From the
We report the identification of a 20-base pair sequence
mediating induced transcription in response to an activated Ha-ras gene and epidermal growth factor (EGF) but not
12-O-tetradecanoylphorbol-13-acetate stimulation. This
signal-specific nuclear target is present in the long terminal repeat
of a mouse VL30 retrotransposon expressed in epidermis. Functional
studies and in vitro binding analyses using cultured
keratinocytes (Balb/MK) reveal that the response element is composed of
two cooperating sequence motifs in juxtaposed position, both of which
are targets for induced binding activity 1-2 h after EGF
stimulation. Of many different activating transcription
factor/cAMP-responsive element binding protein/activating protein 1
factors tested, one part of the sequence selectively binds endogenous
proteins immunologically related to activating transcription factor 3
(ATF3) and Jun isotypes. The other sequence is a target for a nuclear
factor showing binding specificity unrelated to factors known to
mediate EGF- or ras-induced transcription as determined by its
sequence specificity and by antibody experiments. This component has
been characterized and partially purified by gel filtration
chromatography and velocity centrifugation revealing a Stokes radius of
43.6 Å and a sedimentation coefficient of 9.7 S in solution.
Based on these parameters, a molecular mass of 178,000 Da was
calculated. The results indicate that the specific binding of ATF3/Jun
and a previously uncharacterized factor account for signal-specific
transcription in response to EGF or an activated Ha-ras gene
in a cell type in which the cooperative action of an activated
Ha-ras gene and 12-O-tetradecanoylphorbol-13-acetate
cause tumor growth.
Mammalian ras genes (Ha-, Ki-, and N-ras) encode small
GTP-binding proteins (p21) that link the activity of certain plasma
membrane-associated tyrosine kinases, such as the receptor for
epidermal growth factor (EGF),
Owen et al.(16) have identified a RRE in the LTR of another VL30 designated
NVL3. The NVL3 RRE is similar to several previously identified RREs in
that it is composed of an AP1-binding site adjacent to a
PEA3/Ets-binding site. Fig. 2C shows a sequence
alignment of the two RREs that revealed sequence differences both in
the AP1-like site and flanking sequences. Since the NVL3 RRE was
identified using NIH/3T3 cells, we analyzed if the capacity to mediate
Ha-ras-induced transcription differed between the two RREs
when analyzed in Balb/MK and NIH/3T3 cells, respectively. A luciferase
reporter plasmid containing the NVL3 RRE upstream of the thymidine
kinase promoter was constructed and used in co-transfection experiments
(Fig. 2C, NVL3 RRE.Luc). As shown in
Fig. 2C, the NVL3 RRE was found to be functional in both
cell types mediating a 4-fold induction of luciferase activity.
Interestingly, in contrast to the NVL3 RRE, the B10 RRE was found to be
inactive in the fibroblast cell line, whereas it mediated a 4-fold
induction in keratinocytes. This result indicated that the B10 and the
NVL3 RRE mediated ras-induced transcription by different
mechanisms.
The results show that the B10 RRE is
bipartite and contains a 5` AP1-like element and a 3` element having a
sequence unrelated to known transcription factor binding sites. As
judged by mutational studies of the B10 RRE, the presence of both
sequence motifs is essential to confer activated levels of
transcription in response to EGF or mutated ras.
Interestingly, by performing EMSA using nuclear extract prepared at
different time points after EGF stimulation, both sequence motifs are
found to be targets for EGF-induced binding activity.
Owen et
al.(16) have identified a RRE present in another member of
the VL30 family designated NVL3. This response element is similar to
other identified RREs in that it is composed of binding sites for AP1
and PEA3/Ets in juxtaposed position. Our results suggest the NVL3 RRE
and the B10 RRE have evolved to contain different flanking-sequences
juxtaposed to the AP1-like site. These sequence differences most likely
explain the finding that the B10 RRE is a target for a factor unrelated
to PEA3/Ets. The fact that the B10 RRE was unresponsive to a mutated
ras gene in NIH/3T3 cells underscores the difference of the
B10 RRE to other standard RREs as exemplified by the NVL3 RRE. Many
studies have implicated the binding of c-Jun to AP1-sites to be a
common denominator of induced transcription in response to both TPA and
an activated ras gene
(7, 8) . The B10 RRE is
unresponsive to TPA in spite of containing a nuclear factor binding
site that contains a sequence (TGACTCC) nearly identical to a consensus
TPA-responsive element (TGACTCA). Using antibodies specific to c-Fos
and a variety of different ATF/CREB proteins, we have identified the
major binding activity specific for the AP1-like site in the RRE to be
immunologically related to ATF3, c-Jun, JunB, and JunD. This indicates
that the predominant AP1-activity that specifically mediates EGF- and
ras-induced transcription consists of different ATF3/Jun
heterodimers. Support for this conclusion comes from a recent study by
Tan et al. (33). Their study shows that ATF3 and c-Jun
stimulate proenkephalin transcription in a FGF- and Ras-dependent
fashion through a CRE site in neuroblastoma cells
(33) .
