The Ku antigen (KuAg), (
)which consists of two
subunits, p70 and p80 (p86), plays a crucial role in double-stranded
break repair of
DNA(1, 2, 3, 4, 5, 6) .
In this process, its ability to bind to DNA ends nonspecifically is
postulated to be related to subsequent actions such as DNA
recombination or unwinding(1, 2, 3) .
Furthermore, such binding has been reported to be directly coupled with
DNA-dependent protein kinase activity, which is elicited by the
putative catalytic unit of KuAg, p350(7, 8) . On the
other hand, sequence-specific binding of KuAg has been demonstrated in
some genes, such as the small nuclear RNA(9) , T cell
receptor(10) , transferrin receptor(11) ,
collagenase(12) , ribosomal RNA(13, 14) , and
heat shock protein genes(15, 16) . Although expression
of most of these genes is stimulated by KuAg, transcription of the
latter two is repressed by KuAg(14, 15, 16) .
However, neither common KuAg-responsive DNA elements nor detailed
domain structure of KuAg were identified in either type of gene
regulation(9, 10, 11, 12, 13, 14, 15, 16) .
In this regard, it is of note that multimer formation of KuAg with
another proteins was suggested to be involved in some of the above
examples of sequence-specific gene regulation by KuAg (12, 15, 16
We previously reported that
two DNA elements located far upstream of the human parathyroid hormone
gene mediated negative gene regulation by extracellular Ca
(Ca
). These DNA elements
(negative calcium-responsive elements (nCaREs)) bound to common nuclear
proteins (nCaRE binding proteins (nCaREBs)) in a sequence-specific and
Ca
-dependent
manner(17, 18) . We further demonstrated that a redox
factor protein, REF1, was one component of nCaREB by using the
protein-DNA binding (Southwestern) assay(19) . REF1 was first
identified as a mammalian homologue of bacterial apurinic
endonuclease/repair enzyme(20) . Subsequently, it was reported
to potentiate DNA binding activity of several transcription factors
such as AP1 and NF
B by modifying the redox state of these
proteins(21) . In addition to such activities of REF1, we first
reported that it also possessed the sequence-specific transcriptional
repressor function of nCaRE(19) . However, REF1 alone could not
explain all the characteristics of nCaREB activity, and we predicted
the existence of another nuclear protein(s) that functions as nCaREB by
cooperating with REF1(19) . By employing an oligonucleotide
affinity column (22) and amino acid
microsequencing(23) , we demonstrate here that both subunits of
KuAg interact with REF1 to bind to one of the nCaREs and function as
nCaREB.
MATERIALS AND METHODS
Synthetic Oligonucleotides and Plasmid
Constructions
The sequences of oligonucleotides, oligo A and
oligo SP1 site, have seen
described(17, 18, 19) . The sequence of oligo
A4 is

The underlined four base pairs are completely different from
those of oligo A(17) . The method to construct thymidine kinase
promoter-based plasmids has also been shown(17) . For stable
transfection of either subunit of the Ku antigen, a 2.3-kilobase pair BamHI fragment containing full-length p80 cDNA (25) or
a 0.4-kilobase pair EcoRI fragment containing the 5`-coding
sequence of p70 cDNA (2) was subcloned into the respective
unique site of pCB6
neo vector (a generous gift from S.
Xanthoudakis, Roche Institute, Nutley, NJ) in the antisense orientation
(
-p80 and
-p70 plasmids). Because transfection of the latter
p70-based plasmid did not reduce the amount of p70 significantly in the
cultured cells, we also constructed another plasmid, which contained a
1.4-kilobase pair XbaI-Hind fragment of
cDNA
/pGEM7Zf(+) (2) subcloned into the
indicated sites of pRc/CMVneo (Invitrogen). The construction of
pRc/CMV-REF1 plasmid has been reported(19) .
