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J. Biol. Chem., Vol. 280, Issue 12, 10955-10963, March 25, 2005
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Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression*
From the Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
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 
700-bp DNA region 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.
Received for publication, November 12, 2004 , and in revised form, January 12, 2005.
* This work was supported by National Institutes of Health Grant R01 DK67961. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 3130 Litton-Reaves Hall, Virginia Tech, Blacksburg, VA 24061-0306. Tel.: 540-231-1859; Fax: 540-231-3010; E-mail: hojiang{at}vt.edu.
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