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

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 Oh
1
, Eun Kyung Kim
1
, Jung Ho Ko
1
, Seung Hee Yoo
1
, Si Houn Hahn
2
and Ook-Joon Yoo
1
1
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Taejon Korea;
2
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 neur-
onal 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. Electro-
phoretic 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 rela-
ted 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 debilita-
ting 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-trans-

porting 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 mainten-
ance [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 Kusong-
dong, Yusong-gu, Taejon 305-701, Korea. Fax: +82 42 869 8160,
Tel.: +82 42 869 2626, E-mail:
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 2 November 2001, revised 5 February 2002,
accepted 4 March 2002)
Eur. J. Biochem. 269, 2151–2161 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02865.x
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 transcrip-
tion.
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 1 ·
antibiotic–antimycotic solution (Life Technologies, Inc.).
The hamster ovary cell line CHO-K1 and the Ku-80-
deficient cell line Xrs-5 were obtained from the American
Type Culture Collection and maintained Ham’s F12K
medium and alpha minimum essential medium, respect-
ively, that containing 10% fetal bovine serum and 1 ·
antibiotic–antimycotic solution.
Human WD promoter-reporter gene constructs
A 1.6-kb construction of the WD promoter, named pPWD-
Luc, was described previously [7]. Trinucleotide mutations in
eachofthefourMREsoftheWDgenepromoterwere
constructed using the PCR-based quick change site-directed
mutagenesis method (Stratagene). The following oligonucle-
otides were used in the MRE mutagenesis, with the
trinucleotide mutations indicated in lowercase letters
(underlined bases denote the MRE core sequence): MREa
mut, 5¢-GGGCGCC
aatGCCCCCGTTCC-3¢;MREemut,
5¢-GGCCATTGGCTGGCCTT
aatGCACAGCGGATCG
ATTTTC-3¢;MREcmut,5¢-CCAGTACAGTGTCGG
AGCattCCAGCGCGAGGTGGCCG-3¢;MREdmut,
5¢-CGGGAGGACGGCG
GCGCattACTTTGAATCAT
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 1 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. Chemilumi-
nescence 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].
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 · 10
6
cells per
dish) were washed with ice-cold NaCl/P
i
and scraped off the
dish into 5 mL ice-cold NaCl/P
i
. Cell pellets were resus-

pended in 5 vol. hypotonic buffer (10 m
M
Hepes pH 7.9,
1.5 m
M
MgCl
2
,10m
M
KCl, 0.2 m
M
phenylmethanesulfo-
nyl fluoride, 0.5 m
M
dithiothreitol), and the cell suspensions
were homogenized with a Dounce homogenizer (20 strokes,
type B pestle). Nuclear proteins were suspended in 1 vol.
low salt buffer (20 m
M
Hepes pH 7.9, 25% glycerol, 1.5 m
M
MgCl
2
,20m
M
KCl, 0.2 m
M
EDTA, 0.2 m
M
phenyl-

methanesulfonyl fluoride, 0.5 m
M
dithiothreitol), followed
by the addition of 4
M
KCl to a final concentration of 0.3
M
.
Nuclear suspensions were stirred for 30 min on ice and
dialysed against dialysis buffer (20 m
M
Hepes pH 7.9, 20%
glycerol, 100 m
M
KCl, 0.2 m
M
EDTA, 0.2 m
M
phenyl-
methanesulfonyl fluoride, 0.5 m
M
dithiothreitol) for 4 h at
4 °C. Nuclear extracts were frozen in aliquots at )70 °C.
EMSAs
The double-stranded MREa probes were end-labelled with
[c-
32
ATP] (Amersham) and T4 polynucleotide kinase.
HepG2 cell nuclear proteins (10 lg) were incubated in
reaction buffer containing 17 m

M
Hepes pH 7.9, 32 m
M
Tris/HCl pH 7.8, 13% glycerol, 25 m
M
KCl, 0.8 m
M
dithiothreitol, 4 lg poly(dI-dC), and 2–4 fmol
32
P
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 min at 25 °C.
For competition experiments, a molar excess of unlabelled
MRE oligonucleotides were added to the binding reaction
and incubated for 15 min 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
32
P-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 1 lg; Clone 162, Neomarkers Co.) for 5 min, followed by
incubation with
32
P-labelled MRE oligonucleotides for