Oligo A Affinity Column
In brief, 10 ml (50 mg) of
HeLa cell nuclear protein (Hnp) was mixed with 2 mg of sheared herring
DNA for 15 min at 4 °C. Latex particles (25.0 mg) upon which 25
µg of hexamer of oligo A was immobilized were then added to Hnp for
60 min at 4 °C(22) . The binding reaction was terminated by
a brief centrifugation, and the particles were washed extensively five
times with buffer A (20 mM Hepes (pH 7.9), 5 mM MgCl
, 20% glycerol, 0.25 mM EDTA, 0.1%
Nonidet P-40 and 1 mM dithiothreitol) containing 100 mM NaCl. Subsequently, nCaREB was eluted with buffer A containing
stepwise gradient concentrations (0.05-1.0 M) of NaCl,
and nCaREB activity was assayed with an electrophoretic mobility shift
assay (EMSA) using of the eluted protein and the
P-end-labeled oligo A as a probe. 10% of the protein
eluted in 0.5 M NaCl after three consecutive passes over the
column were loaded separately onto an 8% analytical SDS-polyacrylamide
gel, and the proteins were visualized by silver staining. The fraction
in which the two subunits of KuAg were most enriched, the eluate in 0.5 M NaCl after the third pass, was loaded onto an 8% preparative
SDS-polyacrylamide gel, and amino acid microsequencing was performed as
described below.
Transfection and CAT Assay
HeLa cells were grown
in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. For changing Ca
concentrations of the medium, either EGTA (to the same medium) or
calcium chloride (to the calcium-free Dulbecco's modified
Eagle's medium/Ham's F-12 containing 10% fetal calf serum)
was employed. In the CAT assay, 5 µg of the reporter plasmid was
transiently cotransfected with 2.5 µg of both the above
antisense-oriented KuAg expression vectors or with 5 µg of the
vector alone. The method of CAT assay has been
described(17, 18, 19) . The method of cloning
the HeLa cells stably transfected with cDNA encoding either subunit of
KuAg in the antisense orientation was similar to the one employed to
obtain Fer cells (HeLa cells stably transfected with REF1 cDNA in the
antisense orientation; (19) ).
Amino Acid Microsequencing
In the preparative
SDS-polyacrylamide gel, 50 mg of Hnp yielded 400 ng of each of the
purified proteins. The purified proteins were transferred onto a
polyvinylidene difluoride membrane, stained with Ponceau S, and
excised. Then automated amino acid sequencing was carried out. To
obtain the internal sequences, the similarly treated and transferred
proteins were subjected to leucine peptidase digestion, and then the
digested products were eluted and separated by HPLC before automated
amino acid microsequencing(23) . The computer search for the
protein containing the obtained partial amino acid sequence was carried
out by using the Swissprot program.
Preparation of Nuclear Extracts and EMSA
The
method for obtaining nuclear extracts from wild-type HeLa cells or HeLa
cells transfected with the expression vectors and for EMSA have been
described(17, 18, 19) . When purified KuAg (7, 26) was used, 40 ng of it was included in the
reaction. Incubation time was 30 min unless otherwise indicated. In lanes 1, 3, 4, 6, 7, and 9 in Fig. 3A, 10 µg of the nuclear protein
from the indicated cells were used, and in lanes 2, 5, and 8 in the same figure, 5 µg of the proteins
from each of the surrounding lanes were mixed, and
``complementation EMSA'' was performed. When the polyclonal
anti-REF1 and anti-KuAg antibodies were used, 1 µl of each of them
was added in the reaction for the final 10 min. In Fig. 4B, after preincubation of 10 µg of Hnp and 1
µl of the Ab at room temperature for 10 min, 200 ng of both b-REF1
(or its truncated form) and labeled oligo A were included in the
mixture for the final 20 min, and EMSA was carried out. In this
experiment, a 6% nondenaturing polyacrylamide gel was used to examine
whether a complex(es) migrating at the position lower than complex A
would appear. All of the other EMSA in this paper were performed by
using 4% gels.
