Wilson disease (WD), an autosomal recessive inherited disorder of copper transport, is marked by impaired biliary excretion and incorporation of copper into ceruloplasmin. Clinical features of the WD are characterized by hepatic cirrhosis and neuronal degeneration, which result from toxic levels of copper that accumulate in the liver and brain, respectively. To investigate the molecular mechanisms regulating expression of the WD gene, we isolated, sequenced, and characterized ~1.3 kb of the 5'-flanking region of the WD gene from the human genomic library. The ~1.3 kb of the WD sequence directed high level of luciferase activity in HepG2 cells. Interestingly, sequence analysis showed that the 5'-flanking region contained four metal response elements (MREs)-two of four MREs are in reverse orientation- and six MRE-like sequences (MLSs), usually found in the metallothionein gene. It also contained a number of putative regulatory elements such as Sp1, AP-1, AP-2, and E-box, but lacked TATA box near transcription start site. The transcription start site was located at 335 base pairs upstream from the translation initiation site. Further successive 5'-deletion analyses suggested the important role of the 159-base pair region from -811 to -653, which includes MLS2 (-802 to -796) and MLS3 (-785 to -779), as a positive regulatory element during the expression of the WD gene. A negative element was also identified at region -1038 to -812. A protein-MLS complex was identified through electrophoretic mobility shift and competition assay using MLS2/MLS3 and HepG2 cell nuclear proteins.
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-kilodaltons 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 lead to the suppression of the WD gene. We also found that WD promoter activity was decreased in Xrs 5 cells, which unlike the CHO-K1 cells, are defective in the Ku 80 protein. When Ku 80 cDNA was transfected in Xrs 5 and CHO cell, WD promoter activity only was recovered in Xrs 5 cell. In addition, deleted Ku 80 (ΔKu 80) acting as a negative dominant resulted in a decrease of WD promoter activity in HepG2 cell. Taken together, our findings suggest that the Ku 80 subunit is required for constitutive expression of the WD gene.