BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1353-1361
Vol. 186, No. 3, 1992 August 14, 1992
NUCLEAR
PROTEINS
COMPLEXES
FROM
WITH
Barbara Institute
CAPAN-2
CELL
17-IA
ANTIGEN
THE
Siemieniako
LINE
FORM
GENE
SPECIFIC
PROMOTER
and E w a W i l a n d
of H u m a n G e n e t i c s , P o l i s h A c a d e m y of S c i e n c e s , 6 0 - 4 7 9 Pozna6, S t r z e s z y 6 s k a 32, P o l a n d
Received July i, 1992
T o d e t e r m i n e the l o c a t i o n of s i t e s i m p o r t a n t for the f u n c t i o n of the 1 7 - 1 A a n t i g e n g e n e promoter and to c h a r a c t e r i z e the p r o t e i n factors binding to t h e s e sites, f r a g m e n t s of the p r o m o t e r r e g i o n were analysed b y gel r e t a r d a t i o n assay with nuclear extracts f r o m C a p a n 2 cell line. At least two s e p a r a t e r e g i o n s , w h i c h specifically bind nuclear p r o t e i n s , w e r e i d e n t i f i e d w i t h i n the 5'flanking region of the 1 7 - 1 A a n t i g e n gene. These regions have been located between nucleotides -877 to -518 (distal region) and -193 to +3 ( p r o x i m a l region) and presumably p a r t i c i p a t e in regulation of e x p r e s s i o n of the 17-1A a n t i g e n gene. ©1992AcademicPress,Inc.
The
studies
facilitated surface
and
can
characterization (mAbs), but
CO 1 7 - 1 A
also
tumor
its
partial
(Capan),was The
epithelial
17-1A
antigen search
GA733
receptor.
This
a
Monoclonal not
cells.
bind Both,
and
This
sequence
products
of
antibodies
to t u m o r
cells
derived,
glycoprotein,
antigen
was
determined.
purified Finaly,
carcinoma
cell
a line
(2,3).
to the
family
an e v i d e n c e
of
the ~
of
subunit
this
to cell partial
independently
in p a n c r e a t i c
is i n t r i g u i n g
only
cell-surface
(i) .
greatly
bind and
antigens.
belongs
showed
homology
isolation
characterized
gene
been
that
GA 733,
acid
and
have
antibodies
40-kDa
expressed
sequences
the p r o t e i n
antigens
for
antigen
amino
gene,
isolated
database
between
that
these
associated
I7-1A-related
The
used
immunoprecipitate
termed and
be
of
tumor
of monoclonal
and r e l a t e d
to normal
mAbs
human
of
by the use
of
GA 733 genes.
sequence
homology
interleukin-2
and s u g g e s t s
tumor-associated
a
(IL-2)
possibility antigen
gene
ABBREVIATIONS
mAb, m o n o c l o n a l a n t i b o d y ; methyl-sulphonyl fluoride.
IL-2,
inter
leukin
2;
PMSF,
phenyl-
0006-291X/92 $4.00 1353
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
family
may
functlon
structurarly The
related
17-1A
gene
for
a number
and
eukaryotic
ral
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
transcription
and
tumor
there
of of
this
to
(8,9).
antigen gene
action
of p r o t e i n
In t h i s
MATERIALS Preparation
gene
However,
atypical
CAAAT box elements
in
(5) . within
regulation
f o r the
(2,3) . T h e r e f o r e seems bind
varies
studies
in the s t r u c t u r e
expression
seve-
of
(5-7). expression
preliminary
factors which
study we
characterized
retardation
proteins
regulation
of g e n e s
T A T A box,
sequence
implicated
elements
prokaryotic
of
of
to
normal
indicate
5'flanking
observed
the
in
region
differences
regulation depend
to s p e c i f i c
that
of
mainly
in the
on the
sequences within
region.
r e g i o n by gel nuclear
been
been responsible
expression
promoter
cannonical
as an
are no r e a r r a g e m e n t s
gene
the
binding
antigen
factors
response
transcription
as w e l l
17-1A
the
including
a variety
which might have
contains multiple
regulating
have
growth
(2,4).
factors,
17-1A
cells
binding
repeats,
region,
level
IL-2
promoter
genes,
protein
the promoter
The
to
receptors,
of f a c t o r s
8-base direct
Multiple
as
and
of
17-1A
and
METHODS
of
nuclear
assay.
regions
antigen
the We
antigen
identified
that
gene
17-1A
might
gene
sites be
promoter
that
bind
important
for
expression.
extract
N u c l e a r e x t r a c t s w e r e p r e p a r e d by the m o d i f i e d m e t h o d of Dignam et al. (i0) . C a p a n 2 cells, obtained from The Wistar Institute, Philadelphia,PA (USA) were grown as a monolayer to c o n f l u e n c e in L - 1 5 m e d i u m s u p p l e m e n t e d w i t h 10% fetal bovine serum, c o n t a i n i n g g e n t a m y c i n (50mg/l) . T h e c e l l s w e r e harvested with p h o s p h a t e - b u f f e r e d s a l i n e (PBS), c o n t a i n i n g trypsin-EDTA. All m a n i p u l a t i o D s w e r e p e r f o r m e d on ice and all buffers were c h i l e d to 0 - 4UC. Phenyl-methyl-sulfonyl-fluoride (PMSF) and dithiothreitol (DTT) w e r e a d d e d to the b u f f e r s j u s t b e f o r e use. Pelleted cells were s u s p e n d e d in c o l d P B S and c o l l e c t e d b y c e n t r i f u g a t i o n for I0 m i n at 2 0 0 0 rpm. T h e c e l l s w e r e s u s p e n d e d in f i v e p a c k e d cell v o l u m e s of b u f f e r A c o n t a i n i n g 1 0 m M H e p e s p H 7.9, 1 . 5 m M M g C I 2, 1 0 m M KCI, 0.5mM DTT and 0.5m_M P M S F and left on ice for I0 min. C e l l s w e r e c o l l e c t e d as b e f o r e , h o m o g e n i z e d in t w o v o l u m e s of b u f f e r A, and p e l l e t e d at 2 0 0 0 r p m at 4°C. N u c l e a r p e l l e t ( c r u d e nuclei) was resuspended in 20mM Hepes pH 7.9, 25% glycerol, 0.42M NaCI, 1 . 5 m M M g C I 2, 0 . 2 m M EDTA, 0.5mM DTT and 0 . S m M P M S F (buffer C) . T h e s u s p e n s i o n w a s g e n t l y s t i r r e d f o r 30 m i n and c e n t r i f u g e d for 30 m i n at 25 000 x g at 4°C. Then to the supernatant solid (NH4)2SO 4 w a s a d d e d to final concentration of 0.45g/ml. The precipitation was conducted for 30 min on ice-bath with m i x i n g , f o l l o w e d by c e n t r i f u g a t i o n as above. Protein pellet was resuspended in 2 0 m M H e p e s pH 7.9, 20% g l y c e r o l , 0 . 1 M KCI,
1354
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0 . 2 m M EDTA, 0 . 5 m M DTT, 0 . 5 m M P M S F (buffer D) and dialized o v e r n i g h t a g a i n s t the s a m e b u f f e r . T h e d i a l y s a t e w a s centrifuged at 25 000 x g for 30 min, and the supernataDt (nuclear extract) was f r o z e n as a l i q u o t s and s t o r e d at - 8 0 ° C . Preparation
of
restriction
fragments
Targets for DNA binding assay were isolated from MI3 mpl8 phage w h i c h c o n t a i n e d a large fragment of promoter with 5"upstream s e q u e n c e and a small 1 7 - 1 A a n t i g e n g e n e f r a g m e n t , i n s e r t e d betw e e n the Accl and SmaI r e s t r i c t i o n sites. The M I 3 m p l 8 p h a g e w a s cut by restriction enzymes: HinPl, Hinfl, and BspEI. The cut e n d s were filled and labeled w i t h K l e n o w f r a g m e n t of DNA P o l y m e r a s e I. T h e D N A w a s cut a g a i n w i t h Pstl and t h e resulting f r a g m e n t s w e r e s e p a r a t e d by e l e c t r o p h o r e s i s on non-denaturating 5% p o l y a c r y l a m i d e gel, e l u t e d f r o m the gel and p u r i f i e d b y p h e n o l : chloroform extraction and ethanol precipitation.