Figure 3:
Complementation EMSA and effects of
Ca
on the complexes in
EMSA. A, effects of mixing of Hnp in EMSA. Hnp from Fer cells (19) was mixed either with that from
-p80 (lanes
1-6) or with that from
-p70 cells (lanes
7-9). In the lanes denoted as M (lanes
2, 5, and 8), half the amount of Hnp used in the
respective surrounding lanes was mixed and subjected to EMSA. In lanes 1-3 and 7-9 oligo A and in lanes 4-6 SP1 site were used as radiolabeled probes. B, effects of Ca
concentration on the complexes in EMSA. Oligo A was used as
a probe. In both A and B, EMSA was carried out after
10 min of incubation of the reaction
mixture.
Figure 4:
Interactions between KuAg and REF1. A, interactions between the purified KuAg and a truncated
series of b-REF1 tagged with GST. 20 ng of KuAg and 200 ng of each
b-REF1 or GST alone were briefly incubated on ice, and then the mixture
was trapped by the GST-agarose followed by a 10% SDS-polyacrylamide gel
electrophoresis. Equal amounts of the trapped proteins were subjected
either to immunoblotting using the anti-KuAg Ab (upper panel)
or to silver staining (lower panel). In the lower
panel, the positions of each of the truncated or full-length
b-REF1s were indicated by triangles. B, effects of
truncated b-REF1s on the reappearance of complex A after suppression by
the addition of the anti-REF1 Ab(19) . As a control, complex A`
was included. Oligo A was used as a probe. After preincubation of 10
µg of Hnp and 1 µl of the Ab at room temperature for 10 min,
200 ng of each b-REF1 and labeled oligo A were added to the mixture for
the final 20 min, and EMSA was carried out. A 6% nondenaturing
polyacrylamide gel was employed to examine whether a band(s) other than
complex A was formed.
Construction of Truncated REF1 and Protein-Protein
Interaction
REF1 genes truncated at the amino terminus were
constructed by polymerase chain reactions using oligonucleotides
corresponding to fixed 3` and variable 5` end points. Carboxyl-terminal
truncations of REF1 were generated by polymerase chain reaction using
oligonucleotides corresponding to fixed 5` and variable 3` end points
(N120, positions 121-318; C107, positions 1-211). All the
polymerase chain reaction products were digested with BamHI
and Hind. Digested fragments were inserted into
pGEX4`-3(27) . The truncated protein genes of these constructs
were expressed from the GGA triplet of the GGATCC BamHI
recognition sequence as fusion proteins. All resultant plasmids were
introduced into Escherichia coli JM109. The truncation series
(b-REF1) was expressed as glutathione S-transferase (GST)
fusion proteins after
isopropyl-1-thio-
-D-galactopyranoside induction. For the
interaction between the purified KuAg (7, 26) and the
above b-REF1s, 20 ng of the former and 200 ng of the latter were mixed
for 5 min on ice, washed with phosphate-buffered saline in the presence
of GST-agarose three times, and subjected to SDS-polyacrylamide gel
electrophoresis. Immunoblotting with the anti-KuAg antibody (7, 26) was performed by the enhanced
chemiluminescence method, as reported (19) .
RESULTS
Purification of nCaREB
Although nCaRE bound to
purified REF1, nCaREB contained at least one another nuclear
protein(19) . However, we had failed to clone any nCaREB other
than REF1 by the Southwestern method. We assumed that this failure
might be due to the inability of the protein(s) to bind to nCaRE in the
denaturing/renaturing process. Thus, we tried to purify nCaREB in Hnp
by employing a column purification method that used latex particles (22) onto which tandemly ligated oligo A (one of nCaREs; (17) ) had been immobilized. First, the fractions eluted from
the column in 0.05, 0.3, 0.5, and 1.0 M NaCl in a stepwise
manner were tested by EMSA (Fig. 1A). By using of each
eluate, we found that the fraction eluted in 0.5 M NaCl
contained the major nCaRE binding activity (Fig. 1A).
This fraction was analyzed by SDS-polyacrylamide gel electrophoresis.