30 min at 25 °C.
The oligonucleotide sequences used as the
32
P-labelled
probes and competitors were: MREa, 5¢-GGGCGCC
TGCGCCCCCGTTCC-3¢ ()441 to )421); MREa mut1,
5¢-GGGCGCC
AATGCCCCCGTTCC-3¢;MREamut2:
5¢-GGGCGCC
TGCGCCCTTATTCC-3¢; Sp1 : 5¢-ATT
CGATC
GGGGCGGGGCGAGC-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
2152 W. J. Oh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 m
M
Tris/HCl pH 7.5, 100 m
M
NaCl, 1 m
M
EDTA, and 1 m
M
dithiothreitol) for 1 h at room tempera-
ture and then washed twice with TNE-100 buffer (10 m
M
Tris/HCl pH 7.5, 100 m
M
NaCl, 1 m
M
EDTA, 1 m
M
dithiothreitol). The filter-bound proteins were then dena-
tured by incubation in denaturing buffer (7
M
guanidine/
HCl, 50 m
M
Tris/HCl pH 8.0, 50 m
M

dithiothreitol, 2 m
M
EDTA, and 0.25% nonfat milk) for 1 h at room temper-
ature and renatured by incubation in 50 m
M
Tris/HCl
pH 7.5, 100 m
M
NaCl, 2 m
M
dithiothreitol, 2 m
M
EDTA,
0.1% NP-40, 0.25% nonfat milk for 20 h at 4 °C. To
measure the MREa-binding activity of the filter-bound
proteins, the filter was preincubated with TNE-100 con-
taining 10 lgÆmL
)1
poly(dI-dC) for 1 h at room tempera-
ture, and then
32
P end-labelled, double-stranded MREa
oligonucleotide was added (2 · 10
6
c.p.m.ÆmL
)1
)tothe
incubation mixture. After 1 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 oligo-
nucleotide with a biotin head at the 5¢-end. HepG2 nuclear
extracts were incubated with avidin–agarose beads (Sigma)
at 4 °C for 30 min 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 m
M
Hepes/KOH
pH 7.9, 10% glycerol, 40 m
M
KCl, 2 m
M
MgCl
2
,10m
M
dithiothreitol, 125 lgÆmL
)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 min at
25 °C. After elinimating by centrifugation denatured pro-
teins generated during the binding reaction, the mixture was

combined with 20 lL avidin–agarose beads and incubated
at room temperature for 30 min with shaking. The beads
were washed successively with 0.5 mL 0.1
M
KCl buffer III
(20 m
M
Hepes/KOH pH 7.9, 1 m
M
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 min at
4 °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 min at 37 °C, the proteins were
separated by SDS/PAGE and electrotransferred onto
poly(vinylidene difluoride) membrane (Sequi-blot; Bio-
rad). 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 fluores-
cent DNA sequencing (Perkin-Elmer 377). We also con-
structed 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 °Cfor5minand37°C for 60 min followed by 5 min
denaturation at 95 °C in a total volume of 50 lL of reaction
mixture using the RT-PCR kit (Stratagene). PCR amplifi-
cation 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 3 min followed by 18 cycles of
95 °C for 45 s, 55 °C for 45 s, 72 °Cfor45s,and72°Cfor
Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur. J. Biochem. 269) 2153
5 min (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 frag-
ments 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 1.
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. 1. 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,
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 transcrip-
tional 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 MREa-
binding proteins are present in HepG2 cell nuclear extracts.
For the EMSAs we used a
32
P-labelled synthetic oligonu-

Table 1. Oligonucleotide primers and conditions used for competitive RT-PCR analysis.
Target
gene
Forward primer
Reverse primer
PCR products (base pairs) Annealing
temperature
(°C)
Target Competitor
Wilson gene 5¢-TGTTAAGTTTGACCCGGAAATTATC 911 713 55
5¢-CCGGTCAGCCAGCTGCTG
a-actin 5¢-TGATGGTGGGCATGGGTCAG 791 55
5¢-TACATGGTGGTGCCGCCAGA
Fig. 1. 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.
2154 W. J. Oh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
cleotide that contained a copy of MREa (Fig. 2A). We also
performed competition experiments with various oligonu-
cleotide 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
32
P-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 modi-
fication. 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 se-
quenced. 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 anti-
bodies 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 overex-
pressed the Ku proteins (unpublished data), consistent with
previous studies showing that WD proteins are expressed
Fig. 2. Detection of nuclear proteins that bind
specifically to MREa. (A)
32
P-labelled double-
stranded 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 spe-
cific MREa–protein complex and is competed
away with unlabelled MREa. The open
arrowhead denotes a nonspecific band.
Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur. J. Biochem. 269) 2155
Fig. 3. 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
32
P 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 biotin-
avidin 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. 4. 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 proce-