of o l i g o n H c l e o t i d e s
Preparation
Oligonucleotides were synthesized by Applied Biosystems DNA s y n t h e s i z e r , p u r i f i e d a n d s i n g l e s t r a n d s w e r e m i x e d in equimolar a m o u n t s , h e a t e d to 8 8 ° C and c o o l e d slowly to r o o m t e m p e r a t u r e . O l i g o n u c l ~ g t i d e s , u s e d as p r o b e s in some e x p e r i m e n t s w e r e l a b e l e d with Z---P ATP and T4 polynucleotide kinase (Boehringer Mannheim) . 01igonucleotides, containing known consensus sequences f o r t r a n s c r i p t i o n f a c t o r s (e.g. API, AP2, NFI), u s e d in competition experiments, derived from kit (Stratagene Cat.203002) .
Gel
retardation
assay
The standard assa~2 was performed in a v o l u m e of 20 ~ I and a sample containing P labeled probe (2 0 0 0 - 4 000 cpm) , p o l y d l d C as a non-specific competitor ( 0 . 2 - 0 . 4 ~ g per r e a c t i o n ) , b i n d i n g buffer (15mM Hepes/KOH p H 7.5, l l 0 m M NaCI, 2m_M DTT, 250 H g / m l BSA) and n u c l e a r e x t r a c t (0-6 ~ g of p r o t e i n ) . T h e n u c l e a r e x t r a c t was always a d d e d last to the r e a c t i o n mixture. The complete m i x t u r e s w e r e i n c u b a t e d f o r 20 to 30 m i n at r o o m t e m p e r a t u r e and l o a d e d on 5% n a t i v e p o l y a c r y l a m i d e gel in 0.5 x T r i s b o r a t e EDTA (TBE) b u f f e r . T h e e l e c t r o p h o r e s i s w a s c o n d u c t e d at 2 0 0 V f o r 2 to 2.5 h r and the gel a n a l y z e d by a u t o r a d i o g r a p h y at - 8 0 ° C with i n t e n s i f y i n g screen. For competition experiments a I0 to 1 0 0 - f o l d m o l a r e x c e s s of t h e c o r r e s p o n d i n g u n l a b e l e d f r a g m e n t or o l i g o n u c l e o t i d e s were added at the b e g i n n i n g of the b i n d i n g r e a c t i o n .
RESULTS
and
Present
DISCUSSION studies
were
the p r o m o t e r
of the
b l e for h i g h
expression
type
of
chosen these
cells. since
To
of
determine
this gene
fragments,
carcinoma of
identify
specificity covering
cell
about
1355
or
line
antigen
tumor
as w e l l
which
in one,
the
to o t h e r
line S W 948), sequence
to
a n t i g e n gene,
expression
as c o m p a r e d
cell
overlapping
17-1A
Pancreatic
the
cells,
carcinoma
undertaken
cell
elements, might
be r e s p o n s i -
low in the (Capan was
lines
as to n o r m a l of the
within
was
highest
in
(e,g.colorectal cells
detected
i000 bp of
other
2)
the
(2,8,9). complexes, 5"flanking
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS x
A 03
] -1000
-900
-998
-914
I
-800
I I
-700 -800
Hf
i
J
-811 79bp -
4
-
-142
+3
H
-252
- 142 B
359bp
48
-100
H
-518 Hf
-260
-285
~i
-877
-
-360
-5218
H
B
-400
l-.¢n
i
111____ I
-500
(/%0
233bp 8
-
4
P
*'3
1 lObp 8
-4
8
145bp 8
2561op 4 6
-
-
4
6
193bp 4 6
Fig.l. B i n d i n g of p r o t e i n s from the nuclear extract to various fragments s p a n n i n g 5 " u p s t r e a m promoter sequence of 17-1A antigen gene. A. The schematic map of the studied sequence with known consensus s e q u e n c e s present within. R e s t r i c t i o n sites generating fragments are: H-HinPI, Hf-Hinfl, B - B s p E I and P-Pstl. B: Each labeled f r a g m e n t was incubated for 30 m i n w i t h i n d i c a t e d amounts of n u c l e a r e x t r a c t for 30 m i n (binding c o n d i t i o n s were d e s c r i b e d in M a t e r i a l s and Methods). Assays were performed by electrophoresis on n a t i v e 5% polyacrylamide gel f o l l o w e d by autoradiography at -80°C. The figure is a composite of separate gels and therefore fragments of d i f f e r e n t size appear to have the same m o b i l i t y . region assay. most of 359
of
the
Fig.iA
important
binding bp
amounts
in of
gene,
were
shows sites
assays
are
lenght added
individually
assayed
restriction
fragments
recognizing
transcription
shown
in
Fig.lB.
(HinPl-HinPl)
nuclear
extract.
1356
was The
The
by
gel
used,
retardation
as w e l l
factors.
restriction
retarded smaller
256
by bp
as
the
Results fragment, increasing fragment,
Vol. 186, No. 3, 1992
(HinfI-Hinfl), well
resolved
even
located bands.
