-regulatory DNA region involved in this process. By cotransfection analyses of shotgun DNA fragments of a bacterial artificial chromosome sequence containing the entire human IGF-I gene and a large 5'-flanking region, a approximately 700-bp DNA region approximately 75 kb 5' to the IGF-I gene was found to have the ability to enhance gene expression from both heterologous and homologous promoters in the presence of constitutively active STAT5a or STAT5b. This 700-bp DNA region contains two closely located consensus STAT5-binding sites, and its sequence appears to be evolutionarily conserved. Electrophoretic mobility shift assays verified the ability of the two putative STAT5-binding sites to bind to STAT5a and STAT5b. Cotransfection analyses confirmed that both STAT5-binding sites were necessary for the 700-bp DNA region to mediate STAT5a or STAT5b activation of gene transcription. Chromatin immunoprecipitation assays demonstrated that the chromosomal region containing these two STAT5-binding sites was bound by constitutively active STAT5b protein in HepG2 cells and that the binding was accompanied by increased expression of IGF-I mRNA. In reconstituted GH-responsive cells, this 700-bp DNA region was able to mediate GH-induced STAT5a or STAT5b activation of gene expression. These results together suggest that this STAT5-binding site-containing distal 5'-flanking region of IGF-I gene may be an enhancer mediating GH-induced STAT5 activation of IGF-I gene transcription.

Highlights

Insulin-like growth factor-I (IGF-I)1 is an important endocrine and paracrine regulator of cell proliferation and metabolism [1]
Chromatin immunoprecipitation assays demonstrated that the chromosomal region containing these two STAT5-binding sites was bound by constitutively active signal transducer and activator of transcription 5b (STAT5b) protein in HepG2 cells and that the binding was accompanied by increased expression of insulin-like growth factor-I (IGF-I) mRNA
We hypothesized that Growth hormone (GH)-induced STAT5 activation of IGF-I gene expression in humans might be mediated by DNA regions other than or in addition to the IGF-I intron 2 region

Summary

Introduction

Insulin-like growth factor-I (IGF-I) is an important endocrine and paracrine regulator of cell proliferation and metabolism [1]. A homozygous STAT5b gene missense mutation was recently discovered to cause a series of characteristics of growth hormone insensitivity, including reduced serum IGF-I concentration in a human patient [17]. These findings together indicate that STAT5b is a key transcription factor mediating GH regulation of IGF-I gene transcription. Because GH stimulation of IGF-I gene expression may be directly mediated by STAT5, the cis-regulatory DNA regions involved might be the regions that contain STAT5-binding sites. EMSA EMSA chromosomal region containing the human IGF-I gene with constitutively active STAT5 proteins, we have identified an evolutionarily conserved distal 5Ј-flanking region of IGF-I gene that contains two consensus STAT5-binding sites and can mediate GH-induced STAT5 activation of gene expression

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Conclusion