dure; query, sequenced amino acids.
Fig. 5. 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 pro-
teins interacted with mAb N3H10B (anti-Ku-70) and mAb 111 (anti-
Ku-80). The positions of molecular size markers (SM) are indicated at
the left. The Ku-70 and Ku-80 proteins are indicated by solid arrow-
heads. (B) Supershift EMSA of the MREa-Ku complexes with mAb
162 (anti-Ku-70/80 dimer). The
32
P end-labelled MREa probe used in
Fig. 2 was incubated with nuclear extracts from HepG2 for 5 min and
then incubated for an additional 30 min 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.
2156 W. J. Oh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
constitutively [41,42]. To interfere transiently with expres-
sion of the Ku-80 protein in vivo,weperformedRNAi
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 expres-
sion. 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. 7. 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
Fig. 6. 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.
Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur. J. Biochem. 269) 2157
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
(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
It has been proposed that the WD protein acts as a copper-
specific 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
theMTgenetoheavymetals[10,12],thepresenceofMREs
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 determi-
nants 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. 7. 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 con-
taining the same amount of Ku cDNA.
Fig. 8. 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 inde-
pendent 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.
2158 W. J. Oh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 compet-
itors 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 MREa-
binding 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 over-
expression of Ku-80 alone suppresses only MT-I expres-
sion [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 con-
structed 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 com-
pared 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 fac-
tors 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.
Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur. J. Biochem. 269) 2159
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-MM-
01-01-A-01) from the Ministry of Science and Technology through the
BioMedical Research Center at KAIST.
REFERENCES
1. Bull, P.C., Thomas, G.R., Rommens, J.M., Forbes, J.R. & Cox,
D.W. (1993) The Wilson disease gene is a putative copper trans-

porting P-type ATPase similar to the Menkes gene. Nature Genet.
5, 327–337.
2. 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 the MNK locus. Nat. Genet. 8,195–
202.
3. Petrukhin, K., Lutsenko, S., Chernov, I., Ross, B.M., Kaplan,
J.H. & Gilliam, T.C. (1994) Characterization of the Wilson disease
gene encoding a P-type copper transporting ATPase: genomic
organization, alternative splicing & structure/function predictions.
Hum. Mol. Genet. 3, 1647–1656.
4. Vulpe, C., Levinson, B., Whitney, S., Packman, S. & Gitschier, J.
(1993) Isolation of a candidate gene for Menkes disease and evi-
dence that it encodes a copper-transporting ATPase. Nat. Genet. 3,
7–13.
5. Yamaguchi, Y., Heiny, M.E. & Gitlin, J.D. (1993) Isolation and
characterization of a human liver cDNA as a candidate gene for
Wilson disease. Biochem. Biophys. Res. Commun. 197, 271–277.
6. Lutsenko, S. & Kaplan, J.H. (1995) Organization of P-type
ATPases: significance of structural diversity. Biochemistry 34,
15607–15613.
7. Oh, W.J., Kim, E.K., Park, K.D., Hahn, S.H. & Yoo, O.J. (1999)
Cloning and characterization of the promoter region of the Wilson
disease gene. Biochem. Biophys. Res. Comm. 259, 206–211.
8. Stuart, G.W., Searle, P.F. & Palmiter, R.D. (1985) Identifica-
tion of multiple metal regulatory elements in mouse metallothio-
nein-I promoter by assaying synthetic sequences. Nature 317,
828–831.
9. Stuart, G.W., Searle, P.F., Chen, H.Y., Brinster, R.L. & Palmi-