(HinPI-HinPl) smallest
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in size,
the
CAP
quntitively HinPI)
distinct
obtained These
results
fragment,
of
of
two
fragment
described.
shows
The
no b i n d i n g
359 bp
promoter,
in the
with
(HinPl-
several
nuclear
bands.
Two
145 bp f r a g m e n t
C A A A T box. 145
and
ii0 bp
adjacent
the w h o l e
a banding
activity,
qualitatively
labeled
interaction
proximal
binding
t h r e e to f o u r r e t a r d e d
containing
demonstrate
the
both
the
T A T A box and
two fragments
fragments:
showed
233 bp
previously
significant
detected
Ii0 bp f r a g m e n t ,
f o r the
two
an
resolved
containing
f o r the
differed,
Binding
(BspEI-Pstl), the
fragments also
indicated
fragment,
concentration.
bands
complexes were
fragment
within
showed
(Fig.iB) .
(HinPI-Pstl),
part of
(HinPI-HinPl)
retarded
giving well
359 bp
of 256 bp,
restriction
fragment
proteins,
that
protein
site,
the
the
binding pattern
79 bp
at t h e h i g h e s t
although
The
resembles
The remaining to
inside
For
bp
pattern was
the
193 bp
sequence a s u m of
and that
separately.
that m o s t
of
the
(distal r e g i o n )
binding
and
193 bp
occured (proximal
region). Each fragment, specificity 50-fold
by
d i d not
middle
of the
comprising
excess show
bp
DNA
specific
tested
extract
and
233 bp
fragments,
5'GTGGTTTG
H~nPI --
3'
The
was
-- HinP! (110bp) 0 ~0 SO
--
and
binding i0
This
in the region,
containing several
the
binding
H~nP/--Pst11145 bp) 0 I0 S0
Fig. 2. Competition analysis of promoter binding proteln specificity. Labeled restriction fragments, shown in Fig.l, were incubated with nuclear extract in the presence of increasing amounts of the competitor (unlabeled DNA) and analyzed using gel retardation assay The molar excess of the competitor used is shown above the lanes.
1357
or
large r e g i o n ,
identified
although
(7,11)
the
along with
fragments. binding
for
( n u c l e o t i d e s -555 to -252).
sequence
competitor
was
with nuclear
of u n l a b e l e d any
sequence
201
core e n h a n c e r
in F i g . i B ) ,
incubation
molar
which
(shown
Vol. 186, No. 3, 1992 sites
for
number
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
regulatory
of r e t a r d e d
appears nonspecifiC, any s p e c i f i c Results
fragment,
(Fig.l),
since
these bands
and
was
removed
by
As s e e n
fragment was
specifically
molar excess o t h e r hand, the 5 " e n d complex
of
of
the
cases
not
all
lower
set
of
specificity
to the
proximal
bands
were
competed.
bands
within
By
of b i n d i n g seemed
concordant
with
our
crucial box
(12) .
b o x up to - 9 7
contained
proximal
sequence.
specific
box
and
SPI
site,
with
unlabeled
I0,
double
These
to -23.
However,
the b i n d i n g
The
were
and
as
a
appeared
several
mostly
1358
factor.
in m o s t
genes
and
CAP
of
the
of TATA
important
for
the r e g i o n s
of
oligonucleotides 120 bp
used
one,
of
the
as p r o b e s
for
competition TATA
including
-ii0
to
of
The
extract
appropriate
competitor nuclear
nonspecific,
box, CAAAT
-81.
a nuclear
excess
specific by
multiple
box
for
incubated with
retarded
including
containing
second
molar
region
initiation
the
from nucleotide
were
50-fold
1 was
stranded
interactions
extended
that TATA
element
within
the
partially
studied
to n a r r o w
oligonucleotide,
-49
25,
the
oligonucleotides
oligonucleotide
Oligonucleotide
were
efficient
order
showed
transcription
upstream
elements
labeled oligonucleotides along
In
binding
whereas
sequences upstream
two
first
sequence
and
important
DNA-protein The
both
a second (13).
constructed
experiments. comprised
Moreover,
transcription we
If,
specific
transcription
the SPI
the
corresponding
fragment
the
studies had shown
polymerase for
for
both
and
showed
results
since
more
In
one,
the bp
the
generated
sequences
point mutation
encompassing most
These
50-fold On
(145 bp) ,
highest
145
bp)
encompasses
containing
and
binding,
which
two h i g h e r bands,
elements
specific
a
control
Deletions
necessary
by
fragment,
of
145 bp
(Ii0
region,
recognition
by RNA
the
transcription
consensus
of
fragment,
the
expectations,
specific
a n d the G C - r i c h
CAAAT
efficient
the
of
competed
excess
contrast, for
showed
ii0 bp f r a g m e n t ,
an
by
two f r a g m e n t s
unlabeled
The
to be n o n s p e c i f i c .
T A T A box,
site were
of
competed
HinPI-HinPl
for the 3 " e n d
the
presence
to
small
binding
activity
of the p r o m o t e r
seen
lowest one
transcribed
region
that
fragment,
contained
ii0 bp
be
excess
efficiently
than
in t h e
specificity
and
corresponding
binding
pattern
unlabeled
the
the
the
(145 bp),
of the b i n d i n g
binding
a
(not s h o w n ) . that
10-fold molar
in F i g . 2 ,
showed
c o u l d not
demonstrate,
part
a
competitor,
CRE)
In a d d i t i o n ,
HinPI-PstI
A significant
API,
competitor used
in F i g . 2
(ii0 bp)
competition.
(e.g.
bands
or n o n s p e c i f i c
presented
HinPI-HinPl
proteins
(Fig.3) . proteins.
since
these
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS oltgo ~
1.
~,
oligo 2 •
,
S'GGGCAGGTCGGGTAGC~AGAGCC CCCGTCCAGCCCATCGC~ATA~TCTCGG
3'
_ _ S P a _ _
2. S' GGAGGGGGGCGGGATGTGTCACC~AAAT~CC
CC TCCCCCCGCCCTACACAG T G ~ G T T T ~ T G G
3"
Fig.3. N u c l e a r proteins binding to particular oligonucleotides. The o l i g o n u c l e o t i d e s (prepared as described in M a t e r i a l s and Methods), d e p i c t e d s c h e m a t i c a l l y below, were used as p r o b e s and incubated with 6 Hg of the nuclear extract in the p r e s e n c e of increasing amounts of the same oligonucleotide as a competitor.
bands
could
specific competed
by
Binding bands
a
competed
one
and
which
with
of
molar band
excess was
2 showed
two
competed.
1 band
with
oligonucleotide
use
as
respectively.