After three consecutive passes over the same column, two proteins that
migrated with apparent molecular weights of 80 and 70 kDa (indicated by solid arrows, Fig. 1B), as well as several
smaller proteins of fainter intensities (50-65 kDa), were
enriched and visualized by silver staining. Although the
35-37-kDa band indicated by the dotted arrow in lane
3 of Fig. 1B might represent REF1 protein, it
disappeared after the final pass (lane 4, Fig. 1B). On the other hand, the two proteins of 70 and
80 kDa were consistently co-purified through many trials. In the
subsequent preparative SDS-polyacrylamide gel, we found that 50 mg of
Hnp yielded 400 ng of each of the proteins. Table 1shows typical
results of the purification. Amino acid microsequencing ((23) ;
see ``Materials and Methods'') revealed three short peptides
from each band (Fig. 1C), all of which coincided with
partial amino acid sequences of the two subunits of KuAg, p70 and
p80(24, 25) .
Figure 1:
Purification of nCaREB. A, an
elution profile of nCaREB activity. After absorption onto the oligo
A-immobilized latex, Hnp was eluted by the stepwise gradient
concentrations of NaCl as indicated. In EMSA, amount of each eluate
after the first pass over the column was used. Crude Hnp was included
in the first lane (denoted as C). In this figure, only complex
A was visible. F denotes the position of free probe. B, the silver staining of the proteins eluted in 0.5 M NaCl from the oligo A-immobilized latex particles after second (lane 2) and third (final, lane 3) passes over the
column. An 8% SDS-polyacrylamide gel was used. In lane 1 the
molecular mass markers were loaded. The bands indicated by the solid arrows corresponded to two subunits of KuAg, p70 and
p80, respectively. The dotted arrow (see lane 2)
indicates the presumptive REF1 protein that disappeared after the third
pass over the column (lane 3). C, obtained partial
amino acid sequences after microsequencing and their positions within
each subunit of KuAg (top, p70; bottom, p80) are
shown.
Interaction between KuAg and Oligo A
Both subunits
of KuAg were prepared and purified from serum of a patient of
scleroderma/polymyositis by phosphocellulose and double-stranded
DNA-Sepharose chromatography and anion exchange HPLC as
reported(7, 26) . As shown in Fig. 2A,
they could form one discrete complex (complex A`) with oligo A in EMSA.
Complex A` was considered identical to the fastest migrating band
(complex A) among several complexes generated by the interaction
between Hnp and oligo A (Fig. 2A). First, its migrating
position on the gel was identical to that of complex A. The addition of
the purified KuAg to Hnp did not produce another bands even after
longer electrophoresis (not shown). Second, the anti-KuAg antibody (7) abolished complex A formation, whereas preimmune serum did
not. Third, one of the mutants of oligo A, oligo A4, in which the
central four bases of oligo A were completely altered, did not inhibit
complex A formation. Such sequence specificity observed in complex A
formation paralleled that seen in complex A` (Fig. 2A).
However, we noticed that even a 50-fold molar excess of oligo A4 did
not abolish the formation of complex A, although a molar excess greater
than 10-fold eliminated complex A`. Thus, sequence-specific binding of
the purified KuAg, unlike that of Hnp, could be seen only in a narrow
range of a quantitative ratio between KuAg and the probe DNA
(9-16, see ``Discussion'').