ter, R.D. (1984) A 12-base-pair DNA motif that is repeated
several times in metallothionein gene promoters confers metal
regulation to a heterologous gene. Proc. Natl Acad. Sci. USA 81,
7318–7322.
10. Carter, A.D., Felber, B.K., Walling, M.J., Jubier, M.F., Schmidt,
C.J. & Hamer, D.H. (1984) Duplicated heavy metal control
sequences of the mouse metallothionein-I gene. Proc. Natl Acad.
Sci. USA 81, 7392–7396.
11. Czupryn, M., Brown, W.E. & Vallee, B.L. (1992) Zinc rapidly
induces a metal response element-binding factor. Proc. Natl Acad.
Sci. USA 89, 10395–10399.
12. Karin, M., Haslinger, A., Holtgreve, H., Richards, R.I., Krau-
ter, P., Westphal, H.M. & Beato, M. (1984) Characterization of
DNA sequences through which cadmium and glucocorticoid
hormones induce human metallothionein-IIA gene. Nature 308,
513–519.
13. Dalton, T.P., Li, Q., Bittel, D., Liang, L. & Andrews, G.K. (1996)
Oxidative stress activates metal-responsive transcription factor-1
binding activity. Occupancy in vivo of metal response elements in
the metallothionein-I gene promoter. J. Biol. Chem. 271, 26233–
26241.
14. Scriver, C.R., Beaudet, A.L., Sly, W.S. & Valle, D. (1995) The
Metabolic and Molecular Bases of Inherited Disease,Vol.II,
pp. 2211–2236. McGraw-Hill Inc., New York.
15. Imbert, J., Culotta, V., Furst, P., Gedamu, L. & Hamer, D. (1990)
Advances in Inorganic Biochemistry, Vol. 8, pp. 138–164. Elsevier
Publishers, New York.
16. Kagi, J.H. (1991) Overview of metallothionein. Methods Enzymol.
205, 613–626.
17. Vallee, B.L. (1979) Metallothionein: historical review and per-

spectives. Experientia 34(Suppl), 19–39.
18. Dvir,A.,Peterson,S.R.,Knuth,M.W.,Lu,H.&Dynan,W.S.
(1992) Ku autoantigen is the regulatory component of a template-
associated protein kinase that phosphorylates RNA polymerase II.
Proc. Natl Acad. Sci. USA 89, 11920–11924.
19. Boulton, S.J. & Jackson, S.P. (1998) Components of the
Ku-dependent non-homologous end-joining pathway are involved
in telomeric length maintenance and telomeric silencing. EMBO J.
17, 1819–1828.
20. Chu, G. (1997) Double strand break repair. J. Biol. Chem. 272,
24097–24100.
21. Weaver, D.T. (1996) Regulation and repair of double-strand
DNA breaks. Crit. Rev. Eukaryot. Gene Expr. 6, 345–375.
22. Genersch, E., Eckerskorn, C., Lottspeich, F., Herzog, C., Kuhn,
K. & Poschl, E. (1995) Purification of the sequence-specific tran-
scription factor CTCBF, involved in the control of human
collagenIVgenes:subunitswithhomologytoKuantigen.EMBO
J. 14, 791–800.
23. Giffin, W., Torrance, H., Rodda, D.J., Prefontaine, G.G.,
Pope, L. & Hache, R.J.G. (1996) Sequence-specific DNA
binding by Ku autoantigen and its effects on transcription. Nature
380, 265–268.
24. Kim, D., Ouyang, H., Yang, S.H., Nussenzweig, A., Burgman, P.
& Li, G.C. (1995) A constitutive heat shock element-binding
factor is immunologically identical to the Ku autoantigen. J. Biol.
Chem. 270, 15277–15284.
25. Knuth, M.W., Gunderson, S.I., Thompson, N.E., Strasheim, L.A.
& Burgess, R.R. (1990) Purification and characterization of
proximal sequence element-binding protein 1, a transcription
activating protein related to Ku and TREF that binds the prox-

imal sequence element of the human U1 promoter. J. Biol. Chem.
265, 17911–17920.
26. Li, G.C., Yang, S H., Kim, D., Nussenzweig, A., Ouyang, H.,
Wei, J., Burgman, P. & Li, L. (1995) Suppression of heat-induced
hsp70 expression by the 70-kDa subunit of the human Ku auto-
antigen. Proc. Natl Acad. Sci. USA 92, 4512–4516.
27. Messier, H., Fuller, T., Mangal, S., Brickner, H., Igarashi, S.,
Gaikwad, J., Fotedar, R. & Fotedar, A. (1993) p70 lupus auto-
antigen binds the enhancer of the T-cell receptor beta-chain gene.
Proc. Natl Acad. Sci. USA 90, 2685–2689.
28. Roberts, M.R., Han, Y., Fienberg, A., Humihan, L. & Ruddle,
F.H. (1994) A DNA-binding activity, TRAC, specific for the TRA
element of the transferrin receptor gene copurifies with the Ku
autoantigen. Proc. Natl Acad. Sci. USA 91, 6354–6358.
29. Ghoshal, K., Li, Z. & Jacob, S.T. (1998) Overexpression of the
large subunit of the protein Ku suppresses metallothionein-I
induction by heavy metals. Proc.NatlAcad.Sci.USA95, 10390–
10395.
30. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.
31. Lin, S., Yang, S. & Hopkins, N. (1994) LacZ expression in
germline transgenic zebrafish can be detected in living embryos.
Dev Biol. 161, 77–83.
32. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. (1983) Accurate
transcription initiation by RNA polymerase II in a soluble extract
from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–
1489.
33. Dikstein, R., Heffetz, D., Ben-Neriah, Y. & Shaul, Y. (1992) c-abl
has a sequence-specific enhancer binding activity. Cell 69,751–

