Báo cáo Y học: 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 ppt

Eur J Biochem 269, 2151–2161 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02865.x 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 Won Jun Oh1, Eun Kyung Kim1, Jung Ho Ko1, Seung Hee Yoo1, Si Houn Hahn2 and Ook-Joon Yoo1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Taejon Korea; Department of Pediatrics, Ajou University School of Medicine, Suwon, Korea 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 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 [1–5] 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 [8,10–12] 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 function in the maintenance of metal homeostasis and detoxification by forming strong complexes with several types of metal ions [16,17] Given the existence of MREs in the promoter of the WD gene, it seems plausible that metal ions function in the transcriptional regulation of the WD gene via the adjacent MREs The Ku protein is associated with a DNA-dependent protein kinase [18] and is involved in double-stranded DNA break repair, V(D)J recombination, and telomere maintenance [19–21] Sequence-specific DNA binding of Ku family proteins has been reported also for the genes encoding small nuclear RNA, the T-cell receptor, the transferrin receptor, collagenase, and heat shock proteins (HSPs) [22–28] Recently, it was shown that overexpression of the Ku-80 subunit suppresses the MT-I gene, which chelates heavy metals in fibroblast cells [29] In this study, we used mutational analysis to show that MREa ()434 to )438) is the key transcriptional regulatory element of the WD gene We then used electrophoretic mobility shift assays (EMSAs) to detect an MREa-binding activity in HepG2 cell nuclear extracts We purified and characterized the MREa-binding activity, which consisted of two polypeptides of approximately 70 and 82 kDa N-terminal and internal amino acid sequencing and immuno analyses revealed that MREa-binding proteins are either Correspondence to O.-J Yoo, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusongdong, Yusong-gu, Taejon 305-701, Korea Fax: +82 42 869 8160, Tel.: +82 42 869 2626, E-mail: ojyoo@mail.kaist.ac.kr Abbreviations: WD, Wilson Disease; MRE, metal response element; MT, metallothionein; RNAi, RNA interference; IR, inverted repeats; EMSA, electrophoretic mobility shift assay; ZAP, zinc activated proteins (Received November 2001, revised February 2002, accepted March 2002) Keywords: ATP7B gene; Ku antoantigen; RNA interference; site-directed mutagenesis Ó FEBS 2002 2152 W J Oh et al (Eur J Biochem 269) identical or closely related to the human Ku proteins We also performed RNA interference (RNAi) assays and assays using a truncated Ku-80 mutant to study the regulation of WD gene expression by the Ku proteins Finally, we assessed the activity of the WD promoter in the Ku-80-deficient Xrs5 and Ku-80 transfected Xrs5 cell line Our results suggest that the Ku proteins that bind specifically to MREa play an important role as a basal regulator of WD gene transcription MATERIALS AND METHODS Cell culture The human hepatoma cell line HepG2 was obtained from KRIBB and grown in Dulbecco’s modified Eagle’s medium (pH 7.4) containing 10% fetal bovine serum and · antibiotic–antimycotic solution (Life Technologies, Inc.) The hamster ovary cell line CHO-K1 and the Ku-80deficient cell line Xrs-5 were obtained from the American Type Culture Collection and maintained Ham’s F12K medium and alpha minimum essential medium, respectively, that containing 10% fetal bovine serum and · antibiotic–antimycotic solution Transfections using the above constructs were repeated three or more times, and the average result is presented Preparation of HepG2 nuclear extracts Nuclear extracts were prepared as described previously [32], with slight modifications HepG2 cells (9.4 · 106 cells per dish) were washed with ice-cold NaCl/Pi and scraped off the dish into mL ice-cold NaCl/Pi Cell pellets were resuspended in vol hypotonic buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethanesulfonyl fluoride, 0.5 mM dithiothreitol), and the cell suspensions were homogenized with a Dounce homogenizer (20 strokes, type B pestle) Nuclear proteins were suspended in vol low salt buffer (20 mM Hepes pH 7.9, 25% glycerol, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethanesulfonyl fluoride, 0.5 mM dithiothreitol), followed by the addition of M KCl to a final concentration of 0.3 M Nuclear suspensions were stirred for 30 on ice and dialysed against dialysis buffer (20 mM Hepes pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethanesulfonyl fluoride, 0.5 mM dithiothreitol) for h at °C Nuclear extracts were frozen in aliquots at )70 °C EMSAs Human WD promoter-reporter gene constructs A 1.6-kb construction of the WD promoter, named pPWDLuc, was described previously [7] Trinucleotide mutations in each of the four MREs of the WD gene promoter were constructed using the PCR-based quick change site-directed mutagenesis method (Stratagene) The following oligonucleotides were used in the MRE mutagenesis, with the trinucleotide mutations indicated in lowercase letters (underlined bases denote the MRE core sequence): MREa