Nuclear proteins that bind to metal response element a (MREa) in the Wilson disease gene promoter are Ku autoantigens and the Ku-80 subunit is necessary for basal transcription of the WD gene

Wilson disease (WD), an inherited disorder affecting copper metabolism, is characterized by hepatic cirrhosis and neuronal degeneration, which result from toxic levels of copper that accumulate in the liver and brain, respectively. We reported previously that the ≈ 1.3-kb promoter of the WD gene contains four metal response elements (MREs). Among the four MREs, MREa plays the most important role in the transcriptional activation of the WD promoter. Electrophoretic mobility shift assays (EMSAs) using synthetic MREa and an oligonucleotide containing the binding site for transcription factor Sp1 revealed the presence of nuclear factors that bind specifically to MREa. Two MREa-binding proteins of 70 and 82 kDa were purified using avidin-biotin affinity chromatography. Amino acid sequences of peptides from each protein were found to be highly homologous to the Ku proteins. Immunoblot analysis and EMSAs showed that the MREa-binding proteins are immunologically related to the Ku proteins. To study further the functional significance of these Ku-related proteins in transcriptional regulation of the WD gene, we performed RNA interference (RNAi) assays using a Ku-80 inverted-repeat gene to inhibit expression of the Ku-80 gene in vivo. Results of the RNAi assays showed that expression of the Ku-80 protein was suppressed in transfected cells, which in turn led to the suppression of the WD gene. In addition, a truncated Ku-80 (ΔKu-80) mutant inhibited WD promoter activity in HepG2 cells in a dominant-negative manner. We also found that WD promoter activity was decreased in Xrs5 cells, which, unlike the CHO-K1 cells, are defective in the Ku-80 protein. When Ku-80 cDNA was transfected into Xrs5 and CHO cells, WD promoter activity was recovered only in Xrs5 cells. Taken together, our findings suggest that the Ku-80 subunit is required for constitutive expression of the WD gene.