757.
2160 W. J. Oh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
34. Otsuka, F., Iwamatsu, A., Suzuki, K., Ohsawa, M., Hamer, D.H.
& Koizumi, S. (1994) Purification and characterization of a pro-
tein that binds to metal responsive elements of the human
metallothionein IIA gene. J. Biol. Chem. 269, 23700–23707.
35. Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W. &
Wingfield, P.T. (1995) Current Protocol. in Protein Science,Vol.1,
p. 11.4.4. John Wiley and Sons Inc., New York.
36. Tavernarakis, N., Wang, S.L., Dorovkov, M., Ryazanov, A. &
Driscoll, M. (2000) Heritable and inducible genetic interference by
double-stranded RNA encoded by transgenes. Nat. Genet. 24,
180–183.
37. Samson, S.L. & Gedamu, L. (1995) Metal-responsive elements of
the rainbow trout metallothionein-B gene function for basal and
metal-induced activity. J. Biol. Chem. 270, 6864–6871.
38. Bittel, D., Dalton, T., Samson, S.L., Gedamu, L. & Andrews,
G.K. (1998) The DNA binding activity of metal response element-
binding transcription factor-1 is activated in vivo and in vitro by
zinc, but not by other transition metals. J. Biol. Chem. 273, 7127–
7133.
39. Searle, P.F. (1990) Zinc dependent binding of a liver nuclear factor
to metal response element MRE-a of the mouse metallothionein-I
gene and variant sequences. Nucleic Acids Res. 18, 4683–4690.
40. Westin, G. & Schaffner, W. (1988) A zinc-responsive factor
interacts with a metal-regulated enhancer element (MRE) of the
mouse metallothionein-I gene. EMBO J. 7, 3763–3770.
41. Hung, I.H., Suzuki, M., Yamaguchi, Y., Yuan, D.S., Klausner,
R.D. & Gitlin, J.D. (1997) Biochemical characterization of the
Wilson disease protein and functional expression in the yeast

Saccharomyces cerevisiae. J. Biol. Chem. 272, 21461–21466.
42. Schaefer, M., Hopkins, R.G., Failla, M.L. & Gitlin, J.D. (1999)
Hepatocyte-specific localization and copper-dependent trafficking
of the Wilson’s disease protein in the liver. Am.J.Physiol.276,
G639–G646.
43. Montgomery, M.K., Xu, S. & Fire, A. (1998) RNA as a target of
double-stranded RNA-mediated genetic interference in Cae-
norhabditis elegans. Proc.NatlAcad.Sci.USA95, 15502–15507.
44. Tabara, H., Grishok, A. & Mello, C.C. (1998) RNAi in C. elegans:
soaking in the genome sequence. Science 282, 430–431.
45. Timmons,L.&Fire,A.(1998)Specificinterferencebyingested
dsRNA. Nature 395, 854.
46. Samson, S.L., Paramchuk, W.J., Shworak, N.W. & Gedamu, L.
(1995) Functional analyses of the human metallothionein-IG gene.
In vitro and in vivo studies. J. Biol. Chem. 270, 25194–25199.
47. Koizumi, S., Yamada, H., Suzuki, K. & Otsuka, F. (1992) Zinc-
specific activation of a HeLa cell nuclear protein which interacts
with a metal responsive element of the human metallothionein-IIA
gene. Eur. J. Biochem. 210, 555–560.
48. Falzon, M., Fewell, J.W. & Kuff, E.L. (1993) EBP-80, a tran-
scription factor closely resembling the human autoantigen Ku,
recognizes single- to double-strand transitions in DNA. J. Biol.
Chem. 268, 10546–10552.
49. Yaneva, M., Kowalewski, T. & Lieber, M.R. (1997) Interaction of
DNA-dependent protein kinase with DNA and with Ku: bio-
chemical and atomic-force microscopy studies. EMBO J. 16,
5098–5112.
50. Mimori, T. & Hardin, J.A. (1986) Mechanism of interaction
betweenKuproteinandDNA.J. Biol. Chem. 261, 10375–10379.
51. Lees-Miller, S.P., Chen, Y.R. & Anderson, C.W. (1990) Human