mut, 5¢-GGGCGCCaatGCCCCCGTTCC-3¢; MREe mut, 5¢-GGCCATTGGCTGGCCTTaatGCACAGCGGATCG ATTTTC-3¢; MREc mut, 5¢-CCAGTACAGTGTCGG AGCattCCAGCGCGAGGTGGCCG-3¢; MREd mut, 5¢-CGGGAGGACGGCGGCGCattACTTTGAATCAT CCGTG-3¢ Mutations in the MREs were identified and confirmed by automated fluorescent DNA sequencing (Perkin-Elmer 377) Transfection and luciferase assays DNA transfections were performed according to the procedure provided by Life Technologies HepG2 cells were cultured at 40–60% confluence in 6-well dishes and transfected with lg of DNA mixture containing various Ku cDNAs, WD promoter report construct (pPWD-Luc) [7], and pSVb-gal which served as an internal control for transfection efficiency The transfected cells were cultured for a further 24 or 48 h, and the expression levels of luciferase and b-galactosidase were determined Luciferase activity was analysed with the Promega luciferase assay kit The cells were harvested in reporter lysis buffer, and the lysate was spun in a microcentrifuge for 15 s Chemiluminescence was measured with a luminometer (Berthold), and the b-galcatosidase activities were determined as described [30] When determination of the exact number of transfected cells was required, transfected cells were distinguished from nontransfected cell by visualization using X-Gal [31] The double-stranded MREa probes were end-labelled with [c-32ATP] (Amersham) and T4 polynucleotide kinase HepG2 cell nuclear proteins (10 lg) were incubated in reaction buffer containing 17 mM Hepes pH 7.9, 32 mM Tris/HCl pH 7.8, 13% glycerol, 25 mM KCl, 0.8 mM dithiothreitol, lg poly(dI-dC), and 2–4 fmol 32P end-labelled, double-stranded MRE oligonucleotides (50 000 c.p.m per reaction) in a total volume of 20 lL, and the reaction mixture was incubated for 30 at 25 °C For competition experiments, a molar excess of unlabelled MRE oligonucleotides were added to the binding reaction and incubated for 15 prior to the addition of the labelled MRE DNA probe as specified Protein–DNA complexes were separated electrophoretically in a 4% polyacrylamide gel (acrylamide : bisacrylamide, 30 : 0.8 in 0.5 · Tris/borate/EDTA) After electrophoresis, the gel was dried, and the 32P-labelled protein–DNA complexes were detected by autoradiography For supershift EMSAs, nuclear extracts from HepG2 cells were incubated with a mixture of Ku-70/-80 monoclonal antibodies (mAb; 0.2 lg or lg; Clone 162, Neomarkers Co.) for min, followed by incubation with 32P-labelled MRE oligonucleotides for 30 at 25 °C The oligonucleotide sequences used as the 32P-labelled probes and competitors were: MREa, 5¢-GGGCGCC TGCGCCCCCGTTCC-3¢ ()441 to )421); MREa mut1, 5¢-GGGCGCCAATGCCCCCGTTCC-3¢; MREa mut2 : 5¢-GGGCGCCTGCGCCCTTATTCC-3¢; Sp1 : 5¢-ATT CGATCGGGGCGGGGCGAGC-3¢ (underlined bases denote the functional core of the MREs and the Sp1 binding element, and the mutated bases are indicated by italic type) Western blot analysis The MREa-binding proteins purified from HepG2 cells by the avidin–biotin method and lysates (30 lg) of cells Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur J Biochem 269) 2153 transfected with the Ku-80 inverted-repeat (IR) gene were separated by SDS/PAGE on a 10% polyacrylamide gel Proteins were transferred to a nitrocellulose filter using standard procedures The membranes were then incubated with mAbs to the Ku-70 (Clone N3H10, Neomarkers Co.) and Ku-80 (Clone 111, Neomarkers Co.) proteins and with polyclonal antibodies to the WD protein (transfection experiments only) The antigen–antibody complexes were detected using a second antibody conjugated to horseradish peroxidase and the ECL detection system (Amersham) South-Western blotting The South-Western blotting assay was carried out as described by Dikstein [33] HepG2 nuclear extracts (30 lg) were resolved by SDS/PAGE on a 10% polyacrylamide gel and electroblotted onto a nitrocellulose membrane After transfer, the filter was incubated in blocking solution (5% nonfat milk, 50 mM Tris/HCl pH 7.5, 100 mM NaCl, mM EDTA, and mM dithiothreitol) for h at room temperature and then washed twice with TNE-100 buffer (10 mM Tris/HCl pH 7.5, 100 mM NaCl, mM EDTA, mM dithiothreitol) The filter-bound proteins were then denatured by incubation in denaturing buffer (7 M guanidine/ HCl, 50 mM Tris/HCl pH 8.0, 50 mM dithiothreitol, mM EDTA, and 0.25% nonfat milk) for h at room temperature and renatured by incubation in 50 mM Tris/HCl pH 7.5, 100 mM NaCl, mM dithiothreitol, mM EDTA, 0.1% NP-40, 0.25% nonfat milk for 20 h at °C To measure the MREa-binding activity of the filter-bound proteins, the filter was preincubated with TNE-100 containing 10 lgỈmL)1 poly(dI-dC) for h at room temperature, and then 32P end-labelled, double-stranded MREa oligonucleotide was added (2 · 106 c.p.m.