Characterization of proteins binding to E-box/Ku86 sites and function of Ku86 in transcriptional regulation of the human xanthine oxidoreductase gene.
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.
Xu
Ping
P
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.
LaVallee
Patricia A
PA
Lin
Jun J
JJ
Hoidal
John R
JR
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
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