Results
band
specific
for
the
competitor.
specifically
the
of
efficiently
oligonucleotide
oligonucleotides
only
the
be
petitors was
50-fold
highest
oligonucleotide
could
for
by
the
excess
experiments both
even
Only
10-fold
specificity
competition probe
be
experiments
with
binding
not
competitor.
of
specific
and
demonstrated
oligonucleotide
,-_ollgo 1,
we
check
carried i
nonspecific
that 1
retarded To
the
out as
observed
(containing
TATA
~,._oli~o Z
competitor
Fig.4. C o m p e t i t i o n for nuclear proteins binding to the TATA box containing o l i g o n u c l e o t i d e i, with specific and nonspecific competitors (unlabeled o l i g o n u c l e o t i d e 1 and 2). The m o l a r excess of competitors is indicated, above each lane.
1359
a
com-
Vol. 186, No. 3, 1992 box) of
and
that
the b a n d
nonspecific
suggests of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
also
359
the
specific
bands
was
competitor Because
well
as
observed which
stranded
oligonucleotides, protein
fragment) band was CT/NFI
as
sites
by
with
the
(Fig.5) . T h e r e
several ment
binding
might
indicated
protein
in
extensive
the
the
these
was
are
fragment
studies
have
antigen
gene
in t h e indicated promoter,
=NA
decided
same
--
0
10
SO
that
by
exhibit
antigen there may
enhan-
cells.
shift
Anyway role
~n
E
~
~
~
within
the
regulation
A
Fig,5. C o m p e t i t i o n analysis of the distal region b i n d i n g s p e c i f i city, A. Labeled fragment HinPI-HinPI (359bp) was incubated w i t h 6 ~g of nuclear extract in the presence of increasing amounts of specific c o m p e t i t o r (unlabeled fragment). B. Competition experiment using oligonucleotides, c o n t a i n i n g consensus sequences r e c o g n i z e d by transcription factors, indicated above each lane.
1360
of
expression.
part
O
or
binding
putative
sequences
--
as
enhance-
CT/NFI
gene
take
one
little
2
and
oligonu-
with
of
the
are
AP2
band
Capan
upper
whereas
observed
or
API
the
and
dependence
The
API
in
the
API
o r API
(14).
by
additio-
(absent
fragments
from
100
and
as
were double
recognized
that
which
use
efficiency,
"i~PZ-m~P~ ~3SSbp~
cornpet{tor
two
speci-
CT/NFI
to
It w a s f o u n d
to e x p l a i n
that
the the
or
competitors
unaffected
17-1A
of
function
containing
of e i t h e r
extract
binding
promoter,
of
API,CRE
a functional
necessary
the
out
the A P 2 m o t i f
factors
presence
of
unknown
sequences
the
only NFI
nuclear
studies
this distal Our
of
as
we
competitor.
suggesting
cooperation
region
of
is e v i d e n c e
the
(Fig.5).
such
containing
for
to
box.
one
as s p e c i f i c
activity
sites
by t h e m s e l v e s ,
c e r on
17-1A
binding
be d u e
excess
approximatelly
cleotide
This
least
oligonucleotides
expected
(Fig.4).
2)
molar
containing
a nonspecific
competed
excess
At
fragment,
factors,
a 100-fold
10-fold
element
bp
an o l i g o n u c l e o t i d e
might
in d i s t a l
motifs
TATA 359
by
the T A T A
fragment)
sequence
within
nally
by
(unlabeled
the
shift with
located
present
and CT/NFI
band
competition.
additional
even
(oligonucleotide
competed
several
an
competed
interact
fragment,
showed
retarded fic
bp
not
competitor
a protein(s) The
was
Vol. 186, No. 3, 1992
of expression extracts other
sequences
this
or s e p a r a t e
cells
determine
of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
differing the role
of
gene.
Further
factors
in expression factors
for the e x p r e s s i o n
studies
are required
of this gene
recognizing
of the
17-1A
using
purified
and c o m p a r J s i o n with are
specific
antigen
needed
to
promoter
gen~
Ac k howl edg ments We thank Prof. A Horst and Prof. W.H.Trzeciak for reading this paper and helpful discussion, K.G6rczy6ska for technical assistance and Dr. J . W o j c i e r o w s k i of the Medical School in Lublin (Poland) for his k i n d gift of antigen gene promoter, obtained through the c o u r t e s y of D r . A . J . L i n n e n b a c h of the W i s t a r Institute Philadelphia,PA, REFERENCES
l.Ross,A.H., Herlyn,D., lliopoulos,D, and Koprowski,H. (1986) Biochem. Biophys.Res. Commun., 135, 297-303. 2.Linnenbach,A.J., W o j c i e r o w s k i , J . , Wu,S., Pyrc,J.J., Ross,A.H., Dietzschold,B., Speicher,D. and Koprowski,H. (1989) Proc. Natl. Acad. Sci.USA, 86, 27-31. 3 Szala,S., F r o e h l i c h , M . , Scollon,M., Kasai,Y., Steplewski,Z., Koprowski,H. and Linnenbach,A.J. (1990) Proc.Natl.Acad.Sci. USA, 87, 3542-3546. 4.Depper,L.W.J., K a n e h i s a , M . , K r o n k e , M . , Peffer,N.J.,Swetl~k,P.B. Sulivan,M. and Greene,W.C. (1985) Science, 230, 633-639. 5.Benoist,C and Chambon,P. (1981) Nature, 290, 304-310. 6.Concino,M.,Goldman,R.A.,Caruthers,M.M. and Weinmann,R. (1983), J.Biol.Chem., 258, 8493-8496. 7.Khoury,G. and Gruss,P. (1983) Cell, 33, 313-314. 8.Herlyn,M., Steplewski,Z., Her]yn,D. and Koprowski,H. (1986) Hybridoma, 5,3-10. 9.Herlyn,M., S t e p l e w s k i , Z . , Herlyn,D. and Koprowski,H. (1986) Hybridoma, 5, 3-8. 10.Dignam,J.D., Leibovitz,R.M. and Roeder,R.G. (1983) Nucleic Acid. Res., ii, 1475-1489. ll.Wasylyk,B. (1988) Biochim. Biophys. Acta, 951, 17-35. 12.Hen,R.P.,Sassone-Corsi,J.,Corden,M.P.,Gaub,M.P. and Chambon,P. (1982) P r o c . N a t l . A c a d . Sci.USA, 79, 7132-7136. 13.Jove,R. and Manley,J.L. (1984) J.Biol.Chem.,259, 8513-8521. 14.Chong,T.,Chan,W.-K. and Bernard,H.-U. (1990) N u c l e i c Acid Res. 18, 465-470.