ỈmL)1) to the incubation mixture After h of incubation, the filter was washed three times with TNE-100 and subjected to autoradiography Purification of Ku proteins The Ku proteins that bound to MREa were purified from HepG2 nuclear extracts using the avidin–biotin method of Otsuka et al [34] with slight modifications An MREa probe was composed of a chemically synthesized oligonucleotide with a biotin head at the 5¢-end HepG2 nuclear extracts were incubated with avidin–agarose beads (Sigma) at °C for 30 to eliminate materials in the extracts that bind nonspecifically to the beads After removing the beads by filtration, 200 lg of the nuclear extract proteins were incubated in reaction buffer containing 18 mM Hepes/KOH pH 7.9, 10% glycerol, 40 mM KCl, mM MgCl2, 10 mM dithiothreitol, 125 lgỈmL)1 poly(dI-dC), and 50 pmol of the biotinylated MREa probe In addition, 50 pmol MREaMut1 oligonucleotides were added to the binding reaction as a substitute for poly(dI-dC) for the competition assay The binding reaction was carried out for 30 at 25 °C After elinimating by centrifugation denatured proteins generated during the binding reaction, the mixture was combined with 20 lL avidin–agarose beads and incubated at room temperature for 30 with shaking The beads were washed successively with 0.5 mL 0.1 M KCl buffer III (20 mM Hepes/KOH pH 7.9, mM dithiothreitol, 20% glycerol, 0.01% NP-40) and 0.5 mL 0.2 M KCl buffer III, and then the proteins were extracted from the beads by incubation in 0.5 M KCl buffer III (0.5 mL) for 30 at °C The extracts were concentrated to 10% of the original volume of the nuclear extract by trichloroacetic acid precipitation The concentrated sample was analysed by SDS/PAGE (10% polyacrylamide) Amino acid sequencing of purified MREa-binding Ku proteins The Ku proteins described in the previous section were purified from 700 lg HepG2 cells, which yielded  0.5–1 lg of Ku protein After incubation of the purified Ku proteins in SDS sample buffer for 15 at 37 °C, the proteins were separated by SDS/PAGE and electrotransferred onto poly(vinylidene difluoride) membrane (Sequi-blot; Biorad) Internal peptide sequencing of the 70-kDa protein was performed according to the Current Protocol method [35] Concentrated Ku proteins were digested with 50 lL 70% formic acid at 37 °C for 48 h The peptide fragments generated by the formic acid treatment were lyophilized completely and separated by electrophoresis The Ku protein fragments were visualized in the gel by Coomassie blue staining, the bands were sliced out of the gel and subjected to automated amino acid sequencing Construction of the IR Ku-80 gene and the truncated Ku-80 (DKu80) For the double-stranded RNAi assay, the Ku-80 IR vector was constructed according to the method of Tavernarakis et al [36] with slight modification The Ku-80 cDNA (2.2 kb) was amplified by PCR from a Ku-80 clone (a generous gift from Y Sung-Han, Medical College of Georgia, Augusta) The amplified Ku-80 cDNA was digested with EcoRI at a site downstream of the coding region, ligated to generate the IR, digested with BamHI at a site upstream of the coding region, and then inserted into the expression vector pcDNA 3.1(+) (Fig 7A) The IR orientation of Ku-80 was identified by automated fluorescent DNA sequencing (Perkin-Elmer 377) We also constructed a truncated Ku-80 gene, in which the region spanning amino acids 351–416 was deleted; this was used for transfections into HepG2 cells as described above Competitive RT-PCR Competitive RT-PCR assay was performed to determine the amount of transcript of WD gene in HepG2 cells transfected with Ku 80 IR After the Ku 80 IR vector (1 lg) was transfected as described previously, total RNA was isolated from time-dependent HepG2 cells using the RNeasy kit (Qiagen) Reverse transcription was performed at 65 °C for and 37 °C for 60 followed by denaturation at 95 °C in a total volume of 50 lL of reaction mixture using the RT-PCR kit (Stratagene) PCR amplification was performed using LA Taq (Takara, Japan) in a total volume of 50 lL containing 55 pg of competitor At this concentration, the band intensity of the amplified product from the competitor was same as that of the product from the endogenous WD gene Amplification conditions were: 95 °C for followed by 18 cycles of 95 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s, and 72 °C for Ó FEBS 2002 2154 W J Oh et al (Eur J Biochem 269) Table Oligonucleotide primers and conditions used for competitive RT-PCR analysis PCR products (base pairs) Target gene Forward primer Reverse primer Wilson gene 5¢-TGTTAAGTTTGACCCGGAAATTATC 5¢-CCGGTCAGCCAGCTGCTG 5¢-TGATGGTGGGCATGGGTCAG 5¢-TACATGGTGGTGCCGCCAGA a-actin (Perkin-Elmer PCR system 9700) After competitive RT-PCR, a 10-lL aliquot was electrophoresed in a 1% agarose gel and the bands were visualized by staining with ethidium bromide The intensities of the amplified fragments were quantified by densitometric analysis As an internal control 791 base pairs of a-actin was amplified using forward and reverse primers The primers used for competitive RT-PCR and predicted product sizes are given in Table RESULTS Effect of MREa mutations on WD gene promoter activity in HepG2 cells The relative positions of the MREs within the promoter region of the WD gene are indicated in Fig The four MREs are located in the proximal region of the WD gene promoter between )434 and +114, with MREa and MREe in the forward orientation, and MREc and MREd in reverse orientation The effects of the mutiple MREs on promoter activity of the MT gene have been shown to be dependent on the position of the individual MRE [37] For this reason, we assessed the functional contributions of MREa, MREe, Target Competitor Annealing