Cloning and characterization of the promoter region of the Wilson disease gene

Wilson disease (WD), an autosomal recessive disorder of copper transport, i s marked by impaired biliary excretion and incorporation of copper into cer uloplasmin, Molecular mechanism regulating the expression of the WD gene wa s studied. We isolated, sequenced, and characterized similar to 1.3 kb of t he 5'-flanking region of the WD gene from the human genomic library, The si milar to 1.3 kb of the WD sequence directed high level of luciferase activi ty in HepG2 cells. Interestingly, the 5'-flanking region contained four met al response elements (MREs) and six MRE-like sequences (MLSs), usually foun d in the metallothionein genes. It also contained a number of putative regu latory elements such as Sp1, AP-1, AP-2, and E-box, but lacked TATA box. Th e transcription start site was located at 335 base pairs upstream of the tr anslation initiation site, Successive 5'-deletion analyses suggested that t he 159-base pair region from -811 to -653, which includes MLS2 (-802 to -79 6) and MLS3 (-785 to -779), contained one or more positive regulatory eleme nt(s). A negative element was also identified at region -1038 to -812. A pr otein-MLS complex was identified through electrophoretic mobility shift and competition assay using MLS2/MLS3 and HePG2 cell nuclear proteins. (C) 199 9 Academic Press.