Figure 2:
EMSA. A, sequence-specific binding of the purified KuAg (complex A`)
and that of nCaREB in Hnp (complex A). 50-fold (Hnp) and 5-fold (KuAg)
molar excesses of the indicated competitors were used(11) . In
the two right hand lanes, an elimination of complex A by the
addition of the anti-KuAg Ab but not by preimmune serum is shown. B, sequence-specific binding of complex B and time-dependent
shift from complex B to A formation. After 5, 10, 15, and 20 min of
incubation of Hnp with the probe (radiolabeled oligo A), each mixture
was loaded onto the gel. In the two right hand lanes, effects
of the anti-REF1 Ab (19) on complexes A and B are shown. The
band indicated by an asterisk was occasionally seen in some of
EMSA (see also Fig. 3, A and B), and it
probably represented a partially degraded complex derived from
complexes A and/or B. C, immunoblotting of the Hnp derived
from the HeLa cells with the anti-KuAg Ab. Three different sources of
Hnp were used; control HeLa cells (lane 1) and HeLa cells
stably transfected in the antisense orientation with the p70 expression
vector (lane 2) and with the p80 expression vector (lane
3). The amounts of several bands other than p70 or p80 among the
three cell types did not differ significantly, suggesting they were
nonspecific bands recognized by the anti-KuAg Ab. D, effects
of reduced expression of the p70 subunit (lane 2) or p80
subunit (lane 3) on the formation of complex A/B in EMSA. In lane 1, Hnp from control cells was used. In lanes
1-3, oligo A was used as a probe. In lanes
4-6, effects on the SP1 site-binding proteins were similarly
examined. C1 and C2 denote SP1 site-binding proteins.
In all the reactions in this figure, samples were incubated for 10
min.
However, besides complex
A, Hnp produced several slower migrating complexes bound to oligo A.
Shorter incubations generated an uppermost band (complex B) that
behaved similarly to complex A in terms of sequence specificity (Fig. 2B). Time course EMSA experiments revealed that 5
min of incubation generated almost equal intensities of both complexes
A and B (Fig. 2B). Longer incubation led to the
formation of stronger complex A and fainter complex B, resulting in
almost complete disappearance of complex B after 20 min of incubation.
This observation suggested that complex B was first generated and then
converted to complex A.
Effect of Antisense-oriented Expression of the p80
Subunit of KuAg
We next modified HeLa cells by stable
transfection with a p80 subunit expression vector in the antisense
orientation (
-p80 cells) in order to lower the amount of p80 in
the cells. Immunoblot analysis revealed that the amounts of both p80
and p70 were decreased in these cells (Fig. 2C; see
``Discussion''). By densitometoric scanning, we found that
only 20% of p80 and 50% of p70 were retained in these cells compared
with those in control cells. Interestingly, as shown in EMSA in Fig. 2D, like complex A, complex B was markedly
diminished in these cells, whereas complexes between such Hnp and SP1
site (C1 and C2) were not affected.
Interaction between KuAg and REF1 Protein:
Complementation EMSA
Because REF1 protein had nCaREB
activity(19) , generation of these complexes might be affected
after reducing the amount of REF1 protein by a similar antisense
strategy. As shown in Fig. 3A, Hnp from antisense
REF1-transfected cells (lane 1, Fer cells; (19) ) as
well as that from
-p80 cells (lane 3) produced overall
weak EMSA bands. Importantly, mixture of half the amount of the protein
used in each lane (lane 2) conferred more than additive
effects on the two bands. Although the bands between the complexes A
and B behaved similarly to complex A in some experiments, they did not
reproducibly appear. On the other hand, such mixture gave just additive
effects on the binding between the SP1 site and its binding proteins (Fig. 3A, lanes 4-6).Then we
attempted to establish
-p70 cells by stable transfection of a p70
subunit expression vector in the antisense orientation in order to
generate Hnp that would contain reduced amounts of the p70 subunit.
However, all the clones examined yielded only marginally reduced
amounts of the p70 protein (Fig. 2C; see
``Discussion''). Nonetheless, we thought these differences
were significant, because the intensities of the bands other than p70
or p80 were comparable (Fig. 2C, lanes 1 and 2). Further, in EMSA, the intensity of complex B was never
attenuated, and that of complex A was slightly attenuated in Hnp from
these
-p70 cells (Fig. 2D). Perhaps related to
this phenomenon, we observed that a similar mixture of Hnp from any one
of these
-p70 cells with that from Fer cells conferred just
additive but not synergistic effects on complex B formation, whereas
effects on complex A were again synergistic (Fig. 3A, lanes 7-9). These results suggest that even a small
decrease in the amount of p70 in Hnp from our
-p70 cells might be
sufficient for evaluating potential functional cooperativity of p70
with REF1. Taken together, we assume that REF1 and KuAg interact in a
highly cooperative manner but that the p70 subunit might not
participate in the p80-REF1 complex (complex B) especially in the early
time point.