1361
Vol. 186, No. 3, 1992 August 14, 1992
NUCLEAR
PROTEINS
COMPLEXES
FROM
WITH
Barbara Institute
CAPAN-2
CELL
17-IA
ANTIGEN
THE
Siemieniako
LINE
FORM
GENE
SPECIFIC
PROMOTER
and E w a W i l a n d
of H u m a n G e n e t i c s , P o l i s h A c a d e m y of S c i e n c e s , 6 0 - 4 7 9 Pozna6, S t r z e s z y 6 s k a 32, P o l a n d
Received July i, 1992
T o d e t e r m i n e the l o c a t i o n of s i t e s i m p o r t a n t for the f u n c t i o n of the 1 7 - 1 A a n t i g e n g e n e promoter and to c h a r a c t e r i z e the p r o t e i n factors binding to t h e s e sites, f r a g m e n t s of the p r o m o t e r r e g i o n were analysed b y gel r e t a r d a t i o n assay with nuclear extracts f r o m C a p a n 2 cell line. At least two s e p a r a t e r e g i o n s , w h i c h specifically bind nuclear p r o t e i n s , w e r e i d e n t i f i e d w i t h i n the 5'flanking region of the 1 7 - 1 A a n t i g e n gene. These regions have been located between nucleotides -877 to -518 (distal region) and -193 to +3 ( p r o x i m a l region) and presumably p a r t i c i p a t e in regulation of e x p r e s s i o n of the 17-1A a n t i g e n gene. ©1992AcademicPress,Inc.
The
studies
facilitated surface
and
can
characterization (mAbs), but
CO 1 7 - 1 A
also
tumor
its
partial
(Capan),was The
epithelial
17-1A
antigen search
GA733
receptor.
This
a
Monoclonal not
cells.
bind Both,
and
This
sequence
products
of
antibodies
to t u m o r
cells
derived,
glycoprotein,
antigen
was
determined.
purified Finaly,
carcinoma
cell
a line
(2,3).
to the
family
an e v i d e n c e
of
the ~
of
subunit
this
to cell partial
independently
in p a n c r e a t i c
is i n t r i g u i n g
only
cell-surface
(i) .
greatly
bind and
antigens.
belongs
showed
homology
isolation
characterized
gene
been
that
GA 733,
acid
and
have
antibodies
40-kDa
expressed
sequences
the p r o t e i n
antigens
for
antigen
amino
gene,
isolated
database
between
that
these
associated
I7-1A-related
The
used
immunoprecipitate
termed and
be
of
tumor
of monoclonal
and r e l a t e d
to normal
mAbs
human
of
by the use
of
GA 733 genes.
sequence
homology
interleukin-2
and s u g g e s t s
tumor-associated
a
(IL-2)
possibility antigen
gene
ABBREVIATIONS
mAb, m o n o c l o n a l a n t i b o d y ; methyl-sulphonyl fluoride.
IL-2,
inter
leukin
2;
PMSF,
phenyl-
0006-291X/92 $4.00 1353
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
family
may
functlon
structurarly The
related
17-1A
gene
for
a number
and
eukaryotic
ral
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
transcription
and
tumor
there
of of
this
to
(8,9).
antigen gene
action
of p r o t e i n
In t h i s
MATERIALS Preparation
gene
However,
atypical
CAAAT box elements
in
(5) . within
regulation
f o r the
(2,3) . T h e r e f o r e seems bind
varies
studies
in the s t r u c t u r e
expression
seve-
of
(5-7). expression
preliminary
factors which
study we
characterized
retardation
proteins
regulation
of g e n e s
T A T A box,
sequence
implicated
elements
prokaryotic
of
of
to
normal
indicate
5'flanking
observed
the
in
region
differences
regulation depend
to s p e c i f i c
that
of
mainly
in the
on the
sequences within
region.
r e g i o n by gel nuclear
been
been responsible
expression
promoter
cannonical
as an
are no r e a r r a g e m e n t s
gene
the
binding
antigen
factors
response
transcription
as w e l l
17-1A
the
including
a variety
which might have
contains multiple
regulating
have
growth
(2,4).
factors,
17-1A
cells
binding
repeats,
region,
level
IL-2
promoter
genes,
protein
the promoter
The
to
receptors,
of f a c t o r s
8-base direct
Multiple
as
and
of
17-1A
and
METHODS
of
nuclear
assay.
regions
antigen
the We
antigen
identified
that
gene
17-1A
might
gene
sites be
promoter
that
bind
important
for
expression.
extract
N u c l e a r e x t r a c t s w e r e p r e p a r e d by the m o d i f i e d m e t h o d of Dignam et al. (i0) . C a p a n 2 cells, obtained from The Wistar Institute, Philadelphia,PA (USA) were grown as a monolayer to c o n f l u e n c e in L - 1 5 m e d i u m s u p p l e m e n t e d w i t h 10% fetal bovine serum, c o n t a i n i n g g e n t a m y c i n (50mg/l) . T h e c e l l s w e r e harvested with p h o s p h a t e - b u f f e r e d s a l i n e (PBS), c o n t a i n i n g trypsin-EDTA. All m a n i p u l a t i o D s w e r e p e r f o r m e d on ice and all buffers were c h i l e d to 0 - 4UC. Phenyl-methyl-sulfonyl-fluoride (PMSF) and dithiothreitol (DTT) w e r e a d d e d to the b u f f e r s j u s t b e f o r e use. Pelleted cells were s u s p e n d e d in c o l d P B S and c o l l e c t e d b y c e n t r i f u g a t i o n for I0 m i n at 2 0 0 0 rpm. T h e c e l l s w e r e s u s p e n d e d in f i v e p a c k e d cell v o l u m e s of b u f f e r A c o n t a i n i n g 1 0 m M H e p e s p H 7.9, 1 . 5 m M M g C I 2, 1 0 m M KCI, 0.5mM DTT and 0.5m_M P M S F and left on ice for I0 min. C e l l s w e r e c o l l e c t e d as b e f o r e , h o m o g e n i z e d in t w o v o l u m e s of b u f f e r A, and p e l l e t e d at 2 0 0 0 r p m at 4°C. N u c l e a r p e l l e t ( c r u d e nuclei) was resuspended in 20mM Hepes pH 7.9, 25% glycerol, 0.42M NaCI, 1 . 5 m M M g C I 2, 0 . 2 m M EDTA, 0.5mM DTT and 0 . S m M P M S F (buffer C) . T h e s u s p e n s i o n w a s g e n t l y s t i r r e d f o r 30 m i n and c e n t r i f u g e d for 30 m i n at 25 000 x g at 4°C. Then to the supernatant solid (NH4)2SO 4 w a s a d d e d to final concentration of 0.45g/ml. The precipitation was conducted for 30 min on ice-bath with m i x i n g , f o l l o w e d by c e n t r i f u g a t i o n as above. Protein pellet was resuspended in 2 0 m M H e p e s pH 7.9, 20% g l y c e r o l , 0 . 1 M KCI,
1354
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0 . 2 m M EDTA, 0 . 5 m M DTT, 0 . 5 m M P M S F (buffer D) and dialized o v e r n i g h t a g a i n s t the s a m e b u f f e r . T h e d i a l y s a t e w a s centrifuged at 25 000 x g for 30 min, and the supernataDt (nuclear extract) was f r o z e n as a l i q u o t s and s t o r e d at - 8 0 ° C . Preparation
of
restriction
fragments
Targets for DNA binding assay were isolated from MI3 mpl8 phage w h i c h c o n t a i n e d a large fragment of promoter with 5"upstream s e q u e n c e and a small 1 7 - 1 A a n t i g e n g e n e f r a g m e n t , i n s e r t e d betw e e n the Accl and SmaI r e s t r i c t i o n sites. The M I 3 m p l 8 p h a g e w a s cut by restriction enzymes: HinPl, Hinfl, and BspEI. The cut e n d s were filled and labeled w i t h K l e n o w f r a g m e n t of DNA P o l y m e r a s e I. T h e D N A w a s cut a g a i n w i t h Pstl and t h e resulting f r a g m e n t s w e r e s e p a r a t e d by e l e c t r o p h o r e s i s on non-denaturating 5% p o l y a c r y l a m i d e gel, e l u t e d f r o m the gel and p u r i f i e d b y p h e n o l : chloroform extraction and ethanol precipitation.