cells contain a DNA-activated protein kinase that phosphorylates
simian virus 40 T antigen, mouse p53 & the human Ku auto-
antigen. Mol. Cell. Biol. 10, 6472–6481.
52. Reeves, W.H. & Sthoeger, Z.M. (1989) Molecular cloning of
cDNA encoding the p70 (Ku) lupus autoantigen. J. Biol. Chem.
264, 5047–5052.
53. Hoff, C.M. & Jacob, S.T. (1993) Characterization of the factor
E1BF from a rat hepatoma that modulates ribosomal RNA gene
transcriptionanditsrelationshiptothehumanKuautoantigen.
Biochem. Biophys. Res. Comm. 190, 747–753.
54. Kuhn, A., Stefanovsky, V. & Grummt, I. (1993) The nucleolar
transcription activator UBF relieves Ku antigen-mediated
repression of mouse ribosomal gene transcription. Nucleic Acids
Res. 21, 2057–2063.
55. Yaneva, M. & Busch, H. (1986) A 10S particle released from
deoxyribonuclease-sensitive regions of HeLa cell nuclei contains
the 86-kilodalton-70-kilodalton protein complex. Biochemistry 25,
5057–5063.
56. Yang, S H., Nussenzweig, A., Li, L., Kim, D., Ouyang, H.,
Burgman, P. & Li, G.C. (1996) Modulation of thermal induction
of hsp70 expression by Ku autoantigen or its individual subunits.
Mol. Cell. Biol. 16, 3799–3806.
57. Errami, A., Smider, V., Rathmell, W.K., He, D.M., Hendrickson,
E.A., Zdzienicka, M.Z. & Chu, G. (1996) Ku86 defines the genetic
defect and restores X-ray resistance and V (D) J recombination to
complementation group 5 hamster cell mutants. Mol. Cell. Biol.
16, 1519–1526.
58. Marangoni, E., Foray, N., O’Driscoll, M., Douc-Rasy, S.,
Bernier, J., Bourhis, J. & Jeggo, P. (2000) A Ku80 fragment with
dominant negative activity imparts a radiosensitive phenotype to

CHO-K1. Cell 28, 4778–4782.
59. Tai, Y.T., Teoh, G., Lin, B., David, F.E., Chauhan, D., Treon,
S.P.,Raje,N.,Hideshima,T.,Shima,Y.,Podar,K.&Anderson,
K.C. (2000) Ku86 variant expression and function in multiple
myeloma cells is associated with increased sensitivity to DNA
damage. J. Immunol. 165, 6347–6355.
60. Wu, X. & Lieber, M.R. (1996) Protein-protein and protein–DNA
interaction regions within the DNA end-binding protein Ku70-
Ku86. Mol. Cell. Biol. 16, 5186–5193.
61. Cary, R.B., Chen, F., Shen, Z. & Chen, D.J. (1998) A central
region of Ku80 mediates interaction with Ku70 in vivo. Nucleic
Acids Res. 26, 974–979.
62. Osipovich, O., Durum, S.K. & Muegge, K. (1997) Defining the
minimal domain of Ku80 for interaction with Ku70. J. Biol. Chem.
272, 27259–27265.
63. Singleton, B.K., Torres-Arzayus, M.I., Rottinghaus, S.T., Tacci-
oli, G.E. & Jeggo, P.A. (1999) The C terminus of Ku80 activates
the DNA-dependent protein kinase catalytic subunit. Mol. Cell.
Biol. 19, 3267–3277.
64. Wang, J., Dong, X. & Reeves, W.H. (1998) A model for
Ku heterodimer assembly and interaction with DNA. Implica-
tions for the function of Ku antigen. J. Biol. Chem. 273, 31068–
31074.
65. Singleton, B.K., Priestley, A., 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.
66. 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.

Ó FEBS 2002 Ku proteins regulate transcription of the WD gene (Eur. J. Biochem. 269) 2161