Effect of Ca
on the Oligo
A-nCaREB Binding
We next examined whether complex B contained
KuAg. However, we failed to erase or supershift complex B formation by
the anti-KuAg Ab in EMSA (Fig. 2A and data not shown);
this result is in sharp contrast to that following the use of the
anti-REF1 Ab (Fig. 2B). It might reflect a possible
conformational change due to the interaction between p80 and REF1,
which would prevent an access of the anti-KuAg Ab to the complex.
Nonetheless, both complexes A and B were similarly up-regulated by a
rise in Ca
concentration ((17, 18, 19) ; Fig. 3B).
Protein-Protein Interaction between KuAg and REF1:
Binding Domain within REF1 Protein
We next made a truncated
series of bacterially produced REF1 ((19) ; b-REF1s) tagged
with GST(27) . As shown in the upper panel of Fig. 4A, full-length as well as a truncated b-REF1
whose 107 amino acid residues from its COOH terminus were deleted
(C107) retained the ability to bind to KuAg, whereas truncation up to
120 residues from its NH
terminus(N120) lost this ability.
Immunoblot analysis using anti-REF1 Ab revealed that when compared with
C107 or full-length b-REF1, larger amounts of GST and N120 were trapped
by the column (Fig. 4A, lower panel), whereas
these two interacted with much less or only a background level of KuAg (Fig. 4A, upper panel). Further, in EMSA shown
in Fig. 4B, complex A, once abolished by the inclusion
of the anti-REF1 antibody, (19) was revisualized when
full-length REF1 (lane 4) and C107 (lane 6) but not
GST alone (lane 3) or N120 (lane 5) were added back
to the reaction mixture. The polyclonal anti-REF1 Ab used here could
hardly recognize C107 because it was raised against the peptide near
the COOH-terminal portion of REF1(19) . In other cases, huge
excessive amounts of b-REF1s were re-added compared with the amount of
the antibody, thereby eliminating the possibility that the free Ab,
even if it remained, absorbed the added b-REF1s to keep complex A
suppressed. The results in Fig. 4(A and B)
suggest that the NH
-terminal portion of REF1 is crucial for
its synergistic interaction with KuAg, although we could not specify
which subunit of KuAg was involved here, because separation of KuAg
into each subunit was difficult in these experiments.REF1 augments
DNA binding activity of several transcriptional factors through some of
its cysteine residues, especially Cys
, by modifying redox
states of these proteins(21) . Interestingly, Cys65 is
contained within the region capable of interacting with KuAg (Fig. 4, A and B).
Components of nCaREB Required for the Binding to Oligo
A
Although we demonstrated that REF1 was required for formation
of complex A, the migrating position of complex A in EMSA was identical
to that of complex A`, which contains only the two subunits of KuAg (Fig. 2A). However, not only the anti-KuAg antibody but
anti-REF1 antibody impaired complex A formation ((19) ; Fig. 2B). Further, the addition of full-length REF1 as
well as some truncated b-REF1 led to the reappearance of the once
suppressed complex A, but the position of the resultant complex A on
the gel was the same regardless of the heterogeneous size of b-REF1s (Fig. 4B). Therefore, we hypothesize that there are two
distinct steps in complex formation. At first, REF1 and p80, but not
p70, take part in the formation of complex B, although there must be
other unidentified components or multimer forms in complex B, because
it migrates slowly. Then p70 participates in the complex. During EMSA,
REF1 might be detached from the complex, resulting in the formation of
complex A. Such a dissociation of a protein(s) from certain protein-DNA
complexes in EMSA has been reported in several cases ( (28) and (29) ; see ``Discussion'').
Effects of KuAg on nCaRE-mediated Transcription
We
next transiently transfected HeLa cells with the reporter plasmid,
oligo A-thymidine kinase CAT (17, 18) and both of the
antisense-oriented p70 and p80 expression vectors (Fig. 5).