of o l i g o n H c l e o t i d e s
Preparation
Oligonucleotides were synthesized by Applied Biosystems DNA s y n t h e s i z e r , p u r i f i e d a n d s i n g l e s t r a n d s w e r e m i x e d in equimolar a m o u n t s , h e a t e d to 8 8 ° C and c o o l e d slowly to r o o m t e m p e r a t u r e . O l i g o n u c l ~ g t i d e s , u s e d as p r o b e s in some e x p e r i m e n t s w e r e l a b e l e d with Z---P ATP and T4 polynucleotide kinase (Boehringer Mannheim) . 01igonucleotides, containing known consensus sequences f o r t r a n s c r i p t i o n f a c t o r s (e.g. API, AP2, NFI), u s e d in competition experiments, derived from kit (Stratagene Cat.203002) .
Gel
retardation
assay
The standard assa~2 was performed in a v o l u m e of 20 ~ I and a sample containing P labeled probe (2 0 0 0 - 4 000 cpm) , p o l y d l d C as a non-specific competitor ( 0 . 2 - 0 . 4 ~ g per r e a c t i o n ) , b i n d i n g buffer (15mM Hepes/KOH p H 7.5, l l 0 m M NaCI, 2m_M DTT, 250 H g / m l BSA) and n u c l e a r e x t r a c t (0-6 ~ g of p r o t e i n ) . T h e n u c l e a r e x t r a c t was always a d d e d last to the r e a c t i o n mixture. The complete m i x t u r e s w e r e i n c u b a t e d f o r 20 to 30 m i n at r o o m t e m p e r a t u r e and l o a d e d on 5% n a t i v e p o l y a c r y l a m i d e gel in 0.5 x T r i s b o r a t e EDTA (TBE) b u f f e r . T h e e l e c t r o p h o r e s i s w a s c o n d u c t e d at 2 0 0 V f o r 2 to 2.5 h r and the gel a n a l y z e d by a u t o r a d i o g r a p h y at - 8 0 ° C with i n t e n s i f y i n g screen. For competition experiments a I0 to 1 0 0 - f o l d m o l a r e x c e s s of t h e c o r r e s p o n d i n g u n l a b e l e d f r a g m e n t or o l i g o n u c l e o t i d e s were added at the b e g i n n i n g of the b i n d i n g r e a c t i o n .
RESULTS
and
Present
DISCUSSION studies
were
the p r o m o t e r
of the
b l e for h i g h
expression
type
of
chosen these
cells. since
To
of
determine
this gene
fragments,
carcinoma of
identify
specificity covering
cell
about
1355
or
line
antigen
tumor
as w e l l
which
in one,
the
to o t h e r
line S W 948), sequence
to
a n t i g e n gene,
expression
as c o m p a r e d
cell
overlapping
17-1A
Pancreatic
the
cells,
carcinoma
undertaken
cell
elements, might
be r e s p o n s i -
low in the (Capan was
lines
as to n o r m a l of the
within
was
highest
in
(e,g.colorectal cells
detected
i000 bp of
other
2)
the
(2,8,9). complexes, 5"flanking
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS x
A 03
] -1000
-900
-998
-914
I
-800
I I
-700 -800
Hf
i
J
-811 79bp -
4
-
-142
+3
H
-252
- 142 B
359bp
48
-100
H
-518 Hf
-260
-285
~i
-877
-
-360
-5218
H
B
-400
l-.¢n
i
111____ I
-500
(/%0
233bp 8
-
4
P
*'3
1 lObp 8
-4
8
145bp 8
2561op 4 6
-
-
4
6
193bp 4 6
Fig.l. B i n d i n g of p r o t e i n s from the nuclear extract to various fragments s p a n n i n g 5 " u p s t r e a m promoter sequence of 17-1A antigen gene. A. The schematic map of the studied sequence with known consensus s e q u e n c e s present within. R e s t r i c t i o n sites generating fragments are: H-HinPI, Hf-Hinfl, B - B s p E I and P-Pstl. B: Each labeled f r a g m e n t was incubated for 30 m i n w i t h i n d i c a t e d amounts of n u c l e a r e x t r a c t for 30 m i n (binding c o n d i t i o n s were d e s c r i b e d in M a t e r i a l s and Methods). Assays were performed by electrophoresis on n a t i v e 5% polyacrylamide gel f o l l o w e d by autoradiography at -80°C. The figure is a composite of separate gels and therefore fragments of d i f f e r e n t size appear to have the same m o b i l i t y . region assay. most of 359
of
the
Fig.iA
important
binding bp
amounts
in of
gene,
were
shows sites
assays
are
lenght added
individually
assayed
restriction
fragments
recognizing
transcription
shown
in
Fig.lB.
(HinPl-HinPl)
nuclear
extract.
1356
was The
The
by
gel
used,
retardation
as w e l l
factors.
restriction
retarded smaller
256
by bp
as
the
Results fragment, increasing fragment,
Vol. 186, No. 3, 1992
(HinfI-Hinfl), well
resolved
even
located bands.