temperature (°C) 911 713 55 791 55 MREc, and MREd on WD gene promoter activity Trinucleotide mutations were generated in the core sequence of each MRE (Fig 1) and these WD gene promoter variants were fused to a luciferase reporter gene HepG2 cells were transfected with these reporter genes and luciferase activity in the cell extracts was measured after 48 h The mutaton within MREa resulted in a marked decrease in promoter activity (0.5% of wild-type); however, mutations in the other MREs showed no significant changes in luciferase activity compared to the native WD promoter These results show that MREa has the greatest effect on WD gene promoter activity Several studies have reported that MREa in the promoter of the MT gene possesses transcriptional regulatory activity and that a variety of nuclear transcription factors interact specifically with MREa [11,33,38,39] These findings suggest that transcriptional activator proteins that regulate expression of the WD gene may bind to MREa Identification of proteins that interact specifically with MREa We next performed EMSAs to determine whether MREabinding proteins are present in HepG2 cell nuclear extracts For the EMSAs we used a 32P-labelled synthetic oligonu- Fig WD gene promoter fragments used in the transfection analyses and the effects of MRE mutations on WD gene promoter activity (A) Schematic representation of the WD promoter ()1265 to +335) is shown with the position of the MREs indicated (arrow direction conveys MRE orientation) The sequences of the MRE mutants used in this study are shown (B) HepG2 cells were transfected with wild-type and MRE mutant WD gene promoters fused to a luciferase reporter gene in the pGL2 reporter construct [38] The results were normalized using b-galactosidase activity Results are mean ± SD pGL indicates the luciferase activity in cells that had been transfected with the pGL as a negative control pGL promoter (pGLpro.) that contains the SV40 promoter is used as a positive control Luciferase activity for each construct was normalized to b-gal activity, and the relative increases were calculated as the ratio of normalized activity in MREa/mutant transfected cells to that in pGLpro transfected cells Data represent the mean ± SD of three independent transfection assays for each construct N, Nucleotides; R, purine; +1, transcription start site Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur J Biochem 269) 2155 Fig Detection of nuclear proteins that bind specifically to MREa (A) 32P-labelled doublestranded oligonucleotide (Oligo) containing MREa was incubated without (lane 1) and with (lane 2) HepG2 Nuclear extracts (10 lg) (B) Competition experiments were performed in the absence (C, lane 2) or presence of varying concentrations of unlabelled MREa (lanes 3–5), Sp1 (lanes 6–7), MREaMut1 (lane 8), and MREaMut2 (lane 9) Lane 1, oligo only The solid arrowhead indicates the specific MREa–protein complex and is competed away with unlabelled MREa The open arrowhead denotes a nonspecific band cleotide that contained a copy of MREa (Fig 2A) We also performed competition experiments with various oligonucleotide competitors to determine whether the MREa– protein complex displays sequence specificity Binding of the HepG2 nuclear proteins to MREa was inhibited by the addition of a molar excess of unlabelled MREa to the binding reaction (Fig 2B, lanes 3, 4, 5) It has been shown that the MREs overlap with a potential binding site for the transcription factor Sp1 [40] However, addition of a molar excess of oligonucleotide that contained the Sp1 binding site did not affect formation of the MREa–protein complex (Fig 2B, lanes 6, 7) We also constructed two mutant MREa oligonucleotides to analyse the effect of MREa sequence on formation of the MREa–protein complex Mutant competitors were introduced either outside (MREa mut2) or within (MREa mut1) the MREa consensus sequence Neither MREaMut1 nor MREaMut2 affected MREa–protein complex formation (Fig 2B, lanes 8, 9) In addition to MREa-binding complexes, several other bands were detected, possibly formed by nonspecific protein– DNA complexes, as they were not competed out by any of the competitors used in Fig 2B These results suggest that both the proximal and the consensus sequences of MREa function in sequence-specific interaction with the HepG2 nuclear proteins Characterization and purification of MREa-binding factors To determine the molecular mass of the MREa-binding proteins, we performed South-Western analysis A 70-kDa polypeptide was detected only when a 32P-labelled MREa oligonucleotide was used as a probe (Fig 3A) We also observed another band of 82 kDa when the MREa-protein band from the EMSA was excised from the native gel and resolved by SDS/PAGE (unpublished data) We purified the MREa-binding proteins using the avidin–biotin affinity method [34] HepG2 nuclear proteins were incubated with a biotinylated MREa oligonucleotide and trapped by avidin– agarose beads The proteins were extracted from the avidin– agarose beads by washing with a buffer containing 0.5 M KCl (see Materials and methods for details) Two major polypeptides of 70 and 82 kDa were detected by SDS/ PAGE (Fig 3B, lane 3) Also, there was no difference in the quantity of