Ca
-dependent suppression of the CAT
activity driven by oligo A-thymidine kinase CAT was abrogated after
reducing KuAg expression by the antisense strategy, whereas the CAT
activity driven by parental thymidine kinase CAT was unaffected either
by high Ca
concentration (17, 18, 19) or by the antisense-oriented
expression of KuAg. Transfection of either p70 or p80 expression vector
alone in the antisense orientation did not significantly affect
Ca
-mediated suppression of CAT activity
by the oligo A-bearing plasmid (data not shown). These results also
underscore the functional contribution of KuAg to nCaRE-mediated gene
repression by Ca
.
Figure 5:
Effects of cotransfected antisense p70 and
p80 expression vectors on
Ca
-mediated suppression of CAT
activity driven by oligo A as well as parental thymidine kinase CAT (17, 18, 19) . After transfection with the
indicated combinations of plasmids, cells were equally split into two
conditions to avoid difference in transfectional efficiency. Average
CAT activities after five different transfections were represented as
mean ± S.E.(17, 18, 19) , and typical
results are shown.
denotes transfection of the indicated plasmids
in the antisense orientation.
DISCUSSION
KuAg, Alone or in Association with REF1, Binds to Oligo
A in a Sequence-specific Manner
The manner of the binding of
KuAg to oligo A described in this manuscript is clearly distinct from
that of its well characterized nonspecific binding to DNA
ends(1, 2, 3, 4, 5) .
First, unlike the predictions of previous models of the nonspecific DNA
binding of KuAg, such as a sliding model(4) , longer incubation
time generated faster migrating complexes but did not yield slower
migrating nor multiple complexes in EMSA (Fig. 2B).
Second, Hnp protected oligo A, which lies in the central, but not in
the terminal portion of the 110-base pair fragment, from DNase
digestion(17) . Binding to the ends of DNA molecules has been
thought to be a hallmark of sequence-nonspecific binding of KuAg.We
also presented several lines of evidence that Hnp that specifically
bound to oligo A contained not only both subunits of KuAg but also REF1
protein (Fig. 2B, 3A, 4A, and
4B). Thus, as discussed below, KuAg might well bind to oligo A
in a cooperative manner with REF1, thereby giving rise to a reinforced
binding specificity, although the binding of the purified KuAg alone to
oligo A showed less stringent but distinct sequence specificity
(complex A`, Fig. 2A). The sequence of oligo A does not
have significant homology with other reported KuAg-responsive element,
except for the octamer-like sequence within the small nuclear U1 RNA
gene(7) ; oligo A can be considered a degenerate inverted
repeat of the octamer sequence(19) .
nCaREB Consists of KuAg and REF1
We demonstrated
that both of the two nCaRE-nCaREB complexes (A and B) contained KuAg
and REF1. In our transfection experiments, all the clones containing
the p70 subunit in the antisense orientation exhibited a marginal
reduction of the amount of the p70 protein (Fig. 2C).
This is in sharp contrast to the situation in A-p80 cells (Fig. 2C), in which the amount of p80 was markedly
reduced. This finding might reflect a more crucial function of p70 for
the cultured cells compared with that of p80. In any case, we found
that such a minimal but selective reduction of p70 led to reduced
intensity of complex A but not complex B in EMSA (Fig. 2D). Along with this observation, our
complementation EMSA experiments revealed a functional cooperativity of
p70 with REF1 for the formation of complex A but not complex B (Fig. 3A).On the other hand, we suggested that by
the similar complementation EMSA (Fig. 3A), p80 could,
separately from the p70 subunit, bind to and function with REF1 during
complex B formation. Such a dissociation seems to contradict many
reports(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) describing
that many functions of KuAg were elicited by the association of p70
with p80 (and/or p350, a catalytic unit of KuAg). Nevertheless, in the
time course EMSA experiment (Fig. 2B), we demonstrated
a shift of complex formation from B to A, which contained both of the
subunits. Furthermore, an abrogation of oligo A-mediated
transcriptional repression by Ca
was
observed only in the case of combined transfection of the p70 and p80
expression vectors in the antisense orientations (Fig. 5). Thus,
the reassociation of both subunits of KuAg is predicted to lead to
transcriptional suppression by Ca
.