(HinPI-HinPl) smallest
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in size,
the
CAP
quntitively HinPI)
distinct
obtained These
results
fragment,
of
of
two
fragment
described.
shows
The
no b i n d i n g
359 bp
promoter,
in the
with
(HinPl-
several
nuclear
bands.
Two
145 bp f r a g m e n t
C A A A T box. 145
and
ii0 bp
adjacent
the w h o l e
a banding
activity,
qualitatively
labeled
interaction
proximal
binding
t h r e e to f o u r r e t a r d e d
containing
demonstrate
the
both
the
T A T A box and
two fragments
fragments:
showed
233 bp
previously
significant
detected
Ii0 bp f r a g m e n t ,
f o r the
two
an
resolved
containing
f o r the
differed,
Binding
(BspEI-Pstl), the
fragments also
indicated
fragment,
concentration.
bands
complexes were
fragment
within
showed
(Fig.iB) .
(HinPI-Pstl),
part of
(HinPI-HinPl)
retarded
giving well
359 bp
of 256 bp,
restriction
fragment
proteins,
that
protein
site,
the
the
binding pattern
79 bp
at t h e h i g h e s t
although
The
resembles
The remaining to
inside
For
bp
pattern was
the
193 bp
sequence a s u m of
and that
separately.
that m o s t
of
the
(distal r e g i o n )
binding
and
193 bp
occured (proximal
region). Each fragment, specificity 50-fold
by
d i d not
middle
of the
comprising
excess show
bp
DNA
specific
tested
extract
and
233 bp
fragments,
5'GTGGTTTG
H~nPI --
3'
The
was
-- HinP! (110bp) 0 ~0 SO
--
and
binding i0
This
in the region,
containing several
the
binding
H~nP/--Pst11145 bp) 0 I0 S0
Fig. 2. Competition analysis of promoter binding proteln specificity. Labeled restriction fragments, shown in Fig.l, were incubated with nuclear extract in the presence of increasing amounts of the competitor (unlabeled DNA) and analyzed using gel retardation assay The molar excess of the competitor used is shown above the lanes.
1357
or
large r e g i o n ,
identified
although
(7,11)
the
along with
fragments. binding
for
( n u c l e o t i d e s -555 to -252).
sequence
competitor
was
with nuclear
of u n l a b e l e d any
sequence
201
core e n h a n c e r
in F i g . i B ) ,
incubation
molar
which
(shown
Vol. 186, No. 3, 1992 sites
for
number
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
regulatory
of r e t a r d e d
appears nonspecifiC, any s p e c i f i c Results
fragment,
(Fig.l),
since
these bands
and
was
removed
by
As s e e n
fragment was
specifically
molar excess o t h e r hand, the 5 " e n d complex
of
of
the
cases
not
all
lower
set
of
specificity
to the
proximal
bands
were
competed.
bands
within
By
of b i n d i n g seemed
concordant
with
our
crucial box
(12) .
b o x up to - 9 7
contained
proximal
sequence.
specific
box
and
SPI
site,
with
unlabeled
I0,
double
These
to -23.
However,
the b i n d i n g
The
were
and
as
a
appeared
several
mostly
1358
factor.
in m o s t
genes
and
CAP
of
the
of TATA
important
for
the r e g i o n s
of
oligonucleotides 120 bp
used
one,
of
the
as p r o b e s
for
competition TATA
including
-ii0
to
of
The
extract
appropriate
competitor nuclear
nonspecific,
box, CAAAT
-81.
a nuclear
excess
specific by
multiple
box
for
incubated with
retarded
including
containing
second
molar
region
initiation
the
from nucleotide
were
50-fold
1 was
stranded
interactions
extended
that TATA
element
within
the
partially
studied
to n a r r o w
oligonucleotide,
-49
25,
the
oligonucleotides
oligonucleotide
Oligonucleotide
were
efficient
order
showed
transcription
upstream
elements
labeled oligonucleotides along
In
binding
whereas
sequences upstream
two
first
sequence
and
important
DNA-protein The
both
a second (13).
constructed
experiments. comprised
Moreover,
transcription we
If,
specific
transcription
the SPI
the
corresponding
fragment
the
studies had shown
polymerase for
for
both
and
showed
results
since
more
In
one,
the bp
the
generated
sequences
point mutation
encompassing most
These
50-fold On
(145 bp) ,
highest
145
bp)
encompasses
containing
and
binding,
which
two h i g h e r bands,
elements
specific
a
control
Deletions
necessary
by
fragment,
of
145 bp
(Ii0
region,
recognition
by RNA
the
transcription
consensus
of
fragment,
the
expectations,
specific
a n d the G C - r i c h
CAAAT
efficient
the
of
competed
excess
contrast, for
showed
ii0 bp f r a g m e n t ,
an
by
two f r a g m e n t s
unlabeled
The
to be n o n s p e c i f i c .
T A T A box,
site were
of
competed
HinPI-HinPl
for the 3 " e n d
the
presence
to
small
binding
activity
of the p r o m o t e r
seen
lowest one
transcribed
region
that
fragment,
contained
ii0 bp
be
excess
efficiently
than
in t h e
specificity
and
corresponding
binding
pattern
unlabeled
the
the
the
(145 bp),
of the b i n d i n g
binding
a
(not s h o w n ) . that
10-fold molar
in F i g . 2 ,
showed
c o u l d not
demonstrate,
part
a
competitor,
CRE)
In a d d i t i o n ,
HinPI-PstI
A significant
API,
competitor used
in F i g . 2
(ii0 bp)
competition.
(e.g.
bands
or n o n s p e c i f i c
presented
HinPI-HinPl
proteins
(Fig.3) . proteins.
since
these
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS oltgo ~
1.
~,
oligo 2 •
,
S'GGGCAGGTCGGGTAGC~AGAGCC CCCGTCCAGCCCATCGC~ATA~TCTCGG
3'
_ _ S P a _ _
2. S' GGAGGGGGGCGGGATGTGTCACC~AAAT~CC
CC TCCCCCCGCCCTACACAG T G ~ G T T T ~ T G G
3"
Fig.3. N u c l e a r proteins binding to particular oligonucleotides. The o l i g o n u c l e o t i d e s (prepared as described in M a t e r i a l s and Methods), d e p i c t e d s c h e m a t i c a l l y below, were used as p r o b e s and incubated with 6 Hg of the nuclear extract in the p r e s e n c e of increasing amounts of the same oligonucleotide as a competitor.
bands
could
specific competed
by
Binding bands
a
competed
one
and
which
with
of
molar band
excess was
2 showed
two
competed.
1 band
with
oligonucleotide
use
as
respectively.
