Vol.
176,
May
15, 1991
No.
3, 1991
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
SEQUENCE-SPECIFIC THAT INTERACT WHEAT
Hisabumi
April
1593-1600
SINGLE-STRAND DNA-BINDING PROTEINS WITH THE REGULATORY REGIONS OF HISTONE H3 AND H4 GENES
Takase,
Department
Received
AND
Maki
of Botany,
Minami,
and Masaki
Faculty of Science, Kyoto 606, Japan
Iwabuchi
Kyoto
University,
8, 1991
We identified two novel DNA-binding proteins, ssDBP-1 and ssDBP-2, in wheat germ nuclear extract that interact with the proximal sequences of shift the promoter regions of the wheat histone H3 and H4 genes. Mobility and methylation interference assays have demonstrated that these factors specifically bind to the single-strand DNA which partially overlaps the hexamer and octamer cis-elements of the H3 promoter. Both proteins are distinguishable from HBP-la and HBP-lb which specifically bind to the H3 hexamer sequence. These ssDNA-binding proteins are supposed to 0 1991 Academic Pre**, Inc. regulate the transcription of the wheat histone genes. Previously
we found
octamer
(CGCGGATC)
region
of
the
Nakayama
et al., DNA-binding
nuclear
extract,
we
with
the nonamer
distinct
that
(single from
binds
to the
wheat
H3
the
proteins
HBP-la
proteins
ssDBP-1
of
unpublished).
demonstrated
binding
positive
and (2-4).
wheat We
HBP-lb,
HBP-lb
interacting
Moreover,
Here
we describe
strand
DNA-binding
strand
in
the
and H4 (THOll)
proximal
with
(1,
sequence-
in wheat germ
the
are
hexamer,
of cDNA
and clones
bZIP-type
DNA-binding
and
ssDBP-2,
nuclear
proteins.
regulatory
promoters,
(TH012) the
and HBP-2
the novel
three
in the proximal
identified
HBP-lb
protein-l)
(ACGTCA),
gene
in our analyses
and
described
H3
have
HBP-la,
(5,6).
motifs
histone
also
HBP-la
lower
hexamer
(CATCCAACG)
both
the previously
(TH012)
cis-elements:
and nonamer
promoter
specific
HBP-2
three
proteins, which
binds
of
the
to the
0006-291X/91 1593
are
ssDBP-1
sequences
and ssDBP-2
DNA-
$1.50
Copyrighr 0 1991 by Academic Pres.s. Inc. All rights of reproduction in nny fbrrn reserved.
Vol.
176,
No.
upper
BIOCHEMICAL
3, 1991
strand
in
the
overlap
the cis-region
discuss
a possible
transcriptional
same
AND
BIOPHYSICAL
promoters.
These
that has the hexamer function
regulation
of
these
of the H3
RESEARCH
COMMUNICATIONS
protein-binding
and octamer
regions
motifs.
ssDNA-binding
We also
proteins
in
the
gene.
MATERIALS
AND
METHODS
Preparation of nuclear extract: The crude nuclear extract of wheat germ was prepared essentially as described elsewhere (2). Probes and competitor DNA fragments: The DNA fragments used as probes or competitors in the DNA-protein binding assays (Table 1) were synthesized with Cyclone TM Plus (MilliGen/Biosearch). These probes were end-labeled with [y- 32PJATP using T4 polynucleotide kinase. Mobility shift and methylation interference assays: Both the mobility shift and methylation interference assays were conducted as described elsewhere (2, 5). RESULTS
In
spite
binding bind
of the wealth
proteins
HBP-la
specifically
identified.
In
our
search
mobility
probe
double-strand
1.
Table
shift
DISCUSSION
of information
about
and HBP-lb
to the octamer
conducted the
AND
for
motif
Sequence alignments probes and competitors
(3, 5, 6),
no nuclear
of the H3
promoter
octamer-specific
assays DNA
the hexamer-specific
on
wheat
(dsDNA)
proteins have
DNA-binding nuclear
fragment
extract that
DNA-
yet been
proteins, using
contains
of the synthetic oligonucleotides in the DNA mobility shift assay
that
as the both
used as
Sequence
histone
H4
-141/-110
(THOll)
5'-CGGCCACGTCACCGATCCGCGGCATGTCTCCC 3'-GCCGGWGGCTAGGCGCCGTACAGAGGG
Hex (H3) -179/-160
5'-TCGGCCACGTCACCAATCCG (THO12) 3'-AGCCGG-GGTTAGGC
histone
5'-TCGGCCB CCAATCCGCGGCATTC (THO12) 3'-AGCCGG-GGTTAGGCGCCGTAAG
H3 -179/-153
Non-specific -13391-1374
ssDNA 5'-ACTACGAGACACGTCGGACATAGTAG (THOU)
Probes and competitors were used as dsDNA (in Fig. 1) and ssDNA Negative numbers indicate (in Fig. 2). The hexamer motif is underlined. positions in the nucleotides related to the transcriptional initiation site (as +l). 1594
we
the
Vol.
176,
No.
hexamer which
BIOCHEMICAL
3, 1991
and octamer
motifs
has the consensus
octamer
and B2,
when
appeared
2, Fig.
these
shift
BIOPHYSICAL
of the H4
promoter
32P-end-labeled
were
(lane
AND
1A). bands
Prior (lanes
Ii4 *up/low*
(CGCGGATC)
heating
(A)
Two
strands
H4
major
these
H4 up/low’ probe
UPPer
lower
(6) F Bl
aYa4
:-2: -12g
Bl*
**,
.ra
Bl ) B2*
82,
LtQ
.91 I ,xI...-
-128
83, dsDNA ssONA
Free probe probew
ra(L
(C)
m
4W-
12345
6
8 9 10
7
11
12
34
T Ii4
upper
strand
5’-CGGCCACGTCACCdATCCGCGGCATGTCTCCC-3
H4 lower
strand
3’-GCC&TGCAGT&TAGGCGCCGTACAGAGGG-5
Fig. 1. A: Mobility shift and competition assays of wheat germ nuclear extract done with dsDNA fragments containing the regulatory region of 32P-labeled H4 dsDNA fragment wheat histone H4 gene as the probe. probes were incubated with the nuclear extract in the presence or absence of a 20-fold excess of dsDNA fragments as competitors (sequences given in Table 1). Lanes 1-7, the upper and lower strands of the probe were 32P end-labeled (*up/low*); lanes 8 and 9, the upper strand of the probe was 32P-end-labeled (*up/low); lanes 10 and 11, the lower strand of the probe was 32P-end-labeled (up/low*). Lane 7, the nuclear extract was heated at 900 C for 5 min before its addition to the binding mixture. Lanes 1, 8 and 10, no nuclear extract was added to the binding mixture. Arrowheads Methylation indicate probe-protein complexes Bl, B2 and B3. B: interference assays of the Bl and B2 complexes were conducted with the labeled upper strand (lanes 1 and 2) and labeled lower strand (lanes 3 and 4) probes. Lanes 1 and 3 show the respective breakage patterns in the upper and lower strand DNA fragments of the free probes (F). Lanes 2 and 4 show the respective patterns of the probes recovered from the Bl and B2 bands. Arrowheads indicate the positions of methylated guanine residues that interfere with binding. C: Nucleotide sequences of the binding regions of ssDBP-1 and ssDBP-2. The hexamer motif is underlined. 1.595
of
bands really
F 82
Bl *
Bl
the probe
the appearance
that
of
promoter bands,
with
prevented
suggests
Ii4 *up/low probe --
probe
the
was incubated
of the extract
6 and 7), which
both
because
sequence.
extract
COMMUNICATIONS
(THOll);
* probe)
(*up/low
the nuclear
RESEARCH
Vol.
176,
No.
do represent proteins
DNA-protein
in the
probe,
synthetic
prevented
oligonucleotides
promoter
(lane
added.
This
the
mixture
as a competitor,
3),
that the complexes
than
was separable
32P-end-labeled,
not
and an additional
minor
(Fig.
lA,
the complexes
when motif
fragment,
band
of
the
(lane
H3
5) were
the specific
and HBP-lb) this
after
with
DNA-binding
phosphocellulose
Most
lower
(up/low*),
strand
the band
had
been
corresponding
the band corresponding
to B2
appeared
with
strand
probably,
the Bl
(B3)
between
of
through
fact,
whose
lane 11); whereas,
lane 9).
formed
In
H4
double-strand
fragment
and HBP-lb
was used as the probe lA,
unlabeled formation
not
HBP-la
of the
shown).
a dsDNA
(Fig.
from
motif.
HBP-la
(data
when
appeared
but
were formed
(distinct
hexamer
from
chromatography
nuclear
When
DNA
the
(*up/low)
experiments.
coli
other
to Bl
sequences
4) and the Escherichia
sequences
the nuclear
of the
hexamer
protein(s)
COMMUNICATIONS
whether
the
suggests
Interestingly,
determine
containing
of a nuclear
column
RESEARCH
to any
(lane
interaction
protein(s)
bind
to the reaction
was
BIOPHYSICAL
To
specifically competition
was added
complexes
AND
complexes.
extract
we conducted
fragment the
BIOCHEMICAL
3, 1991
single-strand
the labeled
DNA
upper
and B2 bands represent (ssDNA)
fragments
and
proteins.
To confirm
whether
ssDNA
fragments
upper
strand
correspond minor
is so, we conducted
as probes.
was
the
Fig.
probe,
(lane
corresponds
to Bl
experiments
done
2).
two
With
(see Fig. with
shifted
of the B2’
the 1A)
ssDNA
and B3’
bands,
appeared
(lane
9).
formation
strand
of the H3
(Fig.
2A, lanes 12 and 14).
as the
ssDNA
of the Bl’
fragment
band
fragment
1596
the labeled B3’,
which
B3’
being
the
band
Bl’
that
Cross-competition showed
by the lower (Fig.
that strand
2A, lanes 6
was not prevented
nor by the non-specific
These results
When
competitor
bands was not prevented
and 7).
assays with
1A) appeared, strand,
fragments
shift
and
lower
or the non-specific
upper
B2’
labeled
of the H3 fragment Moreover,
mobility
2 shows the results.
to the B2 and B3 bands (see Fig.
band
formation
this
ssDNA
by the fragment
suggest that the upper and lower
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-129
dsoNA probe -
Ilk
1234567
8 9
10 11 12 13 14 12
(C)
H4 upper
strand
S-CGGSfACGTCA$ATCCGCGGCAT&TCTCCC3’
H4 lower
strand
3’-GCCGGTGCAGTGGCTAGGCGCCGTACAGAGGG-3’
34
Fig. 2. A: Mobility shift and competition assays of wheat germ nuclear extract done with ssDNA fragments containing the regulatory region of wheat histone H4 gene as the probe. 32P-labeled H4 ssDNA fragment probes were incubated with the nuclear extract in the presence or absence of a %O-fold excess of ssDNA fragments as competitors (sequences given in Table 1). Lanes 1-7, the upper strand of the probe was 32P-end-labeled (*up); lanes 8-14, the lower strand of the probe was 32P-end-labeled (low*). Lanes 1 and 8, no nuclear extract was added to the binding mixture. Arrowheads indicate the probe-protein complexes Bl’, B2’ and B3’. B: Methylation interference assays of the Bl’ and B2’ complexes were conducted with the labeled upper strand (lanes 1 and 2) and the labeled lower strand (lanes 3 and 4) probes. Lanes 1 and 3 show the respective breakage patterns of the upper and lower strand DNA fragments of the free probes (F). Lanes 2 and 4 show the patterns of the probes recovered from the Bl’ and B2’ bands. Arrowheads indicate the positions of methylated guanine residues that interfere with binding. C: Nucleotide sequences of the binding regions of ssDBP-1 and ssDBP-2. The hexamer motif is underlined.
strands
of the H4 or H3 DNA
fragment
motifs
bind
of
specific lower strand
manner,
of
One
the
that
the
H4
ssDNA
the ssDNA interpretation
dsDNA
fragment before
nuclear
binding
with formation
its
1597
a sequence-
lanes
prevented
is that
anti-ssDNA of
proteins
2A,
and protein
of this
and octamer
of
also
fragment
the hexamer
affinities (Fig.
fragment
possible
competitor
wheat
of the H4 fragment
between
constructed as
the
species
but
than those
complexes 10).
different
containing
the
the
in H3
fragment
5 and 13).
The
formation (Fig. the labeled fragment
protein-probe
of
2A,
anti-
specific
lanes ssDNA
that
are
was
complexes
4 and probe used (free
Vol.
176,
probes
No.
3, 1991
in
lanes
interpretation
4 and
leads
shown
in
Fig.
ssDNA
probe
migrating
BIOCHEMICAL
the
result
dsDNA
that
of
the
the fast-migrating
DNA-binding
ssDNA;
therefore,
(ssDNA
binding
regulatory
proteins we have
the
COMMUNlCATtONS
fragments).
DNA-protein
unannealing
free probe
identified
given
protein-l)
regions
This
complexes
of
complementary
is ssDNA
and the slow-
here
them
which
probably
tentative
binds
are
specific
designations:
to
the
lower
of H3 and H4 genes and ssDBP-2
which
to
ssDBP-1
strand binds
of
the
the upper
of these re,gions.
To locate
the SSDBP-1
we conducted
-137
by methylation
-116 (Figs.
lB,
represent
the
formed
lC,
band
between
the binding
of the guanine
formed
is identical
the upper
binding
(5, 6, Takase
included
in
octamer
proteins
to HBP-la
double-helix
the single-strand motif
considered The
form
of
with our
sequence-specific
ssDNA-binding
systems,
sequence-specific
several
and
of the
moreover,
but,
for
these
when
the DNA change
or HBP-lb
regions
of the open-formed ssDBP-1
and
of
undergoes
of HBP-la
ssDNA-binding
complex
were
relations
motif
In
and
they
ssDBP-1
proteins.
bands
residues
motif,
that
1598
-128
those for HBP-la
yet known;
to the binding
indicate
also
ssDBP-1
guanine
functional
the octamer
these specific
study
The
overlapped
The
and
in terms
unpublished);
to the octamer
strand
at positions
strand
band
are not
including
owing
adjacent
to interact
results
partially
as
-129,
suggests that the B 1 and Bl’
cis-element.
in the region
ssDNA
at positions
residues
B2’
et al.,
and
to the upper
and ssDBP-2.
and HBP-lb
&DNA
residues
the lower
to the
strand
HBP-lb
hexamer
from
of the two proteins
the
This
in the H4 promoter,
strand of the H4 fragment
of ssDBP-2
of the guanine
2B and 2C).
complexes
the B2
to the lower
and the binding
was arrested
regions
assays with
of ssDBP-1
by methylation
and -138
binding
interference
The binding
was prevented -130,
and ssDBP-2
methylation
the probes.
that
RESEARCH
annealed
hypothesis
are the
because
BIOPHYSICAL
one dsDNA.
The
strand
10 indicate
to
1A
AND
and
to
to the
ssDBP-2
are
DNA.
and
ssDBP-2
are
mammalian
and
viral
proteins
have
been
Vol.
176,
No.
reported, DNA
BIOCHEMICAL
3, 1991
some of which double-helix,
transcription. dsDNA
in
(10) proposed
(11) reported
a novel
triple-strand
repressor
elements
capable
nuclear
of c-myc
cis-elements
(12-17).
sequence-specific of genes.
(7,
that
S),
of DNA
and p-interferon
genes
All
this
specifically
evidence
ssDNA-binding
proteins
We are now investigating
information with
suggests in
the
the structures
strand
loop DNA
of
Htun
(H-DNA)
have
and
stretches
of transcription.
a single
interact
(9)
single-strand
containing
that
open the
that
recombination
elements
much
COMMUNlCATfONS
in processes
unpaired,
control
Moreover,
H-DNA.
proteins
to function
RESEARCH
that the H-conformation
type of DNA
of forming
BIOPHYSICAL
replication
are the sites of the actual
and Dahlberg
about
are thought
as
Crick
AND
forms and that stretches
has been reported individual
important
strands functions
of for
transcriptional
regulation
and functions
of ssDBP-
1 and ssDBP-2. ACKNOWLEDGMENTS and K. Mikami for their valuable We are grateful to Drs. T. Nakayama discussions. We also thank Dr. K. Fukuchi of the Research Center, Nisshin This research was Flour Milling Co., Ltd for the gift of wheat germ. supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, by grants from the Naito Foundation, Takeda Science Foundation, and the Research Council, Ministry of Agriculture, Forestry, and Fisheries of Japan, original and creative research project on biotechnology. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
K., Kawata, T., Tabata,T., Kanazawa, Nakayama, T., Ohtsubo, N., Mikami, H., and Iwabuchi, M. (1989) Plant Cell Physiol., 30, 825-832. Mikami, K., Tabata, T., Kawata, T., Nakayama, T., and Iwabuchi, M. (1987) FEBS lett., 223, 273-278. Mikami, K., Takase, H., Tabata, T., and Iwabuchi, M. (1989) FEBS lett., 256, 67-70. Kawata, T., Nakayama, T., Mikami, K., Tabata, T., Takase, H., and Iwabuchi, M. (1988) FEBS lett., 239, 319-323. Tabata, T., Takase, H., Takayama, S., Mikami, K., Nakatsuka, A., Kawata, T., Nakayama, T., and Iwabuchi, M. (1989) Science, 245, 965-967. Tabata, T., Nakayama, T., Mikami, K., and Iwabuchi, M. (1991) EMBO J. in press. Roller, R. J., McCormick, L., and Roizman, B. (1989) Proc. Natl. Acad.Sci. USA, 86, 6518-6522. Traut, W., and Fanning, E. (1988) Mol. Cel. Biol., 8, 903-911. 1599
Vol.
176,
9. 10. 11. 12. 13. 14. 15. 16. 17.
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Edelmann, W., Kroger, B., Goller, M., and Horak, I. (1989) Cell, 57, 937946. Crick, F. (1971) Nature, 234, 25-27. Htun, H., and Dahlberg, J.E. (1990) Science, 241, 1791-1795. Peritz, L.N., Fodor, E.J.B., Silverside, D.W., Cattini, P.A., Baxter, J.D., and Eberhardt, N.L. (1988) J. Biol. Chem., 263, 5005-5007. Rajavashisth, T., Taylor, A.K., Andalibi, A., Svenson, K.L., and Lusis, A.J. (1989) Science, 245, 640643. Lannigan, D. and Notides, A.N. (1989) Proc. Natl. Acad. Sci. USA 86, 863-867. Pan, W.T., Liu, Q., and Bancroft, C. (1990) J. Biol. Chem., 265, 70227028. Wilkison, W-O., Min, H.Y., Claffey, K.P., Satterberg, B. L. and Spiegelman, B.M. (1990) J. Biol. Chem., 265, 477-482. Gailland, C. and Strauss, F. (1990) J. Mol. Biol., 215, 245-255.
1600
176,
May
15, 1991
No.
3, 1991
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
SEQUENCE-SPECIFIC THAT INTERACT WHEAT
Hisabumi
April
1593-1600
SINGLE-STRAND DNA-BINDING PROTEINS WITH THE REGULATORY REGIONS OF HISTONE H3 AND H4 GENES
Takase,
Department
Received
AND
Maki
of Botany,
Minami,
and Masaki
Faculty of Science, Kyoto 606, Japan
Iwabuchi
Kyoto
University,
8, 1991
We identified two novel DNA-binding proteins, ssDBP-1 and ssDBP-2, in wheat germ nuclear extract that interact with the proximal sequences of shift the promoter regions of the wheat histone H3 and H4 genes. Mobility and methylation interference assays have demonstrated that these factors specifically bind to the single-strand DNA which partially overlaps the hexamer and octamer cis-elements of the H3 promoter. Both proteins are distinguishable from HBP-la and HBP-lb which specifically bind to the H3 hexamer sequence. These ssDNA-binding proteins are supposed to 0 1991 Academic Pre**, Inc. regulate the transcription of the wheat histone genes. Previously
we found
octamer
(CGCGGATC)
region
of
the
Nakayama
et al., DNA-binding
nuclear
extract,
we
with
the nonamer
distinct
that
(single from
binds
to the
wheat
H3
the
proteins
HBP-la
proteins
ssDBP-1
of
unpublished).
demonstrated
binding
positive
and (2-4).
wheat We
HBP-lb,
HBP-lb
interacting
Moreover,
Here
we describe
strand
DNA-binding
strand
in
the
and H4 (THOll)
proximal
with
(1,
sequence-
in wheat germ
the
are
hexamer,
of cDNA
and clones
bZIP-type
DNA-binding
and
ssDBP-2,
nuclear
proteins.
regulatory
promoters,
(TH012) the
and HBP-2
the novel
three
in the proximal
identified
HBP-lb
protein-l)
(ACGTCA),
gene
in our analyses
and
described
H3
have
HBP-la,
(5,6).
motifs
histone
also
HBP-la
lower
hexamer
(CATCCAACG)
both
the previously
(TH012)
cis-elements:
and nonamer
promoter
specific
HBP-2
three
proteins, which
binds
of
the
to the
0006-291X/91 1593
are
ssDBP-1
sequences
and ssDBP-2
DNA-
$1.50
Copyrighr 0 1991 by Academic Pres.s. Inc. All rights of reproduction in nny fbrrn reserved.
Vol.
176,
No.
upper
BIOCHEMICAL
3, 1991
strand
in
the
overlap
the cis-region
discuss
a possible
transcriptional
same
AND
BIOPHYSICAL
promoters.
These
that has the hexamer function
regulation
of
these
of the H3
RESEARCH
COMMUNICATIONS
protein-binding
and octamer
regions
motifs.
ssDNA-binding
We also
proteins
in
the
gene.
MATERIALS
AND
METHODS
Preparation of nuclear extract: The crude nuclear extract of wheat germ was prepared essentially as described elsewhere (2). Probes and competitor DNA fragments: The DNA fragments used as probes or competitors in the DNA-protein binding assays (Table 1) were synthesized with Cyclone TM Plus (MilliGen/Biosearch). These probes were end-labeled with [y- 32PJATP using T4 polynucleotide kinase. Mobility shift and methylation interference assays: Both the mobility shift and methylation interference assays were conducted as described elsewhere (2, 5). RESULTS
In
spite
binding bind
of the wealth
proteins
HBP-la
specifically
identified.
In
our
search
mobility
probe
double-strand
1.
Table
shift
DISCUSSION
of information
about
and HBP-lb
to the octamer
conducted the
AND
for
motif
Sequence alignments probes and competitors
(3, 5, 6),
no nuclear
of the H3
promoter
octamer-specific
assays DNA
the hexamer-specific
on
wheat
(dsDNA)
proteins have
DNA-binding nuclear
fragment
extract that
DNA-
yet been
proteins, using
contains
of the synthetic oligonucleotides in the DNA mobility shift assay
that
as the both
used as
Sequence
histone
H4
-141/-110
(THOll)
5'-CGGCCACGTCACCGATCCGCGGCATGTCTCCC 3'-GCCGGWGGCTAGGCGCCGTACAGAGGG
Hex (H3) -179/-160
5'-TCGGCCACGTCACCAATCCG (THO12) 3'-AGCCGG-GGTTAGGC
histone
5'-TCGGCCB CCAATCCGCGGCATTC (THO12) 3'-AGCCGG-GGTTAGGCGCCGTAAG
H3 -179/-153
Non-specific -13391-1374
ssDNA 5'-ACTACGAGACACGTCGGACATAGTAG (THOU)
Probes and competitors were used as dsDNA (in Fig. 1) and ssDNA Negative numbers indicate (in Fig. 2). The hexamer motif is underlined. positions in the nucleotides related to the transcriptional initiation site (as +l). 1594
we
the
Vol.
176,
No.
hexamer which
BIOCHEMICAL
3, 1991
and octamer
motifs
has the consensus
octamer
and B2,
when
appeared
2, Fig.
these
shift
BIOPHYSICAL
of the H4
promoter
32P-end-labeled
were
(lane
AND
1A). bands
Prior (lanes
Ii4 *up/low*
(CGCGGATC)
heating
(A)
Two
strands
H4
major
these
H4 up/low’ probe
UPPer
lower
(6) F Bl
aYa4
:-2: -12g
Bl*
**,
.ra
Bl ) B2*
82,
LtQ
.91 I ,xI...-
-128
83, dsDNA ssONA
Free probe probew
ra(L
(C)
m
4W-
12345
6
8 9 10
7
11
12
34
T Ii4
upper
strand
5’-CGGCCACGTCACCdATCCGCGGCATGTCTCCC-3
H4 lower
strand
3’-GCC&TGCAGT&TAGGCGCCGTACAGAGGG-5
Fig. 1. A: Mobility shift and competition assays of wheat germ nuclear extract done with dsDNA fragments containing the regulatory region of 32P-labeled H4 dsDNA fragment wheat histone H4 gene as the probe. probes were incubated with the nuclear extract in the presence or absence of a 20-fold excess of dsDNA fragments as competitors (sequences given in Table 1). Lanes 1-7, the upper and lower strands of the probe were 32P end-labeled (*up/low*); lanes 8 and 9, the upper strand of the probe was 32P-end-labeled (*up/low); lanes 10 and 11, the lower strand of the probe was 32P-end-labeled (up/low*). Lane 7, the nuclear extract was heated at 900 C for 5 min before its addition to the binding mixture. Lanes 1, 8 and 10, no nuclear extract was added to the binding mixture. Arrowheads Methylation indicate probe-protein complexes Bl, B2 and B3. B: interference assays of the Bl and B2 complexes were conducted with the labeled upper strand (lanes 1 and 2) and labeled lower strand (lanes 3 and 4) probes. Lanes 1 and 3 show the respective breakage patterns in the upper and lower strand DNA fragments of the free probes (F). Lanes 2 and 4 show the respective patterns of the probes recovered from the Bl and B2 bands. Arrowheads indicate the positions of methylated guanine residues that interfere with binding. C: Nucleotide sequences of the binding regions of ssDBP-1 and ssDBP-2. The hexamer motif is underlined. 1.595
of
bands really
F 82
Bl *
Bl
the probe
the appearance
that
of
promoter bands,
with
prevented
suggests
Ii4 *up/low probe --
probe
the
was incubated
of the extract
6 and 7), which
both
because
sequence.
extract
COMMUNICATIONS
(THOll);
* probe)
(*up/low
the nuclear
RESEARCH
Vol.
176,
No.
do represent proteins
DNA-protein
in the
probe,
synthetic
prevented
oligonucleotides
promoter
(lane
added.
This
the
mixture
as a competitor,
3),
that the complexes
than
was separable
32P-end-labeled,
not
and an additional
minor
(Fig.
lA,
the complexes
when motif
fragment,
band
of
the
(lane
H3
5) were
the specific
and HBP-lb) this
after
with
DNA-binding
phosphocellulose
Most
lower
(up/low*),
strand
the band
had
been
corresponding
the band corresponding
to B2
appeared
with
strand
probably,
the Bl
(B3)
between
of
through
fact,
whose
lane 11); whereas,
lane 9).
formed
In
H4
double-strand
fragment
and HBP-lb
was used as the probe lA,
unlabeled formation
not
HBP-la
of the
shown).
a dsDNA
(Fig.
from
motif.
HBP-la
(data
when
appeared
but
were formed
(distinct
hexamer
from
chromatography
nuclear
When
DNA
the
(*up/low)
experiments.
coli
other
to Bl
sequences
4) and the Escherichia
sequences
the nuclear
of the
hexamer
protein(s)
COMMUNICATIONS
whether
the
suggests
Interestingly,
determine
containing
of a nuclear
column
RESEARCH
to any
(lane
interaction
protein(s)
bind
to the reaction
was
BIOPHYSICAL
To
specifically competition
was added
complexes
AND
complexes.
extract
we conducted
fragment the
BIOCHEMICAL
3, 1991
single-strand
the labeled
DNA
upper
and B2 bands represent (ssDNA)
fragments
and
proteins.
To confirm
whether
ssDNA
fragments
upper
strand
correspond minor
is so, we conducted
as probes.
was
the
Fig.
probe,
(lane
corresponds
to Bl
experiments
done
2).
two
With
(see Fig. with
shifted
of the B2’
the 1A)
ssDNA
and B3’
bands,
appeared
(lane
9).
formation
strand
of the H3
(Fig.