purified MREa-binding proteins whether the MREa mut1 oligonucleotide was present in the reaction or not (Fig 3C) This result confirmed that these proteins interacted with MREa in sequence-dependent manner, as shown in Fig 2B (lanes 6, 7) To perform N-terminal amino-acid sequencing on the two proteins, the purified protein bands were blotted onto a polyvinylidene difluoride membrane, and the individual proteins were subjected to N-terminal sequencing The N-terminus of the 70-kDa protein was blocked by modification Therefore, the protein was digested with 70% formic acid, which cleaves Asp–Pro (D–P) peptide bonds (located at amino-acid positions 342 and 343 in the 70-kDa protein), and the resulting peptide fragments were sequenced The sequences of the peptides from the 82 kDa and 70-kDa proteins were found to be almost (90%) identical to peptide sequences from the Ku-80 and Ku-70 subunits of the human Ku autoantigen (Fig 4) To confirm that the MREa-binding protein is homologous with the Ku proteins, immuonoblot analyses and supershift EMSAs were performed using mAbs to Ku-70 and Ku-80 The purified 70- and 82-kDa MREa-binding proteins reacted with antibodies to Ku-70 (Clone N3H10) and Ku-80 (Clone 111), respectively (Fig 5A) EMSAs performed with HepG2 nuclear extract in the presence or absence of a mixture of Ku-70/-80 mAbs (Clone 162) revealed that the MREa–protein complex was supershifted when the antibodies were added to the binding reaction (Fig 5B) These results indicate the two MREa-binding proteins are closely related or identical to the Ku proteins The effects of RNAi of the Ku-80 subunit and the truncated Ku-80 mutant on WD gene expression To examine the effects of the Ku protein on the regulation of WD gene expression, we measured the amount of WD protein expressed in HepG2 cells where the Ku-70 or/and Ku-80 subunits were transiently overexpressed We observed no significant difference in the amount of WD protein expressed in wild-type cells and cells that overexpressed the Ku proteins (unpublished data), consistent with previous studies showing that WD proteins are expressed 2156 W J Oh et al (Eur J Biochem 269) Ó FEBS 2002 Fig Characterization and purification of the MREa-binding protein (A) South-Western analysis of the MREa-binding protein Nuclear extracts (30 lg) from Cos cells (lane1) and HepG2 cells (lane 2) were separated by SDS/PAGE (10% polyacrylamide) followed by electrotransfer to a nitrocellulose membrane Blots were incubated with a 32P end-labelled MREa probe as described in Materials and methods and subjected to autoradiography A 70-kDa polypeptide is indicated by solid arrowhead (B) SDS/PAGE analysis of MREa-binding proteins purified by the biotinavidin method (see Materials and methods) Lane 1, proteins eluted by 0.1 M KCl; lane 2, proteins eluted by 0.2 M KCl; lane 3, proteins eluted by 0.5 M KCl; lane 4, avidin–agarose beads (B) After elution by 0.5 M KCl (C) Confirmation of sequence-specific binding of MREa to the two proteins (see Materials and methods) Purified proteins without (lane 1) and with (lane 2) 50 pmol of mutant competitor (MREa mut 1) The positions of molecular size marker (SM) are indicated at the left of each panel The purified protein bands are indicated by solid arrowheads Fig Microsequencing analysis of the MREa-binding proteins (A) The N-terminal sequence of the 82-kDa band was identical to amino-acid positions 6–16 of the Ku-80 protein (B) The 70-kDa band did not yield an N-terminal sequence, probably due to modification Internal sequencing of a peptide generated by formic acid digestion of the 70 kDa protein was identical to an internal sequence of the Ku-70 protein X, Residues not properly identified by the sequencing procedure; query, sequenced amino acids Fig Confirmation that MREa-binding proteins are related to the Ku proteins (A) Immunoblot analysis of proteins eluted from the avidin– agarose beads as described in Fig 3B Both the 70- and 82-kDa proteins interacted with mAb N3H10B (anti-Ku-70) and mAb 111 (antiKu-80) The positions of molecular size markers (SM) are indicated at the left The Ku-70 and Ku-80 proteins are indicated by solid arrowheads (B) Supershift EMSA of the MREa-Ku complexes with mAb 162 (anti-Ku-70/80 dimer) The 32P end-labelled MREa probe used in Fig was incubated with nuclear extracts from HepG2 for and then incubated for an additional 30 either in the absence (lane 1) or presence (lane 2, 3) of varying concentrations of mAb 162 Bands shifted by the antibodies are indicated by solid arrowheads Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur J Biochem 269) 2157 Fig Construction of a Ku-80 IR gene for RNAi assays and the expression pattern of the WD and Ku-80 proteins after transfection (A) Ku-80 cDNA was amplified using two primers that introduced EcoRI and BamHI sites at the ends of the Ku-80 gene The EcoRI site was used to generate the IR, and the BamHI site was used to ligate the Ku-80 IR gene to pcDNA3.1(+) to yield pc80IR Expression of the Ku-80 IR gene was driven by the cytomegalovirus (CMV) promoter (B) Immunoblot analysis showing that the WD and Ku proteins were expressed in transiently transfected HepG2 cells HepG2 cells were transfected with 0.5 lg of pc80IR and 0.5 lg of