Results
band
specific
for
the
competitor.
specifically
the
of
efficiently
oligonucleotide
oligonucleotides
only
the
be
petitors was
50-fold
highest
oligonucleotide
could
for
by
the
excess
experiments both
even
Only
10-fold
specificity
competition probe
be
experiments
with
binding
not
competitor.
of
specific
and
demonstrated
oligonucleotide
,-_ollgo 1,
we
check
carried i
nonspecific
that 1
retarded To
the
out as
observed
(containing
TATA
~,._oli~o Z
competitor
Fig.4. C o m p e t i t i o n for nuclear proteins binding to the TATA box containing o l i g o n u c l e o t i d e i, with specific and nonspecific competitors (unlabeled o l i g o n u c l e o t i d e 1 and 2). The m o l a r excess of competitors is indicated, above each lane.
1359
a
com-
Vol. 186, No. 3, 1992 box) of
and
that
the b a n d
nonspecific
suggests of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
also
359
the
specific
bands
was
competitor Because
well
as
observed which
stranded
oligonucleotides, protein
fragment) band was CT/NFI
as
sites
by
with
the
(Fig.5) . T h e r e
several ment
binding
might
indicated
protein
in
extensive
the
the
these
was
are
fragment
studies
have
antigen
gene
in t h e indicated promoter,
=NA
decided
same
--
0
10
SO
that
by
exhibit
antigen there may
enhan-
cells.
shift
Anyway role
~n
E
~
~
~
within
the
regulation
A
Fig,5. C o m p e t i t i o n analysis of the distal region b i n d i n g s p e c i f i city, A. Labeled fragment HinPI-HinPI (359bp) was incubated w i t h 6 ~g of nuclear extract in the presence of increasing amounts of specific c o m p e t i t o r (unlabeled fragment). B. Competition experiment using oligonucleotides, c o n t a i n i n g consensus sequences r e c o g n i z e d by transcription factors, indicated above each lane.
1360
of
expression.
part
O
or
binding
putative
sequences
--
as
enhance-
CT/NFI
gene
take
one
little
2
and
oligonu-
with
of
the
are
AP2
band
Capan
upper
whereas
observed
or
API
the
and
dependence
The
API
in
the
API
o r API
(14).
by
additio-
(absent
fragments
from
100
and
as
were double
recognized
that
which
use
efficiency,
"i~PZ-m~P~ ~3SSbp~
cornpet{tor
two
speci-
CT/NFI
to
It w a s f o u n d
to e x p l a i n
that
the the
or
competitors
unaffected
17-1A
of
function
containing
of e i t h e r
extract
binding
promoter,
of
API,CRE
a functional
necessary
the
out
the A P 2 m o t i f
factors
presence
of
unknown
sequences
the
only NFI
nuclear
studies
this distal Our
of
as
we
competitor.
suggesting
cooperation
region
of
is e v i d e n c e
the
(Fig.5).
such
containing
for
to
box.
one
as s p e c i f i c
activity
sites
by t h e m s e l v e s ,
c e r on
17-1A
binding
be d u e
excess
approximatelly
cleotide
This
least
oligonucleotides
expected
(Fig.4).
2)
molar
containing
a nonspecific
competed
excess
At
fragment,
factors,
a 100-fold
10-fold
element
bp
an o l i g o n u c l e o t i d e
might
in d i s t a l
motifs
TATA 359
by
the T A T A
fragment)
sequence
within
nally
by
(unlabeled
the
shift with
located
present
and CT/NFI
band
competition.
additional
even
(oligonucleotide
competed
several
an
competed
interact
fragment,
showed
retarded fic
bp
not
competitor
a protein(s) The
was
Vol. 186, No. 3, 1992
of expression extracts other
sequences
this
or s e p a r a t e
cells
determine
of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
differing the role
of
gene.
Further
factors
in expression factors
for the e x p r e s s i o n
studies
are required
of this gene
recognizing
of the
17-1A
using
purified
and c o m p a r J s i o n with are
specific
antigen
needed
to
promoter
gen~
Ac k howl edg ments We thank Prof. A Horst and Prof. W.H.Trzeciak for reading this paper and helpful discussion, K.G6rczy6ska for technical assistance and Dr. J . W o j c i e r o w s k i of the Medical School in Lublin (Poland) for his k i n d gift of antigen gene promoter, obtained through the c o u r t e s y of D r . A . J . L i n n e n b a c h of the W i s t a r Institute Philadelphia,PA, REFERENCES
l.Ross,A.H., Herlyn,D., lliopoulos,D, and Koprowski,H. (1986) Biochem. Biophys.Res. Commun., 135, 297-303. 2.Linnenbach,A.J., W o j c i e r o w s k i , J . , Wu,S., Pyrc,J.J., Ross,A.H., Dietzschold,B., Speicher,D. and Koprowski,H. (1989) Proc. Natl. Acad. Sci.USA, 86, 27-31. 3 Szala,S., F r o e h l i c h , M . , Scollon,M., Kasai,Y., Steplewski,Z., Koprowski,H. and Linnenbach,A.J. (1990) Proc.Natl.Acad.Sci. USA, 87, 3542-3546. 4.Depper,L.W.J., K a n e h i s a , M . , K r o n k e , M . , Peffer,N.J.,Swetl~k,P.B. Sulivan,M. and Greene,W.C. (1985) Science, 230, 633-639. 5.Benoist,C and Chambon,P. (1981) Nature, 290, 304-310. 6.Concino,M.,Goldman,R.A.,Caruthers,M.M. and Weinmann,R. (1983), J.Biol.Chem., 258, 8493-8496. 7.Khoury,G. and Gruss,P. (1983) Cell, 33, 313-314. 8.Herlyn,M., Steplewski,Z., Her]yn,D. and Koprowski,H. (1986) Hybridoma, 5,3-10. 9.Herlyn,M., S t e p l e w s k i , Z . , Herlyn,D. and Koprowski,H. (1986) Hybridoma, 5, 3-8. 10.Dignam,J.D., Leibovitz,R.M. and Roeder,R.G. (1983) Nucleic Acid. Res., ii, 1475-1489. ll.Wasylyk,B. (1988) Biochim. Biophys. Acta, 951, 17-35. 12.Hen,R.P.,Sassone-Corsi,J.,Corden,M.P.,Gaub,M.P. and Chambon,P. (1982) P r o c . N a t l . A c a d . Sci.USA, 79, 7132-7136. 13.Jove,R. and Manley,J.L. (1984) J.Biol.Chem.,259, 8513-8521. 14.Chong,T.,Chan,W.-K. and Bernard,H.-U. (1990) N u c l e i c Acid Res. 18, 465-470.
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