title: Characterization of proteins binding to E-box/Ku86 sites and function of Ku86 in transcriptional regulation of the human xanthine oxidoreductase gene.
abstract: 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.
authors.affiliation: 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, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
authors.forename: Patricia A
authors.initials: PA
authors.lastname: LaVallee
authors.forename: Jun J
authors.initials: JJ
authors.lastname: Lin
authors.forename: John R
authors.initials: JR
authors.lastname: Hoidal
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics* / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024721:E-Box Elements / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010088:Oxidoreductases / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014970:Xanthines / Q000378:metabolism; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D014970:Xanthines; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; D010088:Oxidoreductases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen