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Nucleic
Acids
Research,
Vol.
19,
No.
15
4225-4231
A
nuclear
factor
binds
to
the
metal
regulatory
elements
of
the
mouse
gene
encoding
metallothionein-l
Simon
Labbe,
Jacinthe
Prevost,
Paolo
Remondelli1,
Arturo
Leone1
and
Carl
Seguin*
Centre
de
Recherche
en
Cancerologie
de
l'Universite
Laval,
L'H6tel-Dieu
de
Quebec,
Quebec
GiR
2J6
and
Departement
de
Physiologie,
Faculte
de
Medecine,
Universite
Laval,
Quebec
GlK
7P4,
Canada
and
1Dipartimento
di
Biochimica
e
Biotecnologie
Mediche,
11
Facolta
di
Medicina
e
Chirurgia,
Universita
di
Napoli,
Naples
80131,
Italy
Received
March
26,
1991;
Revised
and
Accepted
July
3,
1991
ABSTRACT
The
ability
of
vertebrate
metallothionein
(MT)
genes
to
be
induced
by
heavy
metals
is
controlled
by
metal
regulatory
elements
(MREs)
present
in
the
promoter
in
multiple,
non-identical
copies.
The
binding
specificity
of
the
mouse
L-cell
nuclear
factor(s)
that
interact
with
the
element
MREd
of
the
mouse
MT-I
gene
was
analyzed
by
in
vitro
footprinting,
protein
blotting,
and
UV
cross-linking
assays.
In
vitro
footprinting
analyses
revealed
that
synthetic
oligodeoxynucleotides
(oligomers)
corresponding
to
the
metal
regulatory
elements
MREa,
MREb,
MREc,
MREd
and
MREe
of
the
mouse
MT-I
gene,
as
well
as
the
MRE4
of
the
human
MT-IIA
gene
and
the
MREa
of
the
trout
MT-B
gene,
all
competed
for
the
nuclear
protein
species
binding
to
the
MREd
region
of
the
mouse
MT-I
gene,
the
MREe
oligomer
being
the
weakest
competitor.
In
addition,
protein
blotting
experiments
revealed
that
a
nuclear
protein
of
108
kDa,
termed
metal
element
protein-1
(MEP-1),
which
specifically
binds
with
high
affinity
to
mouse
MREd,
binds
with
different
affinities
to
the
other
mouse
MRE
elements,
mimicking
their
relative
transcriptional
strength
in
vivo:
MREd
2
MREa
=
MREc
>
MREb
>
MREe
>
MREf.
Similarly,
human
MRE4
and
trout
MREa
bind
to
MEP-1.
A
protein
similar
in
size
to
MEP-1
was
also
detected
in
HeLa-cell
nuclear
extracts.
In
UV
cross-linking
experiments
the
major
protein
species,
complexed
with
mouse
MREd
oligomers,
migrated
on
a
denaturating
gel
with
an
apparent
Mr
of
115
000
and
was
detected
using
each
of
the
mouse
MRE
oligomers
tested.
These
results
show
that
a
mouse
nuclear
factor
can
bind
to
multiple
MREs
in
mouse,
trout,
and
human
MT
genes.
INTRODUCTION
Metallothioneins
(MTs)
are
small
cysteine-rich
proteins
that
bind
heavy
metal
ions
such
as
Cd2+,
Zn2+
and
Cu2
+
(1,2).
The
genes
encoding
MTs
are
inducible
at
the
transcriptional
level
by
the
same
metal
ions
that
the
MTs
bind.
Heavy
metal
activation
of
MT
gene
transcription
is
dependent
on
the
presence
of
short
cis-acting
elements,
termed
metal
regulatory
elements
(MREs),
which
are
present
in
six
non-identical
copies
(MREa
through
MREf)
in
the
5'
flanking
region
of
the
mouse
MT-I
gene
(3-7).
Synthetic
copies
of
four
of
the
mouse
MT-I
MRE
sequences
are
able
to
confer
heavy-metal
regulation
on
a
heterologous
promoter.
MREe
and
MREf
elements
are
apparently
inactive
while
MREd
is
the
strongest
element
for
metal
induction
(6,8).
In
addition,
the
MREd
element
has
the
capacity
to
respond
to
the
same
spectrum
of
metal
ions
(Cd2+,
Zn2+
and
Cu2+)
as
does
the
complete
MT
gene
promoter,
suggesting
that
all
MRE
elements
are
responsive
to
the
different
metals
and
together
act
to
facilitate
a
strong
induction
response
(9).
Competition
experiments
suggested
that
one
or
more
positively
acting
transcription
factors
interact
with
these
elements
(10,11).
The
mechanism
by
which
these
factors act
as
positive
regulatory
proteins
in
the
presence
of
metals
is
still
unknown.
It
has
been
postulated
that
heavy
metals
increase
transcription
by
inducing
the
binding
of
a
regulatory
factor
to
the
MREs
(3-7,
12-14).
However,
it
is
not
yet
known
whether
metal
induction
of
MT
gene
transcription
involves
different
factors
binding
to
different
MRE
sequences
or
a
single
factor
binding
to
different
MREs
with
different
affinities.
In
vivo,
dimethylsulphate
protection
has
been
observed
on
all
MREs
upon
stimulation
both
in
the
mouse
(7)
and
the
rat
(14).
In
vitro,
metal-dependent
binding
of
factors
has
been
observed
both
to
the
mouse
MREd
(13,15)
and
MREa
(8)
elements
as
well
as
to
the
rat
MT
gene
promoter
(16).
Imbert
et
al.
(17)
have
shown
that
MBF-1,
a
mouse
nuclear
factor
of
74
kDa
purified
to
near
homogeneity,
can
bind
to
the
MREa
element
of
the
trout
MT-B
gene
as
well
as
to
MREd
and
MREe
of
the
mouse
MT-I
gene.
However,
this
factor
was
reported
not
to
bind
to
the
other
MREs
of
the
mouse
gene.
We
have
previously
shown
that
a
mouse
nuclear
factor
of
108
kDa,
here
designated
MEP-1,
binds
with
high
affinity
to
the
MREd
of
the
MT-I
gene
(18).
To
determine
if
different
MRE
elements
can
bind
common
nuclear
factors,
we
performed
competition
experiments
in
an
exonuclease
III
(ExolII)
footprinting
assay.
In
addition,
we
performed
protein
blotting
and
UV
cross-linking
experiments
to
assess
the
binding
properties
*
To
whom
correspondence
should
be
addressed
.=)
1991
Oxford
University
Press
4226
Nucleic
Acids
Research,
Vol.
19,
No.
15
of
MEP-1
to
different
MRE
elements.
Our
results
show
that
the
protein(s)
binding
to
the
MREd
region
of
the
mouse
MT-I
gene
can
bind with
different
affinities
to
other
MRE
elements.
Moreover,
MEP-1
can
bind
to
the
different
MREs
present
in
the
promoter
of
the
mouse
MT-I
gene
as
well
as
to
the
MREa
of
the
trout
MT-B
gene
and
the
MRE4
of
the
human
MT-HA
gene.
MATERIALS
AND
METHODS
Cell
culture
and
footprinting
analyses
HeLa
(Strain
S3,
American
Type
Culture
Collection)
and
heavy
metal-resistant
mouse
L
cells
(L50)
(generously
provided
by
D.H.
Hamer)
were
grown
in
suspension
in
SMEM
supplemented
with
glutamine,
antibiotics
and
10%
fetal
calf
serum.
L50
cells
were
grown
in
the
continuous
presence
of
15
,uM
CdCl2
(15)
while
HeLa
cells
were
treated
with
5
ytM
CdCl2
for
10
hours
before
harvesting
by
centrifugation.
All
buffers
used
to
prepare
crude
nuclear
extracts
contained
5
AtM
CdCl2
(13).
ExoHl
footprinting
analyses
were
performed
as
described
by
Seguin
and
Hamer
(13).
The
MT
fragment
used
as
the
probe
was
5'
end-labelled
at
position
+64
and
extends
to
position
-200
relative
to
the
start
point
of
transcription.
Gel-purified
oligodeoxynucleotide
(oligomer)
competitors
(Fig.
1B)
were
annealed
to
form
double-stranded
DNA
in
medium
salt
buffer
(19)
by
slow
cooling
from
95°C
to
room
temperature.
The
concentration
of
each
single
stranded
oligomer
DNA
was
measured
by
spectrophotometry.
The
percentage
of
double
stranded
molecules
was
evaluated
by
gel
electrophoresis
and
was
always
greater
than
95%.
Competitors
were
added
together
with
the
probe
and
binding
was
allowed
to
proceed
for
20
min
at
24°C.
The
samples
were
loaded
and
electrophoresed
at
a
constant
current
of
30
mAmp
on
5
%
sequencing
gels
(38:2
acrylamide:bisacrylamide).
The
gels
were
dried
and
autoradiographed
by
standard
procedures.
Densitometry
was
performed
on
multiple
exposures
of
each
gel
using
a
Joyce-Loebl
Chromoscan
3
densitometer.
Protein
transfer
analyses
The
protein
blotting
procedure
(South
Western)
was
performed
with
double
stranded
oligomers
(Fig.
iB)
freshly
labelled
with
polynucleotide
kinase
and
[-y32P]-ATP
at
a
specific
activity
of
2000
to
2500
cpm/fmol
(18).
Typically,
30
Al
of
crude
nuclear
B
-54
-40
4
RZ
4
1M5a:
5'-
GATCCCTT_gGGGACTA
-3'
GGAAACGCGGGCCTGATCTAG
GGCCTGAT
-56
-70
NREb:
5
-
GATCCGTTTgfC,&GCAGA
-3'
GCAAACGTGGGTCGTCTCTAG
GGTCGTCT
-132
-118
aKc:
5'-
GATCCAAG
_OGCTCA
-3'
GTTCACGCGAGCCGAGTCTAG
AGCCGAGT
UNREdl:
5'-
-
CGATGGCAGdTC=GLOYCGCCCGAAAAGTGC-_GAT
--3'
TACCCTCGAGACGTGAGGCGGGCTTTTCACGCTAGC
-150
000oe-
ea*TTTTCACGCT
Spl
MREc
-136
41
4
dAMds:
5'-
cGATCTcT&qg
cGCCCGA
-3'
TAGAGACGTGAGGCGGGCTGC
GGCGGGCT
-175
-161
4
41
UNRE:
5'-
GATCCCTGZgkTGGCGA
-3'
GGACACGTGTGACCGCTCTAG
TGACCGCT
-94
-80
NREf:
5'-
GATCCCTA2gGCTGGA
-3'
GGATACGCACCCGACCTCTAG
MREe
MRE
d
MRE
c
-*
-o
- b -
-175
-150
-132
MRE
f
MREb
MRE
a
-94
-70
-54
hNRE4:
5'-
-154
-133
4-MR4
r
3-
-63
-49
4
4
tNRa:
5'-
CGATT
CCAV
AGGCACAT
-3'
TAAAACGTGTGCCGTGTAGC
Figure
1.
A)
Arrangement
of
the
six
metal
regulatory
elements
(arrows)
of
the
mouse
MT-I
gene,
the
binding
sites
for
the
transcription
factor
SpI
(7,23)
the
G-rich
sequence
interacting
with
the
transcription
factor
MLTF
(22)
and
the
TATA
box.
The
hatched
box
indicates
the
protected
region
around
MREd
in
ExoIll
footprinting
experiments.
B)
Sequences
of
the
synthetic
oligomers.
The
underlined
nucleotides
correspond
to
the
conserved
MRE
core
sequence
TGCRCNC
(R,
purine;
N,
any
nucleotide).
On
the
mMREdl
oligomer,
dots
below
the
sequence
indicate
similarity
with
the
consensus
Spl
binding
site;
black
dots
indicate
agreements
with
unique
nucleotides,
white
dots
indicate
agreements
with
ambiguous
nucleotides
and
the
asterix
indicates
disagreement
at
an
ambiguous
nucleotide.
Vertical
arrows
indicate
the
boundaries
(-153
and
-127)
of
the
protected
region
in
an
ExoHII
footprinting
analysis
(13).
The
arrows
on
the
other
MRE
elements
indicate
the
region
covering
the
MRE
consensus
sequences
(6).
Brackets
indicate
the
positions
of
MREd
and
MREc
consensus
sequence
on
the
mMREdl
oligomer
and
the
positions
of
MRE4
and
MRE3
core
sequence
on
the
hMRE4
synthetic
DNA.
Shorter
italicized
DNA
sequences
beneath
each
paired
MRE
element
correspond
to
the
primer
used
to
generate
the
probe
for
the
UV
cross-linking
analyses.
MT
sequences
extend
to
positions
-155
and
-126
on
mMREdl
and
to
position
-134
on
mMREds.
The
mMREb
oligomer
is
in
the
inverse
orientation.
MRE
oligomers
were
synthesized
with
a
BamHI
(mMREa,
mMREb,
mMREc,
mMREe
and
mMREf)
or
a
Clal
(mMREdl,
mMREds,
hMRE4
and
tMREa)
site
on
both
ends.
The
trout
element
is
from
the
MT-B
gene
and
the
human
element
from
the
MT-IIA
gene.
-153
MREd
4
-127
A
~j
L-
p
~ W
I
I
Spl
Spi
MLTF
+1
-28
)
EC.,
--LmT-i
TATAAA
CGAGGCCCAGNCCAQT
-:
TACGCGGGCCGGGTCACGCGCGTAGC
*3
,
Nucleic
Acids
Research,
Vol.
19,
No.
15
4227
extracts
(10
mg
protein/ml,
as
determined
by
the
BioRad
protein
assay)
were
electrophoresed
on
NaDodSO4/PAGE
using
8%
acrylamide-bisacrylamide
(50:1)
separating
gels
and
transferred
to
Immobilon-PVDF
membranes
(Millipore).
Filter
strips
were
incubated
for
4h
in
binding
buffer containing
105
cpm
of
32p-
labelled
DNA
probe
per
ml.
The
relative
intensity
of
the
signal
obtained
with
the
different
MRE
probes
was
evaluated
by
laser
densitometry
and
a
value
of
one
was
arbitrarily
assigned
to
the
signal
obtained
with
the
mMREdl
oligomer.
UV
cross-linking
A
UV
cross-linking
protocol
to
determine
the
molecular
weight
of
the
MRE-binding
protein(s)
in
crude
nuclear
extracts
was
developed,
based
on
those
described
by
others
(17,20,21).
The
probe
was
prepared
by
hybridizing
a
21-base
oligomer
of
the
coding
strand
of
the
different
MRE
elements
from
the
mouse
MT-I
gene
to
an
8-
or
10-base
complementary
primer
(Fig.
1B).
These
oligomers
were
made
completely
doubled
stranded
by
incubation
with
the
Klenow
fragment
of
DNA
polymerase
I
in
the
presence
of
the
appropriate
[a32P]-dNTP
and
the
three
other
unlabelled
nucleotides.
The
[a
32P]-labelled
nucleotide
was
dC-
TP
for
MREa
and
MREds,
dTTP
for
MREb,
MREdl
and
MREe,
and
dATP
for
MREc.
Each
reaction
mixture
contained
20
fmol
of
oligomer
probe
(5
x
104
to
5
x
I05
cpm),
10
Atg
of
yeast
tRNA
(Gibco-BRL),
1
,Ag
of
p(dN)5
(Pharmacia),
0.5
ltg
of
MspI
digest
of
lambda
DNA
and
20
pd
of
crude
L50-cell
nuclear
extracts
(10
mg/ml)
in
buffer
IH
(13).
The
final
reaction
volume
was
brought
to
50
yl
with
binding
buffer
(20
mM
HEPES,
pH
7.9;
50
mM
NaCl;
1
mM
DTT;
5
mM
MgCl2;
5%
glycerol;
0.1
mM
EGTA).
After
DNA
protein
binding
(20
min,
24°C),
reaction
mixtures
were
irradiated
for
20
min
at
4°C
with
a
254
nM
UV
lamp
(600
,oW/cm).
When
specified,
DNase
I
(Worthington
Diagnostics,
2064
units/mg)
was
added
as
indicated
in
the
figure
legends
and
the
mixture
incubated
at
37°C
for
15
min.
Fractions
were
resolved
by
8%
NaDodSO4-polyacrylamide
(acrylamide/bisacrylamide:
30-0.8)
gel
electrophoresis
and
analyzed
by
autoradiography.
RESULTS
Exonuclease
III
footprinting
competition
experiments
Fig.
1A
shows
the
arrangement
of
the
six
MRE
elements
on
the
mouse
MT-I
gene
(3,5,7,12),
the
G-rich
sequence
that
interacts
with
the
major
late
transcription
factor
MLTF
(22)
and
the
two
Spl
sites
(7,23).
Using
an
in
vitro
ExoIII
footprinting
assay,
we
have
previously
shown
(13)
that
one
or
more
nuclear
proteins
bind
to
the
MREd
region
between
nucleotides
-127
and
-
153
relative
to
the
start
point
of
transcription
(Fig.
lA,
hatched
box).
The
DNA
sequence
recognized
by
this
factor
is
the
same
as
that
required
for
in
vivo
transcriptional
activity
of
MREd
(9),
since
substitution
of
residues
G
or
C
of
mMREds
(nucleotides
-146
and
-143,
see
Fig.
IB)
by
A
(MUT2)
or
T
respectively,
completely
abolished
binding
activity
(15).
Zinc
ions
are
required
for
specific
in
vitro
DNA
binding
of
the
MREd-binding
protein(s)
the
latter
which
is
different
from
the
transcription
factor
Spl
(15,18,
Labbe
and
Seguin,
unpublished
results).
To
determine
if
the
MREd-binding
protein(s)
present
in
mouse
extracts
can
also
bind
to
the
other
MREs
of
the
mouse
MT-I
gene
or
to
MRE
elements
present
in
MT
genes
from
other
species,
we
performed
competition
experiments
with
synthetic
oligomers
corresponding
to
the
individual
MREs.
The
sequences
of
the
different
oligomers
used
are
shown
in
Fig.
IB:
mMREdl
corresponds
to
the
protected
region,
as
assayed
by
ExollI
footprinting,
in
the
mouse
MREd
region,
namely
the
entire
MREd
sequence
and
the
5'
end
portion
of
MREc
(nucleotides
-127
to
-153);
mMREds
contains
only
the
MREd
consensus
sequence
(nucleotides
-134
to
-150);
mMREa,
mMREb,
mMREc,
mMREe
and
mMREf
correspond
to
the
different
MREs
present
in
the
mouse
MT-I
gene.
Oligomers
corresponding
to
the
MRE3-MRE4
region
of
the
human
MT-
HA
gene
(hMRE4)
and
MREa
of
the
trout
MT-B
gene
(tMREa)
were
also
synthesized.
All
of
the
MREs
tested
could
compete
with
the
protein(s)
binding
to
the
mouse
MREd
region
(Fig.
2),
indicating
that
the
same
cellular
factor
binds
to
all
of
these
MRE
elements;
mMREdl,
mMREds,
mMREa,
mMREb
and
mMREc
showed
B
A
mMTI
MREd
(-200,+64)
MREa
MREc
MREe
0
0
0
0
0
E-4
H
EA
C)
0.
0.
_m
W.
u
......-
_w
*-
-
-_
A
AMN
"M
.1
_
gm
.m
-.O.-
mMRE&
-0-
MMREdl
-_-
mMREb
-0-
MiREe
-A-
MREC
0,
0,
U
(XMTg
a
I
+
mMREds
-
m-
WMT-I
-{-
hMRE4
mMREdl
--~-tMREa
1.2
1
0.
8
0.6-
0.42
0
20 40 60
80
1O0
COMPETITOR
(ng)
Figure
2.
A)
ExoIII
footprinting
assay
of
a
representative
competition
experiment
using
crude
nuclear
extracts
prepared
with
heavy
metal-resistant
mouse
L
cells
(L50)
grown
in
the
continuous
presence
of
15
IoM
CdCI2,
as
described
in
Material
and
Methods.
Competition
was
performed
with
double-stranded
unlabelled
oligomers
corresponding
to
the
various
mouse
MRE
elements,
as
indicated,
or
with
the
same
MT
DNA
fragment
(mMTI)
(-200
to
+64)
used
as
a
probe
in
the
ExolIl
assay.
The amount
of
competitor
oligomer
used
in
each
reaction
is
shown
above
the
lanes.
The
probe
was
used
at
a
concentration
of
approximately
1
ng
per
assay.
The
MREd
oligomer
used
in
this
experiment
is
mMREdl.
The
arrow
indicates
the
ExoII
stop
at
the
-
153
boundary.
B)
Graphic
representations
of
ExoIII
footprinting
competition
experiments
performed,
as
in
Fig.
2A,
with
induced
L50-cell
nuclear
extracts
and
unlabelled
oligomers
corresponding
to
the
indicated
MRE
elements.
The
DNA
competitor
coMTgal
is
a
plasmid
in
which
most
of
the
MT
promoter
sequences
have
been
replaced
by
a
fragment
of
the
human
co-globin
gene
(10);
no
MRE
element
is
present
in
this
DNA.
The
intensity
of
the
band
at
-153
(arrow
in
Fig.
2A)
relative
to
that
with
no
competitor
DNA
(relative
activity)
is
shown
as
a
function
of
the
concentration
of
competitor
DNA.
The
intensity
of
the
bands
was
measured
by
scanning
the
films
of
multiple
exposures
of
each
gel
with
a
transmission
densitometer.
4228
Nucleic
Acids
Research,
Vol.
19,
No.
15
similar
competition
strength,
while
mMREe
was
al
50%
weaker.
Competition
experiments
were
also
pe
synthetic
oligomers
corresponding
to
the
MREa
of
th
gene
and
to
a
region
of
the
human
MT-IIA
gene
MRE4
element
and
the
5'
end
portion
of
MRE3
-
133
to
-154),
a
region
interacting
with
mouse
nuclear
proteins
as
assayed
by
Dnase
I
footprinting
P.,
Seguin,
C.
and
Leone,
A.,
unpublished
results)
oligomers
competed
equally
well
and
as
strongly
(Fig.
2B).
The
heterologous
competitor
DNA,
a
Simian
Virus
40-based
plasmid,
containing
hun
promoter
sequences
fused
to
a
minimal
MT
promot
-34
(10);
this
construct
does
not
contain
any
M
and
did
not
compete
for
the
MREd-binding
protein(
A
B
C
D
E
*
f!
::
}
..
.
f<
~~~~~~~~~~~~~~~~~~~Jlf-
_en
Figure
3.
Binding
of
oligomer
MRE
variants
to
L50-cell
nuclear
proteins
as
assayed
by
the
protein
blotting
procedure
(South-Western)
A)
Mouse
MRE
oligomer
probes
MREdl
to
MREe
as
designated
above
the
lanes.
A
shorter
exposure
of
the
band
corresponding
to
the
108
kDa
protein
species
(MEP-1)
is
shown
at
the
bottom
of
the
figure.
M:
14C
labelled
protein
markers.
B)
Binding
of
the
mouse
MREds
(lane
1)
and
the
MUT2
(lane
2)
oligomer
probes
to
MEP-1.
MUT2
is
a
nonfunctional
mutant
in
which
the
G
at
position
-146
has
been
replaced
by
an
A
(15).
C)
Binding
of
the
tMREa
oligomer
probe
to
L50-cell
nuclear
proteins
(lane
1).
The
gel
was
loaded
with
300
jig
of
extract.
D)
Relative
binding
affinity
of
the
mouse
MREf
oligomer.
The
mouse
MRE
probes
are
designated
above
the
lanes.
E)
Analysis
of
the
binding
of
human
and
mouse
MRE
oligomers
to
nuclear
proteins
using
hMRE4
(lanes
I
-5)
or
mMREdl
(lanes
6-10)
as
the
ligand.
Nuclear
extracts:
lanes
1
and
6,
L50-cell
crude
nuclear
extracts;
lanes
2-5
and
7-
10,
HeLa-cell
crude
nuclear
extracts.
The
indicated
amount
of
protein
(in
jig)
was
loaded
on
the
gel.
Arrows
correspond
to
MEP-1.
pproximately
rformed
with
e
trout
MT-B
covering
the
(nucleotides
Dh
-aE
"A
le'IX"I
Thus,
the
protein(s)
binding
to
the
mouse
MREd
region
can
bind
the
other
MREs
present
in
the
mouse
gene
as
well
as
MREs
from
MT
genes
of
other
species.
South
Western
analyses
(Remonudelli
We
have
previously
shown,
using
a
protein-blotting
procedure
All
of
these
(South
Western),
that
a
mouse
protein
of
108
kDa
(p1O8),
distinct
Al
REl
ofthese
from
Spi,
binds
with
high
affinity
to
the
mMREdl
oligomer
(18).
as
mMREdl
To
assess
more
directly
the
binding
properties
of
the
different
MTgal,
is
a
MREs,
South
Western
analyses
were
performed
with
the
same
nan
cz-globin
MRE
oligomers
used
in
the
Exolll
footprinting
competition
ter
at
position
experiments.
The
oligomers
mMREdl,
mMREa,
mMREc
'RE
elements
(Fig.
3A)
and
mMREds
(Fig.
3B,
lane
1)
bound
strongly
to
the
(s)
(Fig.
2B).
108
kDa
protein
species,
while
the
binding
was
3
fold
lower
for
mMREb,
6
fold
lower
for
mMREe
and
more
than
20
fold
lower
for
mMREf
(Fig.
3A
and
3D).
The
nonfunctional
core
mutant
MUT2,
which
is
inactive
in
vivo
(9),
does
not
compete
for
the
MREd-binding
protein(s)
in
an
Exolll
footprinting
assay
(15),
and
did
not
bind
to
p108
(Fig.
3B,
lane
2).
These
results
correlate
very
well
with
the
relative
strength
as
metal-dependent
cis-acting
transcriptional
elements
of
these
MREs
in
vivo.
Strong
binding
.A;.
_
of
p108
to
hMRE4
(Fig.
3E,
lane
1)
and
tMREa
(Fig.
3C)
probes
was
also
detected.
As
shown
in
Fig.
3E,
the
mMREdl
and
hMRE4
oligomers
also
bound
to
a
HeLa-cell
nuclear
protein
of
108
kDa.
The
p108
MRE-binding
protein
detected
by
the
protein
blotting
procedure
will
be
henceforth
referred
as
to
MEP-
1
(metal
element
protein-1).
Detection
of
MEP-1
by
UV
cross-linking
analyses
A
mouse
nuclear
protein
of
74
kDa,
MBF-
1,
binds
to
the
mMREds
oligomer,
as
assayed
by
UV
cross-linking
(17).
However,
MBF-
1
was
not
detected
by
the
protein-blotting
procedure
used
in
this
study.
To
compare
the
molecular
weight
of
the
MRE-binding
protein
species
detected
with
the
two
assays,
UV
cross-linking
experiments
were
performed
with
crude
L50-cell
nuclear
extracts.
The
major
protein
species
complexed
with
the
mMREdl
(Fig.
4A,
lane
1
and
4B,
lanes
1
-7,
arrows)
or
mMREds
(Fig.
4B,
lane
8)
oligomers
(and
cross-linked
by
UV
irradiation)
migrated
on
a
denaturating
gel
with
an
apparent
Mr
of
115
000.
When
probe
DNA
was
irradiated
in
the
absence
of
protein
or
when
UV
irradiation
was
omitted,
no
labelled
species
were
generated
(data
not
shown).
Moreover,
using
this
UV
irradiation
assay,
all
of
the
mouse
MRE
elements
tested
were
able
to
form
the
same
complex
of
115
kDa
(Fig.
5A
and
SB).
Since
the
covalent
attachment
of
short
oligomers
to
proteins
has
only
a
minor
effect
on
the
mobility
of
these
proteins
in
SDS-
polyacrylamide
gel
electrophoresis
(20,21),
these
experiments
indicate
that
a
protein
of
approximately
115
kDa
binds
to
the
MRE-binding
sites.
The
molecular
weight
of
this
protein
is
consistent
with
that
of
MEP-1.
The
amount
of
label
in
this
115
kDa-species
increased
with
increasing
amount
of
probe
and,
at
high
probe
concentrations,
some
lower
molecular
weight
proteins
became
radiolabelled
(Fig.
5);
these
protein
species
were
not
further
analyzed.
Furthermore,
while
the
chelating
agent
EDTA
led
to
a
45
percent
inhibition
of
complex
formation
with
the
mMREdl
oligomer,
Zn2+
could
restore
the
binding
activity
of
chelated
extracts
to
135
percent
of
the
control
(data
not
shown).
Finally,
the
sensitivity
of
the
complex
to
DNase
digestion
was
analyzed.
In
contrast
to
the
complexes
formed
with
MBF-1,
the
UV-irradiated
protein-DNA
complex
of
115
kDa
detected
in
these
experiments
was
very
sensitive
to
DNase
I
digestion
and
10
units
----*
ltw
:1.1
%*
0-
Nucleic
Acids
Research,
Vol.
19,
No.
15
4229
of
DNase
I
was
sufficient
to
destroy
most
of
the
complex
(Fig.
4A,
lane
2
and
Fig.
4B,
lanes
5-7).
DISCUSSION
We
have
used
an
ExoIl
footprinting
assay
to
ask
whether
the
mouse
nuclear
protein
binding
to
the
MREd
region
of
the
mouse
MT-I
gene
promoter
can
bind
to
other
MRE
elements.
We
have
demonstrated
that
all
of
the
mouse
MRE
elements
tested,
as
well
as
MREs
present
in
human
and
trout
MT
genes,
are
able
to
compete
for
the
protein
species
binding
to
the
mouse
MREd
region.
In
addition,
protein
blotting
experiments
demonstrated
that
MEP-1
binds
with
different
affinities
to
the
six
MRE
elements
of
the
mouse
MT-I
gene
as
well
as
to
a
human
and
a
trout
element.
MEP-1
was
also
detected
in
HeLa
cells
using
either
a
mouse
or
a
human
oligomer
as
probe.
By
UV
cross-linking
analyses,
we
have
detected
a
DNA-protein
complex
migrating
on
SDS-polyacrylamide
gels
with
a
size
of
approximately
115
kDa,
which
is
consistent
with
the
size
of
MEP-1.
Using
synthetic
mouse
MRE
sequences,
it
has
been
shown
that,
in
vivo,
MREd
is
the
strongest
element,
MREa
and
MREc
are
50
to
80
percent
weaker,
MREb
is
90
percent
weaker
and
MREe
is
apparently
non-functional
(6).
Here,
we
have
shown
that,
in
footprinting
assays,
the
mMREdl,
mMREds,
mMREa,
mMREb,
mMREc,
hMRE4,
and
tMREa
oligomers
all
competed
with
similar
strength
for
the
protein
species
binding
to
the
mouse
MREd
region
(Fig.
2).
Only
mMREe,
a
non
functional
element,
clearly
showed
a
weaker
competitive
activity.
Moreover,
the
binding
affinity
of
MEP-
1,
as
assayed
by
South
Western
analysis,
is
equivalent
for
the
mMREdl,
mMREds,
mMREa,
mMREc,
hMRE4
and
tMREa
oligomers,
while
it
is
3
fold
lower
for
mMREb
and
6
to
20
fold
lower
for
mMREe
and
mMREf
(Fig.
3).
The
low
binding
affinity
of
mMREf
is
in
agreement
with
the
inability
of
this
element
to
confer
regulatory
activity
in
vivo
(8).
Overall,
there
is
a
good
correlation
between
the
binding
affinity
of
MEP-1
toward
the
different
mouse
MREs,
as
assayed
in
vitro
by
South
Western
analyses,
and
their
relative
strength
A
B
-
200
kDa
-92.5
for
transcriptional
metal
induction
in
vivo
(6).
This
suggests
a
central
role
of
MEP-1
in
the
regulation
of
MT
genes
transcription.
Two
kinds
of
evidence
suggest
that
MEP-1
is
one
of
the
proteins
which
is
competed
out
of
the
MREd
region
by
the
different
MRE
oligomers,
as
assessed
by
footprinting
(Fig.
2).
First,
MEP-
1
binds
to
MREd
probes
with
high
affinity.
Second,
the
relative
binding
affinity
of
any
MRE
oligomer
for
MEP-1
corresponds
reasonably
well
to
the
relative
ability
of
that
oligomer
to
compete
with
the
MREd-binding
proteins.
In
addition,
mutants
such
as
MUTd,
which
has
all
five
of
the
most
strongly
conserved
nucleotides
of
the
MRE
consensus
sequence
changed
(18),
or
MUT2
which
contains
a
transition
at
the
nucleotide
-146
in
the
core
MRE
consensus
region
(15)
do
not
bind
MEP-1
(18
and
Fig.
3B)
and
do
not
compete
for
the
MREd-binding
protein(s)
(15,18).
Thus,
nucleotides
important
for
competing
out
the
factors
binding
to
the
MREd
region
are
also
required
for
binding
to
MEP-1.
Consequently,
it
is
likely
that
MEP-1
contributes
to
the
footprint
observed
in
the
MREd
region.
However,
further
experiments
will
be
required
to
provide
definitive
proof
of
this.
Three
other
nuclear
proteins
have
been
reported
to
bind
the
mouse
MREd
element,
namely
MBF-1
(17),
ZAP
(8)
and
MTF-1
(24).
MBF-1
is
a
mouse
nuclear
factor
of
74
kDa
which
has
been
detected
by
UV
cross-linking
analyses,
using
a
mouse
MREd
oligomer
as
probe,
and
was
purified
to
near
homogeneity
employing,
as
an
affinity
reagent,
the
trout
MREa
element.
However,
it
is
not
yet
known
whether
MBF-1
can
bind
directly
to
MRE
sequences
other
than
the
trout
MREa.
Imbert
et
al.
(17)
have
observed
only
a
weak
footprint
over
the
trout
MREb
element
and
a
strong
footprint
over
the
mouse
MREe
region
but
no
apparent
binding
over
the
other
MRE
regions
of
the
mouse
promoter.
It
is
not
clear
why
MBF-
1
was
not
detected
by
any
of
the
binding
assays
used
in
this
study
since
the
mouse
MREd
oligomer
used
to
detect
MBF-1
by
UV
cross-linking
analyses
was
identical
to
mMREds
which
bound
MEP-1
as
shown
both
by
South
Western
(Fig.
3B)
and
UV
cross-linking
(Fig.
4B)
analyses.
A
lower
affinity
of
MBF-1
for
the
MREd
sequences
could
explain
why
it
was
not
detected
in
the
South
Western
assay
0
0
0
0
10
5
2.5
0
Units
20 50
100
200
P9
H
4lI
J-I
_
__
__
._
-_
-
69
-
46
2
3
4
5
6
7
8
2
M
Figure
4.
MRE
affinity
labelling
performed
by
UV
cross-linking
as
described
in
Materials
and
Methods
A)
DNA
affinity
labelling
of
mMREdl-binding
factors
in
a
L50-cell
crude
nuclear
extract
incubated
for
20
min
at
24°C
followed
by
an
incubation
in
the
presence
of
10
mM
MgCl2
and
0
(lane
1)
or
20
(lane
2)
units
of
DNase
I.
The
band
at
the
bottom
of
lane
1
corresponds
to
the
free
oligomer
probe.
B)
The
effect
of
increasing
the
amount
of
nuclear
proteins
or
incubating
with
different
amounts
of
DNase
I.
The
amount
of
protein
extract
in
the
binding
reaction
(20-200
jog)
and
the
amount
(0-
10
units)
of
DNase
I
are
shown
above
the
lanes.
Incubation
with
DNase
I
was
for
15
min
at
37'C.
The
oligomer
probe
was
mMREdI
(lanes
1-7)
or
mMREds
(lane
8).
Arrows
indicate
the
approximately
115
kDa
DNA-protein
complex.
4230
Nucleic
Acids
Research,
Vol.
19,
No.
15
A
B
i
Figure
5.
UV
cross-linking
experiments
using
the
different
mouse
MRE
elements
as
probes.
Oligomer
probes.
A)
Lanes:
1-3,
mMREdl;
4-6,
mMREa;
7-9,
mMREe;
B)
Lanes:
10-12,
mMREdl;
13-15,
mMREb;
16-18,
mMREc.
Increasing
amounts
(1.2,
2.5
and
5.0x
105
cpm)
of
labelled
oligomers
where
incubated
with
150
ltg
of
a
L50-cell
nuclear
extract.
The
film
in
B)
was
exposed
twice
as
long
as
in
A)
while
differences
in
the
binding
buffer,
the
assay
conditions
and
the
cell
types
used
to
prepare
the
nuclear
extracts
could
explain
the
difference
in
binding
activities
detected
by
us
and
by
Imbert
et
al.,
(17)
in
UV
cross-linking
experiments.
Nevertheless,
these
results
show
that
MRE
elements
like
mMREd
and
tMREa
can
bind
at
least
two
protein
species,
namely
MBF-1
and
MEP-1.
There
is
strong
evidence
that
MBF-1
is
distinct
from
MEP-1
and
does
not
represent
a
degradation
product
of
MEP-
1
and
that
MEP-1
does
not
represent
a
dimerization
complex
of
MBF-1.
Indeed,
MBF-1
has
been
reported
not
to
bind
to
the
mouse
MREa,
MREb
or
MREc
elements
(17)
while
MEP-l
clearly
does
(Figs.
3
and
5).
Moreover,
in
contrast
to
MBF-
I
which
interacts
strongly
with
the
mouse
MREe
region,
MEP-1
shows
little
affinity
for
the
mMREe
oligomer
(Fig.
3A
and
3D).
In
addition,
the
mMREd
oligomer-protein
complexes
formed
by
MBF-
1
and
MEP-1
are
different
with
respect
to
their
sensitivity
to
DNase.
While
MBF-I
complexes
were
resistant
to
1000
units
of
DNase
I
(17),
10
units
was
sufficient
to
destroy
most
of
the
MEP-1
complexes
(Fig.
4B).
Two
factors
have been
detected
in
nuclear
extracts
which
bind
to
mouse
MRE
elements
in
a
Zn2+-dependent
manner.
MTF-1
(24)
is
present
in
HeLa
cells
and
binds
to
the
mouse
MREd
element
while
ZAP
(8)
is
found
in
rat
liver
cells
and
binds
to
the
mouse
MREa
and
MREd
elements.
ZAP
has
been
proposed
to
be
the
rat
equivalent
of
MTF-
1
(8).
While
the
molecular
weight
of
MTF-l
has
not
been
reported,
preliminary
protein
blots
suggest
a
size
for
ZAP
similar
to
that
of
MEP-
1
(8).
However,
more
studies
will
be
required
to
established
the
precise
relation
between
MEP-1,
ZAP
and
MTF-1.
Recently,
Andersen
et
al.
(16),
using
gel
retardation
and
UV
cross-linking
procedures
have
demonstrated
Cd2
+-dependent
binding
of
a
39
kDa
factor
(p39),
present
in
nuclear
extracts
of
rat
Fao
hepatoma
cells,
to
an
oligomer
containing
the
MREc
and
MREd
elements
of
the
rat
MT-I
gene.
Contrary
to
what
we
observed
for
MEP-1,
a
single
MRE
is
insufficient
for
efficient
binding
of
this
39
kDa
protein
and
at
least
two
MREs
are
apparently required
for
binding.
In
Saccharomyces
cerevisiae,
Cu2
+-inducible
transcription
of
the
yeast
MT
gene
CUP]
requires
Cu2+-dependent
binding
of
the
ACE]
protein
to
specific
sites
in
the
promoter
region
(25-31);
in
addition,
a
gene
designated
ACE2,
plays
a
role
in
regulating
basal-level
expression
of
CUP]
in
Cu2
+
-independent
fashion
(32).
The
phenomenon
of
multiple
factors
that
can
bind
to
the
same
DNA
site
appears
to
be
common
in
higher
eukaryotes
(33)
and
a
number
of
proteins
that
bind
to
ATF
sites
(34),
CC-
AAT/enhancer
binding
sites
(35),
octamer
sites
(36),
Spl
sites
(37,38)
and
homeotic
protein-binding
sites
(39)
have been
identified.
The
apparent
overlapping
DNA
binding
specificities
of
MBF-
1
and
MEP-
1
at
the
level
of
MREd
is
intriguing
and
raises
the
problem
of
assessing
the
role
of
the
different
proteins
in
the
regulation
of
MT
genes
expression
by
heavy
metals.
Moreover,
the
role
of
the
different
MREs
and
the
nature
of
the
interaction
between
the
factors
that
bind
to
them
are not
clear.
Two
possible
mechanisms
can be
envisioned.
(i)
Both
MEP-1
and
MBF-1
play
a
role
in
regulating
induced-level
expression
of
MT
transcription
or
alternatively,
(ii)
MEP-
1
and
MBF-
1
may
modulate
induced-
and
basal
transcription
in
a
way
similar
to
ACE]
and
ACE2
in
yeast.
These
hypotheses
do
not
exclude
the
involment
of
other
putative
MRE-binding
proteins,
such
as
p39
(16),
or
non
DNA-binding
factor(s)
acting
as
'co-activator'
proteins
(40).
Analysis
of
the
action
of
MEP-
1
on
MT
transcription
and
its
potential
interactions
with
MBF-
1
will
require
precise
definition
of
the
recognition
sequence
of
both
proteins
and
in
vitro
transcription
reconstitution
experiments
using
purified
MBF-I
and
MEP-l
and
specific
antibodies.
ACKNOWLEDGEMENTS
We
thank
W.Waithe,
A.Anderson
and
A.Maresca
for
helpful
suggestions
and
discussion,
L.Larouche
for
her
expert
technical
assistance
and
M.Gagnon
for
excellent
typing.
This
work
was
supported
by
grants
from
the
Medical
Research
Council
(MRC)
to
C.
S.
Work
at
the
Universita
di
Napoli
was
supported
by
a
grant
from
the
Consiglio
Nazionale
delle
Ricerche
(C.N.R.)
P.F.
'Ingegneria
genetica'
to
A.L..
S.L.
is
supported
by
a
Studentship
from
the
Natural
Sciences
and
Engineering
Research
Council
of
Canada,
C.S.
by
a
Scholarship
from
MRC
and
P.R.
from
the
Italian
Association
for
Research
on
Cancer
(A.I.R.C.)
during
his
visit
to
the
laboratory
of
C.S.
REFERENCES
1.
Hamer,D.H.
(1986)
Annu.
Rev.
Biochem.
55,
913-951.
2.
Kagi,J.H.R.
and
Kojima,Y.
(eds.)
(1987)
Metallothionein
II,
Birkhauser
Verlag,
Basel.
3.
Carter,A.D.,
Felber,B.K.,
Walling,M.J.,
Jubier,M.F.,
Schmidt,C.J.
and
Hamer,D.H.
(1984)
Proc.
Natl.
Acad.
Sci.
USA
81,
7392-7396.
4.
Karin,M.,
Haslinger,A.,
Holtgreve,H.,
Richards,R.I.,
Krauter,P.,
Westphal,H.M.
and
Beato,M.
(1984)
Nature
308,
513-519.
5.
Stuart,G.W.,
Searle,P.F.,
Chen,H.Y.,
Brinster,R.L.
and
Palmiter,R.D.
(1984)
Proc.
NatI.
Acad.
Sci.
USA
81,
7318-7322.
6.
Stuart,G.W.,
Searle,P.F.
and
Palmiter,R.D.
(1985)
Nature
317,
828-831.
7.
Mueller,P.R.,
Salser,S.J.
and
Wold,B.
(1988)
Genes
Dev.
2,
412-427.
8.
Searle,P.F.
(1990)
Nucleic
Acids
Res.
18,
4683-4690.
9.
Culotta,V.C.
and
Hamer,D.H.
(1989)
Mol.
Cell. Biol.
9,
1376-1380.
Nucleic
Acids
Research,
Vol.
19,
No.
15
4231
10.
Seguin,C.,
Felber,B.K.,
Carter,A.D.
and
Hamer,D.H.
(1984)
Nature
312,
781
-785.
11.
Scholer,H.,
Haslinger,A.,
Heguy,A.,
Holtgreve,H.
and
Karin,M.
(1986)
Science
232,
76-80.
12.
Searle,P.F.,
Stuart,G.W.
and
Palmiter,R.D.
(1985)
Mol.
Cell.
Biol.
5,
1480-1489.
13.
Sdguin,C.
and
Hamer,D.H.
(1987) Science
235,
1383-1387.
14.
Andersen,R.D.,
Taplitz,S.J.,
Wong,S.,
Bristol,G.,
Larkin,B.
and
Herschman,H.R.
(1987)
Mol.
Cell.
Biol.
7,
3574-3581.
15.
Seguin,C.
(1991)
Gene
97,
295-300.
16.
Andersen,R.D.,
Taplitz,S.J.,
Oberbauer,A.M.,
Calame,K.L.
and
Herschman,H.R.
(1990)
Nucleic
Acids
Res.
18,
6049-6055.
17.
Imbert,J.,
Zafarullah,M.,
Culotta,V.C.,
Gedamu,L.
and
Hamer,D.H.
(1989)
Mol.
Cell.
Biol.
9,
5315-5323.
18.
Seguin,C.
and
Prevost,J.
(1988)
Nucleic
Acids
Res.
16,
10547-10560.
19.
Maniatis,T.,
Fritsch,E.F.
and
Sambrook,J.
(1989)
Molecular
cloning.
A
laboratory
manual,
Cold
Spring
Harbor
Laboratory
Press,
Cold
Spring
Harbor.
20.
Safer,B.,
Cohen,R.B.,
Garfinkel,S.
and
Thompson,J.A.
(1988)
Mol.
Cell.
Biol.
8,
105-113.
21.
Chodosh,L.A.
(1988)
In
Ausubel,F.A.,
Brent,R.,
Kingston,R.E.,
Moore,D.D.,
Seidman,J.G.,
Smith,J.A.
and
Struhl,K.
(eds.).
Current
protocols
in
molecular
biology.
Greene
Publishing
and
Wiley-Interscience.
New-York:
pp.
12.5.1-12.5.8.
22.
Carthew,R.W.,
Chodosh,L.A.
and
Sharp,P.A.
(1987)
Genes
Dev.
1,
973-980.
23.
Kadonaga,J.T.,
Jones,T.A.
and
Tjian,R.
(1986)
Trends.
Biochem.
11,
20-23.
24.
Westin,G.
and
Schaffner,W.
(1988)
EMBO
J.
7,
3763-3770.
25.
Thiele,D.J.
(1988)
Mol.
Cell.
Biol.
8,
2745-2752.
26.
Szczypka,M.S.
and
Thiele,D.J.
(1989)
Mol.
Cell.
Biol.
9,
421-429.
27.
Culotta,V.C.,
Hsu,T.,
Hu,S.,
Furst,P.
and
Hamer,D.H.
(1989)
Proc.
Natl.
Acad.
Sci.
USA
86,
8377-8381.
28.
Furst,P.,
Hu,S.,
Hackett,R.
and
Hamer,D.H.
(1988)
Cell
55,
705-717.
29.
Furst,P.
and
Hamer,D.H.
(1989)
Proc.
Natl.
Acad.
Sci.
USA
86,
5267-5271.
30.
Huibregtse,J.M.,
Engelke,D.R.
and
Thiele,D.J.
(1989)
Proc.
Natl.
Acad.
Sci.
USA
86,
65-69.
31.
Buchman,C.,
Skroch,P.,
Welch,J.,
Fogel,S.
and
Karin,M.
(1989)
Mol.
Cell.
Biol.
9,
4091-4095.
32.
Butler,G.
and
Thiele,D.J.
(1991)
Mol.
Cell.
Biol.
11,
476-485.
33.
Johnson,P.F.
and
McKnight,S.L.
(1989)
Annu.
Rev.
Biochem.
58,
799-839.
34.
Hai,T.,
Liu,F.L.,
Coukos,W.J.
and
Green,M.R.
(1989)
Genes
Dev.
3,
2083
-2090.
35.
Roman,C.,
Platero,J.S.,
Shuman,J.
and
Calame,K.
(1990)
Genes
Dev.
4,
1404-1415.
36.
Scholer,H.R.,
Hatzopoulos,A.K.,
Balling,R.,
Suzuki,N.
and
Gruss,P.
(1989)
EMBO
J.
8,
2543-2550.
37.
Chavrier,P.,
Vesque,C.,
Galliot,B.,
Vigneron,M.,
Dolle,P.,
Duboule,D.
and
Charnay,P.
(1990)
EMBO
J.
9,
1209-1218.
38.
Nardelli,J.,
Gibson,T.J.,
Vesque.C.
and
Charnay,P.
(1991)
Nature
349,
175-178.
39.
Han,K.,
Levine,M.S.
and
Manley,J.L.
(1989)
Cell
56,
573-583.
40.
Pugh,B.F.
and
Tjian,R.
(1990)
Cell
61,
1187-1197.
