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

Abstract

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 (DKu-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.Keywords: ATP7B gene; Ku antoantigen; RNA interference; site-directed mutagenesis.Wilson disease (WD) is an autosomal recessive disorder characterized by defect in copper transport. Hepatic cirrhosis and neuronal degeneration are the most debilitating symptoms of WD and are caused by the impairment of biliary copper excretion and the accumulation of toxic concentration of copper. The WD gene product shares a high degree of sequence similarity with the cation-transporting P-type ATPases [1][2][3][4][5] and functions in the binding and translocation of copper [6].Interestingly, multiple copies of metal-response elements (MREs) are located in the 1.3-kb promoter of the WD gene [7]. Five or more nonidentical MREs are present in the 5¢-flanking region of the vertebrate metallothionein (MT) gene [8,9] and are believed to mediate the transcriptional activation of the MT gene by heavy metals [8,[10][11][12] and oxidative stress [13,14]. The MREs consist of 12-base pair sequences that contain a seven-nucleotide core sequence (TGCRCNC) surrounded by less well-conserved flanking nucleotides [15]. MTs are small (6-7 kDa), cysteine-rich, metal-binding proteins that funct...

Function and Regulation of Human Copper-Transporting ATPases

“…IIIE), and recently, the first steps have been made towards dissecting the underlying mechanisms. The promoter regions of ATP7A and ATP7B have been mapped to 1.3-to 2.2-kb segments of the genomic DNA upstream of the coding sequence and the first information on promoter organization began to emerge (97,154,166,198,199). Oh et al (199) identified a region ϳ1.3 kb upstream of the 5Ј-flanking region of the ATP7B gene, which when analyzed in a reporter assay had high levels of luciferase activity in HepG2 cells.…”

“…MREa was found to bind 70and 82-kDa proteins that were purified using avidin-biotin affinity chromatography and identified as Ku-related proteins. Subsequent functional studies produced evidence that the Ku-80 subunit is required for constitutive expression of the ATP7B gene (198).…”

Identification and functional expression of ctaA, a P-type ATPase gene involved in copper trafficking in Trametes versicolor

“…The transcriptional regulation of homologous proteins has yet to be investigated in any detail. For example, four metal response elements (MREs) detected in the promoter of the WND gene (Oh et al, 2002) have not yet been tested for copper induction. The P-type ATPases can probably be considered household genes that are often regulated at the protein level.…”

Genome Scanning by RNA Interference

“…RNAi technique was successfully applied to neuroscience studies performed with mammalian cells in vitro and mammals in vivo to study gene function in fundamental neurobiological processes such as intracellular signaling, embryonic development, formation of cellular junctions, cell cycle, programmed cell death, neural plasticity, axon guidance, and membrane trafficking. Effective gene silencing using RNAi screening has been demonstrated in neuronal cell lines [174 ], neural stem cells [175 ], primary mammalian neurons [176 ], astrocytes [177 ], and Schwann cells [178 ]. Moreover, gene silencing by adenovirus vector shRNA expression has also been demonstrated in mouse brain in vivo [179 ].…”