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J. Biol. Chem., Vol. 279, Issue 21, 22228-22235, May 21, 2004

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Identification of a Novel PDX-1 Binding Site in the Human Insulin Gene Enhancer^*

John Le Lay, Taka-aki Matsuoka, Eva Henderson, and Roland Stein

From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37215

Islet cell type-specific transcription of the insulin gene is regulated by a number of cis-acting elements found within the proximal 5'-flanking region. The control sequences conserved between mammalian insulin genes are acted upon by transcription factors, like PDX-1 and BETA-2, that are also involved in islet cell function and formation. In the current study, we investigated the contribution to human insulin expression of the GG2 motif found between nucleotides -145 and -140 relative to the transcription start site. Site-specific mutants were generated within GG2 that displayed a parallel increase (i.e. -144 base pair) or decrease (i.e. -141 base pair) in insulin enhancer-driven reporter and gel shift binding activity in cells consistent with human GG2 being under positive regulatory control. In contrast, the corresponding site in the rodent insulin gene, which only differs from the human at nucleotides -144 and -141, is negatively regulated by the Nkx2.2 transcription factor (Cissell, M. A., Zhao, L., Sussel, L., Henderson, E., and Stein, R. (2003) J. Biol. Chem. 278, 751-756). Human GG2 activator binding activity was present in nuclear extracts prepared from human islets and enriched in those from rodent cell lines. The human GG2 activator binding factor(s) was shown to be 38-40 kDa and distinct from other size-matched islet-enriched transcription factors, including Nkx2.2, Pax-4, Cdx2/3, and Isl-1. Combined DNA chromatographic purification and mass spectrometry analysis revealed that the GG2 activator was PDX-1. These results demonstrate that the GG2 element, despite its divergence from the core homeodomain consensus binding motif, is a site for PDX-1 activation in the human insulin gene.

Received for publication, November 19, 2003 , and in revised form, March 9, 2004.

^* This work was supported by National Institutes of Health Grant RO1 DK50203 (to R. S.), Juvenile Diabetes Research Foundation International Grants 12002775 (to R. S.) and 32001678 (to T.-A. M.), and partial support from the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public Health Service Grant P60 DK20593 from the National Institutes of Health). 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.

Present address: Dept. of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan 565-0871.

To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 723 Light Hall, Nashville, TN 37215. Tel.: 615-322-7026; Fax: 615-322-7236; E-mail: roland.stein{at}mcmail.vanderbilt.edu.

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