the pRSVb-gal reporter gene After transfection, cells were incubated for 0.5, 4, 8, 12, 24, 30, 38 h and harvested in 200 lL lysis buffer Whole-cell lysates (30 lg) were resolved by SDS/PAGE (10% polyacrylamide), and immunoblotting was performed with the antibodies described in Fig 5A The control (C) was a whole cell lysate of untransfected HepG2 cells The WD protein is indicated by the open arrowhead, and the Ku-70 and -80 proteins are indicated by closed arrowheads In addition, the asterisks represent reappearance of the WD proteins after 24 h (upper panel) The levels of actin protein are not significantly different at all time points (lower panel) (C) WD mRNA expression as quantified by competitive RT-PCR in HepG2 cells transfected Ku-80 IR Results of each values were expressed as the following ratio: intensity of WD cDNA/intensity of competitor and then divided by intensity of amplified actin cDNA PC, the result of competitive RT-PCR using mRNA isolated from nontransfected HepG2 cells as a positive control constitutively [41,42] To interfere transiently with expression of the Ku-80 protein in vivo, we performed RNAi assays (Fig 6A) Double-stranded RNAi is an effective method for disrupting expression of specific genes in higher organisms, especially Caenohabditis elegans [43–45] Recently, it was reported that RNAi caused by in vivo expression of IR versions of specific genes has advantages over RNAi by directly introduced double-stranded RNA [36] We constructed a Ku-80 IR vector that was able to synthesize hairpin double-stranded RNA and transfected the vector into HepG2 cells Immunoblot analysis showed that expression levels of the Ku-80 and WD proteins were significantly reduced from 0.5 to 12 h after transfection (Fig 6B, lanes 2, 3, 4, 5), and then recovered to wild-type levels after 24 h as cellular division proceeded (Fig 6B, lanes 6, 7, 8) Ku-80 gene expression recovered before WD gene expression, suggesting that the Ku-80 protein is a constitutive transcriptional regulator of WD gene expression On the other hand, there was no significant change in the concentration of Ku-70 protein, although it seemed that there was a slight reduction from 0.5 to 12 h after transfection, especially at 0.5 h In addition, the effect of Ku-80 on the WD gene expression was examined by competitive RT-PCR In consistency with the immunoblot analysis result, the mRNA level of WD gene was signifi- cantly diminished from 0.5 to 12 h after transfection and then completely restored to wild-type level by 40 h after transfection For further investigation on the role of Ku-80 protein in the basal transcription of WD disease gene, a truncated Ku-80 (DKu-80) cDNA, in which an internal region harbouring a part of the dimerization domain was deleted, was constructed as shown in Fig The DKu-80 cDNA and/or Ku-70 were cotransfected with pPWD-Luc into HepG2 cells There was no significant change in WD promoter activity in Ku-70-transfected cells In contrast, the promoter activity was remarkably reduced to a 50% of normal activity in DKu-80-transfected and Ku70/DKu-80-transfected cells (Fig 7) It is implied that the lack of the dimerization domain in the DKu-80 cDNA decreased the WD promoter activity in a dominant-negative manner These results suggest that Ku-80 protein is an important factor in the regulation of WD promoter activity Comparison of WD promoter activity in Ku-deficient Xrs-5 cells to the activity in CHO cells To verify that Ku-80 is an essential regulatory factor in WD gene expression, pPWD-Luc was transfected into both a Ku-80 normal-cell line, CHO-K1, and a Ku-80-mutant cell Ó FEBS 2002 2158 W J Oh et al (Eur J Biochem 269) (Fig 8A) To examine whether the WD promoter activity was recovered after expression of Ku-80 protein in Xrs5 and CHO cells, the Ku-80 cDNA and the pPWD-Luc were cotransfected After 48 h, the promoter activity in Xrs5 cells transfected with Ku-80 was recovered to about 27% higher than that observed in nontransfected Xrs-5 cells In contrast, there were no significant changes after transfection of the Ku-80 in CHO cells (Fig 8B) These results clearly showed that the Ku-80 protein plays a key role in constitutive expression of the WD gene DISCUSSION Fig Construction of DKu-80 protein and pattern of promoter activity in HepG2 cells after transfection of DKu-80 The full-length wild-type Ku-80 protein and the truncated Ku-80 (DKu-80) are represented schematically, and the location of a deleted region is indicated The dimerization domain is indicated by solid box, and the region involved in DNA binding activity is by diagonally cross-hatched box (upper panel) Changes of WD promoter activity were assayed in 48 h after cotransfection of the pGL, Ku-70, DKu-80, and Ku-70/DKu-80 cDNA into HepG2 cells (lower panel) PC (positive control), promoter activity in HepG2 cells transfected with pBluescript SK DNA containing the same amount of Ku cDNA line, Xrs5 The activity of the WD gene promoter was reduced by 50% in Xrs5 cells 24 h after transfection, compared to WD gene promoter activity in CHO-K1 cells It has been proposed that the WD protein acts as a copperspecific pump that mediates the export of copper from the cytosol, similar to the P-type ATPase [14] On the