REF1 Is a Labile but Distinct Component of
nCaREB
Then how is REF1 involved in nCaREB function? We
previously showed that REF1 alone, either in the form of bacterially
produced REF1 (b-REF1) or that of one component of Hnp, was able to
bind to nCaRE in a sequence-specific manner(19) . However, its
binding intensity was rather weak, and its specificity was not
stringent(19) . In contrast, through heteromultimer formation
with KuAg, REF1 formed complexes A and B, both of which exhibited a
high degree of the binding specificity to oligo A (Fig. 2, A and B). The supershift (Fig. 2B) and
complementation (Fig. 3A) in EMSA convincingly
suggested that REF1 affected the formation of both complexes. Although
we could clearly show that the purified KuAg and REF1 interacted with
each other (Fig. 4A), mixture of both of them in EMSA
did not create such a band as complex B migrating more slowly than
complex A` (data not shown).REF1 may be easily detached from the
REF1-KuAg complex during electrophoresis in nondenaturing conditions.
This hypothesis could explain why complex A migrated at the identical
position with complex A` in EMSA (Fig. 2A). Further,
removal of REF1 by electrophoresis could explain how the addition of
some of the b-REF1s reversed the suppression of complex A without
changing the mobility of complex A, irrespective of the heterologous
size of b-REF1s (Fig. 4B).
The relatively short
half-life of complex B (Fig. 2B) might also reflect
labile binding of REF1 protein during electrophoresis. Perhaps another
unidentified protein(s), such as TATA box binding protein, which has
been reported to interact with KuAg to regulate the expression of the
collagen gene(12) , can stabilize complex B transiently. Such a
dissociation during electrophoresis in EMSA has been described for
NF
B (28) and Phox (29) proteins.
Domain Structures of REF1-KuAg Binding
We found
that the amino-terminal portion of REF1 bound to KuAg to function as
nCaREB (Fig. 4, A and B). Further, at the
putative leucine zipper motif (residues 395-399) of the p70
subunit of KuAg, there is a sequence, AALCR (residues 395-399),
that is similar to the consensus site AA(K/E/R)CR needed for REF1
interaction(21) . This consensus sequence found in JUN protein
has been shown to be important for its binding to REF1. Also, a
cysteine preceded by lysine was found in the putative leucine zipper
region of p80 (residues 156 and 157).Although functional separation
of KuAg into separate subunits is
difficult(1, 2, 3, 4, 5, 6) ,
our observation raises the possibility that some form of REF1-KuAg
interaction really occurs in vivo. This finding is of
particular interest because both REF1 and KuAg have been reported to be
engaged in the basic transcriptional activity coupled with DNA
repair/recombination
processes(1, 2, 3, 4, 5, 6, 20) .
We had previously shown that not only oligo A but also oligo B
functioned as nCaREs. Oligo B forms a unique palindromic sequence, and
its sequence is widely conserved among several genes to achieve
negative calcium responsiveness(18) . Although oligo B, like
oligo A, bound to REF1 protein(17, 18, 19) ,
oligo B-specific binding to REF1-KuAg complexes could not be
demonstrated (data not shown). The mechanism underlying this
distinction is currently unknown.
A rise in
Ca
concentration augmented the binding of
nCaREB to nCaRE ( (18) and (19) ; Fig. 3B). In response to a rise in
Ca
concentration, the amounts of both
mRNA and protein of REF1 were elevated(19) , whereas those of
KuAg were not significantly altered (not shown). We are currently
examining the possibility that post-transcriptional modifications of
KuAg by Ca
also play some role in
potentiating nCaRE-nCaREB interactions, although it is possible that
the rate-limiting step is Ca
-dependent
change in the activity of REF1 protein alone.