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J. Biol. Chem., Vol. 279, Issue 16, 16057-16063, April 16, 2004

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Characterization of Proteins Binding to E-box/Ku86 Sites and Function of Ku86 in Transcriptional Regulation of the Human Xanthine Oxidoreductase Gene^*

Ping Xu, Patricia A. LaVallee, Jun J. Lin, and John R. Hoidal

From the Department of Internal Medicine, Division of Respiratory, Critical Care and Occupational Medicine, University of Utah Health Sciences Center and Veterans Affairs Medical Center, Salt Lake City, Utah 84132

We reported previously that E-box and TATA-like elements repress human xanthine oxidoreductase gene (hXOR) expression. In the present investigation, we determined the means by which the E-box site functions in this basal repression. DNA affinity purification demonstrated that at least five proteins are involved in the nuclear protein complex binding to the E-box and adjacent Ku86-binding sites. Amino acid sequence analysis demonstrated that three proteins, DNA-PK catalytic subunit, Ku86, and Ku70 are components of DNA-dependent protein kinase (DNA-PK). By electrophoretic mobility shift assays, gel-shift, and site-directed mutagenesis, we confirmed Ku86 binding to the Ku86 site. Studies indicated that the other two proteins of the complex are AREB6-like proteins binding to the E-box. Pull-down and immunoprecipitation analyses demonstrated the binding of Ku86 to AREB6-like proteins. The functional loss of Ku86 increases hXOR promoter activity and transcript expression. Based on the findings, we propose that DNA-PK/AREB6-like proteins play a central role in repression of basal hXOR activity. AREB6-like proteins specifically bind to the E-box, whereas Ku86 binds an adjacent site and recruits DNA-PK catalytic subunit and Ku70 proteins. A working model is presented to account for the role of DNA-PK and AREB6-like proteins in regulating hXOR activity.

Received for publication, June 4, 2003 , and in revised form, December 17, 2003.

^* This work was supported by National Institutes of Health Grant 5RO1HL40665 and the Veterans Administration Research Service. 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: Pulmonary Division, Wintrobe Building, Rm. 743A, 50 N. Medical Dr., University of Utah Health Sciences Center, Salt Lake City, UT 84132. Tel.: 801-581-7806; Fax: 801-585-3355.

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