basis of the fact that MREs mediate the transcriptional response of the MT gene to heavy metals [10,12], the presence of MREs in the promoter of the WD gene suggests that MREs and their cognate binding proteins function in the regulation of WD gene expression Because the T1 and C3 nucleotides within the MRE consensus heptamers are major determinants in the sequence-specific binding of zinc activated protein (ZAP) [39], and because single point mutations may not obliterate MRE function [46], we mutated the first three nucleotides within the core sequence of MREs and tested the effect of these mutations on WD gene transcription We found that of the four MREs, MREa was the most significant element in the transcriptional regulation of WD gene Previous studies report that MREa is also the most crucial MRE for transcription of the MT gene and that there is a relationship between the distance from each MRE to the TATA box and their influence on promoter activity of the MT gene, the proximal element MREa exhibiting the strongest effect In addition, it is known that function of distal MREs is dependent on MREa in proximal promoter region [46] In contrast with the above findings, MREa, the Fig Difference of WD promoter activity between Xrs-5 and CHO cells (A) A decrease in WD promoter activity in Xrs5 cells CHO-K1 and Xrs5 cells were transiently transfected with a WD promoter-luciferase reporter gene (pPWD) [38] Twenty-four hours after transfection, the cells were assayed for luciferase activity (B) Restoration of promoter activity after transfection of Ku-80 gene in Xrs5 cells, but not in CHO cells Ku-80 cDNA and pPWD were transiently expressed in Xrs5 (upper panel) and CHO cells (lower panel) Data represent means ± SD of three independent transfection assays for each construct pGL indicates the luciferase activity in cells that had been transfected with the pGL as a negative control NC, Negative control transfected with the same amount of pBluescript SK instead of the Ku-80 cDNA Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur J Biochem 269) 2159 most distal cis-element from transcription start, played the most important role in WD gene promoter activity Further investigation is needed to elucidate the exact reason why the most distal MREa from transcription start site is important for activity, especially in a TATA-less promoter such as the WD gene Several proteins that bind specifically to MREs, MTF-1, ZAP, and ZRF (zinc regulatory factor), have been identified in mouse and human cells [38,40,47] EMSAs performed with unlabelled MREa or the Sp1 binding site as competitors revealed that binding of the Ku protein to the MREa of the WD gene is sequence specific Excess Sp1 oligonucleotide reduced slightly the amount of the MREa–Ku protein complex (Fig 2B, lane 7), possibly due to the ability of the Ku protein to bind DNA in a sequence-independent manner [48,49] In the MREa–protein complex detected by EMSA (Fig 2A), both the 82- and 70-kDa proteins were visualized by silver staining (unpublished data) In contrast, only the 70 kDa subunit was observed by South-Western blot analysis (Fig 3A) This is consistent with previous studies showing that the Ku-70 protein interacts with DNA without requiring Ku-80 [50] Using the avidin–biotin system, we purified two MREabinding proteins from HepG2 cell nuclear extracts It was clearly shown that the two proteins bound to MREa in a sequence-specific manner, by using competitors during the purification process (Fig 3C) as well as EMSA (Fig 2B) to exclude the possibility of sequence-independent binding of Ku protein to double-stranded DNA ends The purified proteins were of the same molecular sizes as those shown by silver staining of an SDS/polyacrylamide gel after EMSAs N-terminal sequencing of the 70-kDa protein was inhibited by modification, consistent with previous reports showing that the amino terminus of the human Ku-70 protein is blocked and therefore inaccessible to Edman degradation [51,52] EMSA supershift assays (Fig 5B) and immunoblot analyses (Fig 5A) using mAbs specific to human Ku-70 and Ku-80 revealed that the MREa-binding proteins described herein and the Ku proteins are immunologically related The Ku protein complex is known to contain equimolar amounts of the 70- and 80-kDa polypeptides, which form heterodimers [50,53,54] Various transcription factors such as CHBF, CTCBF, TREF, and PSE1, each of which recognize specific promoters elements, are known to be identical or related to the Ku protein [22,24,25,55] The Ku proteins have been shown to inhibit the expression of stress-responsive proteins For example, overexpression of Ku-70 and Ku-80 or Ku-70 alone specifically inhibits HSP70 expression [56], whereas overexpression of Ku-80 alone suppresses only MT-I expression [29] We examined the effect of the Ku proteins on the transcriptional regulation of the WD gene First, we overexpressed Ku protein in HepG2 cells, and the results indicated that overexpression of Ku protein did not alter the concentration of WD protein in the cell (unpublished data) We also inhibited expression of the Ku-80 protein using the RNAi IR gene method In vivo RNAi with an IR gene successfully inactivates a specific gene in established cell