2A, lanes 12 and 14).
as the
ssDNA
of the Bl’
fragment
band
fragment
1596
the labeled B3’,
which
B3’
being
the
band
Bl’
that
Cross-competition showed
by the lower (Fig.
that strand
2A, lanes 6
was not prevented
nor by the non-specific
These results
When
competitor
bands was not prevented
and 7).
assays with
1A) appeared, strand,
fragments
shift
and
lower
or the non-specific
upper
B2’
labeled
of the H3 fragment Moreover,
mobility
2 shows the results.
to the B2 and B3 bands (see Fig.
band
formation
this
ssDNA
by the fragment
suggest that the upper and lower
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-129
dsoNA probe -
Ilk
1234567
8 9
10 11 12 13 14 12
(C)
H4 upper
strand
S-CGGSfACGTCA$ATCCGCGGCAT&TCTCCC3’
H4 lower
strand
3’-GCCGGTGCAGTGGCTAGGCGCCGTACAGAGGG-3’
34
Fig. 2. A: Mobility shift and competition assays of wheat germ nuclear extract done with ssDNA fragments containing the regulatory region of wheat histone H4 gene as the probe. 32P-labeled H4 ssDNA fragment probes were incubated with the nuclear extract in the presence or absence of a %O-fold excess of ssDNA fragments as competitors (sequences given in Table 1). Lanes 1-7, the upper strand of the probe was 32P-end-labeled (*up); lanes 8-14, the lower strand of the probe was 32P-end-labeled (low*). Lanes 1 and 8, no nuclear extract was added to the binding mixture. Arrowheads indicate the probe-protein complexes Bl’, B2’ and B3’. B: Methylation interference assays of the Bl’ and B2’ complexes were conducted with the labeled upper strand (lanes 1 and 2) and the labeled lower strand (lanes 3 and 4) probes. Lanes 1 and 3 show the respective breakage patterns of the upper and lower strand DNA fragments of the free probes (F). Lanes 2 and 4 show the patterns of the probes recovered from the Bl’ and B2’ bands. Arrowheads indicate the positions of methylated guanine residues that interfere with binding. C: Nucleotide sequences of the binding regions of ssDBP-1 and ssDBP-2. The hexamer motif is underlined.
strands
of the H4 or H3 DNA
fragment
motifs
bind
of
specific lower strand
manner,
of
One
the
that
the
H4
ssDNA
the ssDNA interpretation
dsDNA
fragment before
nuclear
binding
with formation
its
1597
a sequence-
lanes
prevented
is that
anti-ssDNA of
proteins
2A,
and protein
of this
and octamer
of
also
fragment
the hexamer
affinities (Fig.
fragment
possible
competitor
wheat
of the H4 fragment
between
constructed as
the
species
but
than those
complexes 10).
different
containing
the
the
in H3
fragment
5 and 13).
The
formation (Fig. the labeled fragment
protein-probe
of
2A,
anti-
specific
lanes ssDNA
that
are
was
complexes
4 and probe used (free
Vol.
176,
probes
No.
3, 1991
in
lanes
interpretation
4 and
leads
shown
in
Fig.
ssDNA
probe
migrating
BIOCHEMICAL
the
result
dsDNA
that
of
the
the fast-migrating
DNA-binding
ssDNA;
therefore,
(ssDNA
binding
regulatory
proteins we have
the
COMMUNlCATtONS
fragments).
DNA-protein
unannealing
free probe
identified
given
protein-l)
regions
This
complexes
of
complementary
is ssDNA
and the slow-
here
them
which
probably
tentative
binds
are
specific
designations:
to
the
lower
of H3 and H4 genes and ssDBP-2
which
to
ssDBP-1
strand binds
of
the
the upper
of these re,gions.
To locate
the SSDBP-1
we conducted
-137
by methylation
-116 (Figs.
lB,
represent
the
formed
lC,
band
between
the binding
of the guanine
formed
is identical
the upper
binding
(5, 6, Takase
included
in
octamer
proteins
to HBP-la
double-helix
the single-strand motif
considered The
form
of
with our
sequence-specific
ssDNA-binding
systems,
sequence-specific
several
and
of the
moreover,
but,
for
these
when
the DNA change
or HBP-lb
regions
of the open-formed ssDBP-1
and
of
undergoes
of HBP-la
ssDNA-binding
complex
were
relations
motif
In
and
they
ssDBP-1
proteins.
bands
residues
motif,
that
1598
-128
those for HBP-la
yet known;
to the binding
indicate
also
ssDBP-1
guanine
functional
the octamer
these specific
study
The
overlapped
The
and
in terms
unpublished);
to the octamer
strand
at positions
strand
band
are not
including
owing
adjacent
to interact
results
partially
as
-129,
suggests that the B 1 and Bl’
cis-element.
in the region
ssDNA
at positions
residues
B2’
et al.,
and
to the upper
and ssDBP-2.
and HBP-lb
&DNA
residues
the lower
to the
strand
HBP-lb
hexamer
from
of the two proteins
the
This
in the H4 promoter,
strand of the H4 fragment
of ssDBP-2
of the guanine
2B and 2C).
complexes
the B2
to the lower
and the binding
was arrested
regions
assays with
of ssDBP-1
by methylation
and -138
binding
interference
The binding
was prevented -130,
and ssDBP-2
methylation
the probes.
that
RESEARCH
annealed
hypothesis
are the
because
BIOPHYSICAL
one dsDNA.
The
strand
10 indicate
to
1A
AND
and
to
to the
ssDBP-2
are
DNA.
and
ssDBP-2
are
mammalian
and
viral
proteins
have
been
Vol.
176,
No.
reported, DNA
BIOCHEMICAL
3, 1991
some of which double-helix,
transcription. dsDNA
in
(10) proposed
(11) reported
a novel
triple-strand
repressor
elements
capable
nuclear
of c-myc
cis-elements
(12-17).
sequence-specific of genes.
(7,
that
S),
of DNA
and p-interferon
genes
All
this
specifically
evidence
ssDNA-binding
proteins
We are now investigating
information with
suggests in
the
the structures
strand
loop DNA
of
Htun
(H-DNA)
have
and
stretches
of transcription.
a single
interact
(9)
single-strand
containing
that
open the
that
recombination
elements
much
COMMUNlCATfONS
in processes
unpaired,
control
Moreover,
H-DNA.
proteins
to function
RESEARCH
that the H-conformation
type of DNA
of forming
BIOPHYSICAL
replication
are the sites of the actual
and Dahlberg
about
are thought
as
Crick
AND
forms and that stretches
has been reported individual
important
strands functions
of for
transcriptional
regulation
and functions
of ssDBP-
1 and ssDBP-2. ACKNOWLEDGMENTS and K. Mikami for their valuable We are grateful to Drs. T. Nakayama discussions. We also thank Dr. K. Fukuchi of the Research Center, Nisshin This research was Flour Milling Co., Ltd for the gift of wheat germ. supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, by grants from the Naito Foundation, Takeda Science Foundation, and the Research Council, Ministry of Agriculture, Forestry, and Fisheries of Japan, original and creative research project on biotechnology. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
K., Kawata, T., Tabata,T., Kanazawa, Nakayama, T., Ohtsubo, N., Mikami, H., and Iwabuchi, M. (1989) Plant Cell Physiol., 30, 825-832. Mikami, K., Tabata, T., Kawata, T., Nakayama, T., and Iwabuchi, M. (1987) FEBS lett., 223, 273-278. Mikami, K., Takase, H., Tabata, T., and Iwabuchi, M. (1989) FEBS lett., 256, 67-70. Kawata, T., Nakayama, T., Mikami, K., Tabata, T., Takase, H., and Iwabuchi, M. (1988) FEBS lett., 239, 319-323. Tabata, T., Takase, H., Takayama, S., Mikami, K., Nakatsuka, A., Kawata, T., Nakayama, T., and Iwabuchi, M. (1989) Science, 245, 965-967. Tabata, T., Nakayama, T., Mikami, K., and Iwabuchi, M. (1991) EMBO J. in press. Roller, R. J., McCormick, L., and Roizman, B. (1989) Proc. Natl. Acad.Sci. USA, 86, 6518-6522. Traut, W., and Fanning, E. (1988) Mol. Cel. Biol., 8, 903-911. 1599
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No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Edelmann, W., Kroger, B., Goller, M., and Horak, I. (1989) Cell, 57, 937946. Crick, F. (1971) Nature, 234, 25-27. Htun, H., and Dahlberg, J.E. (1990) Science, 241, 1791-1795. Peritz, L.N., Fodor, E.J.B., Silverside, D.W., Cattini, P.A., Baxter, J.D., and Eberhardt, N.L. (1988) J. Biol. Chem., 263, 5005-5007. Rajavashisth, T., Taylor, A.K., Andalibi, A., Svenson, K.L., and Lusis, A.J. (1989) Science, 245, 640643. Lannigan, D. and Notides, A.N. (1989) Proc. Natl. Acad. Sci. USA 86, 863-867. Pan, W.T., Liu, Q., and Bancroft, C. (1990) J. Biol. Chem., 265, 70227028. Wilkison, W-O., Min, H.Y., Claffey, K.P., Satterberg, B. L. and Spiegelman, B.M. (1990) J. Biol. Chem., 265, 477-482. Gailland, C. and Strauss, F. (1990) J. Mol. Biol., 215, 245-255.
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