lines as well as in nematodes [44,45] We found that transfection of a Ku-80 IR gene into cells inhibited the expression of the Ku-80 protein and the WD protein (Fig 6B and C) In addition, the concurrent decrease in the level of Ku-70 from 0.5 to 12 h after transfection of Ku-80 IR gene is consistent with previous report that the stability of Ku-70 is compromised by the absence of Ku-80 [57] This is the first report to show that Ku-80 expression is suppressed effectively through cell-line transfection In several studies, it has been reported that cells expressing truncated Ku-80 protein exhibit increased sensitivity to radiation and diminished DNA repair [58,59], although there are still some arguments in the exact locations of domains in Ku-80 [60–63] Based on the facts that amino acids 371–510 of Ku-80 mediate dimerization with Ku-70 protein, and that amino acids 179–510 of Ku-80 are involved in Ku-80-dependent DNA binding [64], we constructed a Ku-80 deletion mutant (DKu-80) (Fig 7) Expression of the DKu-80 cDNA in HepG2 cells resulted in decreased WD promoter activity, suggesting that DKu-80 protein inhibits the formation of endogenous Ku protein complex and its binding to the WD promoter by competing with native Ku-80 for Ku-70 protein We then verified that Ku-80 is required for transcriptional regulation of the WD gene in the Ku-80-deficient Xrs5 cell line (Fig 8) These findings suggest that Ku-80 binds to MREa and may be an essential component of the transcription machinery of the WD gene However, it is not known if or how Ku-70 functions in the regulation of WD gene expression The reduction of luciferase activity in Xrs5 compared to CHO-K1 was smaller than the reduction of luciferase activity caused by the MREa mutation as shown in Fig 1B It is possible that a low level of Ku-80 transcript present in the Xrs5 cell line [65] recovers the luciferase activity of the WD promoter to some degree, while a mutation within MREa is able to confer a completely negative effect on WD gene promoter activity It remains to be elucidated why WD promoter activity in Ku-80-expressing Xrs5 cells was not restored completely to the activity observed in CHO cells It could be that human Ku-80, whose amino acid sequence is 21% diverged from Chinese hamster Ku-80, rescues less efficiently in hamster species Also, it is consistent with previous report that CHO mutant cells transfected with Syrian hamster Ku-80 exhibit reduced X-ray resistance and V(D)J recombination compared with wild-type CHO cells [57] To rule out this possibility, we transfected the Chinese hamster Ku-80 clone into Xrs-5 cells However, there was no difference in the recovery of the promoter activity between human and Chinese hamster Ku-80 proteins The exact reason for this lack of difference is not clearly understood and further characterization is required The Ku proteins have been shown to be components of the mammalian DNA-depedent protein kinase, which regulates other DNA-binding factors such as Sp1 and p53 through phosphorylation [66], and directly modulates RNA polymerase I-mediated transcription [25,54] It will be of interest to determine how this kinase influences WD gene expression and to decipher the mechanism by which it interacts functionally with constitutive transcriptional factors like Sp1 and Ku It will also be of interest to examine whether additional proteins bind to the other three MREs in the WD promoter, and if so, to determine the precise mechanisms by which such proteins modulate WD gene expression 2160 W J Oh et al (Eur J Biochem 269) ACKNOWLEDGEMENTS We thank S.-H Yoo for providing Ku-70 and -80 cDNAs and K.-D Park for critically reading the manuscript We also thank our colleagues in Dr Yoo’s laboratory for useful discussions This work was supported by the Molecular Medicine Research Group Program grant (98-MM01-01-A-01) from the Ministry of Science and Technology through the BioMedical Research Center at KAIST REFERENCES Bull, P.C., Thomas, G.R., Rommens, J.M., Forbes, J.R & Cox, D.W (1993) The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene Nature Genet 5, 327–337 Kaler, S.G., Gallo, L.K., Proud, V.K., Percy, A.K., Mark, Y., Segal, N.A., Goldstein, D.S., Holmes, C.S & Gahl, W.A (1994) Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at 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Steingrimsdottir, H., Gell, D., Blunt, T., Jackson, S.P., Lehmann, A.R & Jeggo, P.A (1997) Molecular and biochemical characterization of xrs mutants defective in Ku80 Mol Cell Biol 17, 1264–1273 Gottlieb, T.M & Jackson, S.P (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen Cell 72, 131–142  ... 5B) and immunoblot analyses (Fig 5A) using mAbs specific to human Ku- 70 and Ku- 80 revealed that the MREa-binding proteins described herein and the Ku proteins are immunologically related The Ku. .. transcriptional regulatory activity and that a variety of nuclear transcription factors interact specifically with MREa [11,33,38,39] These findings suggest that transcriptional activator proteins. .. methods and subjected to autoradiography A 70-kDa polypeptide is indicated by solid arrowhead (B) SDS/PAGE analysis of MREa-binding proteins purified by the biotinavidin method (see Materials and