Furthermore, ATF3 is implicated in the induction of gene expression
associated with the G
How is the specificity of the B10 RRE achieved?
Recent reports have shown that the selective dimerization of different
bZIP members determine the binding specificity toward polymorphic AP1-
and CRE/ATF-sites
(36, 37) . Consequently, different
bZIP-homo- or hetero-dimers with distinct binding specificity and
sensitivity to intracellular signals may determine the regulatory
identity of a certain type of AP1- or CRE/ATF-site. One might therefore
speculate that the lack of TPA responsiveness of the here-characterized
RRE is a consequence of a low affinity of this site to AP1 activity
mediating TPA-induced transcription. Support for this hypothesis comes
from a study that shows that a TGACTCC (i.e. the same sequence
as in the B10 RRE) motif in the JE gene is unresponsive to TPA and does
not bind in vitro-translated c-Jun homodimers or c-Jun/c-Fos
heterodimers
(5) . Moreover, introduction of nucleotide
substitutions in the consensus AP1-binding site present in the
collagenase gene to form a TGACTCC sequence abolishes both
TPA-inducible enhancer activity as well as binding of affinity-purified
AP1
(36) .
It is known that in vitro translated
ATF3/Jun heterodimers display a dual binding specificity in that these
can bind to and regulate transcription from both AP1 and CRE/ATF
sites
(37, 34) . Accordingly, the finding that both
consensus AP1 and CRE/ATF sites compete for binding to the AP1-like
site in the B10 RRE support the notion that this site exhibits
specificity to ATF3/Jun heterodimers. The involvement of an ATF3/Jun
heterodimer in Ras-induced transcription and the dual binding
specificity of this heterodimer is interesting in view of the number of
RREs that have been identified that contain either a CRE/ATF or an AP1
site
(38, 39) .
In this study, we show that induced
binding to a single ATF3/Jun-site is not sufficient to confer
ras-responsiveness to an heterologous promoter. The presence
of a juxtaposed nuclear factor binding site is essential for
functionality. Interestingly, this sequence is target for induced
binding activity in response to EGF and does not exhibit any affinity
to AP1, CRE, ETS, NF-
In
conclusion, we have identified a novel type of response element that
mediates signal-specific transcriptional induction in response to EGF
and an activated ras gene. The finding that this RRE is
unresponsive to the tumor promoter TPA and encompasses cooperating
binding sites that are targets for EGF-induced binding of ATF3/Jun and
a previously uncharacterized factor now provide means to further
analyze transcriptional mechanisms activated specifically by an
initiation event in a model system extensively used to define the
concepts of tumor initiation and promotion.
We thank Dr. Derek Tobin for critical reading of the
manuscript and Dr. Giannis Spyrou for the generous gift of
anti-Jun-antibodies. We also thank Lena Möller and Dr. Johan Lund
for expertise help with gel permeation chromatography.
(
)
to changes in
gene expression via a phosphorylation cascade involving Raf kinase and
mitogen-activated protein kinases
(1, 2) . The mutational
activation of the Ha-ras gene in keratinocytes has been shown
to occur during the initiation step in the mouse skin model for
multistage carcinogenesis
(3) . Skin tumor promoters such as
12-O-tetradecanoylphorbol-13-acetate (TPA) stimulate a
selective outgrowth of initiated keratinocytes to form benign tumors
(papillomas)
(4) . The mechanisms that determine the
cooperativity between an activated Ha-ras and a tumor promoter
remain largely unknown. However, it is assumed that altered gene
expression caused by the presence of an activated ras oncogene
in conjunction with TPA-induced protein kinase C activity results in
imbalance between keratinocyte growth and terminal differentiation. In
order to elucidate differences and similarities in signaling elicited
by TPA and an activated Ha-ras gene in keratinocytes, we have
used the LTR of a VL30 retrotransposon (B10) expressed in mouse
epidermis to identify sequences and transcription factors activated in
keratinocytes during these two phases of tumor development. Accumulated
data implicate that the protein kinase C- and Ras-pathways converge at
the DNA level
(5) . Both TPA and oncogenic Ras protein induce
transcription of a set of cellular and viral genes by activation of the
Jun/Fos (AP1) family of transcription factors, which bind to the TPA
response element (TGACTCA)
(6, 7, 8) . Members
within the Jun family can also bind and activate transcription via the
related cAMP response element (CRE; TGACGTCA) or variants thereof,
either as homodimers or heterodimers with CREB/ATF proteins
(9) .
Several identified DNA elements mediating induced transcription in
response to both an activated ras gene and TPA contain two
cooperating binding sites in juxtaposed positions that are recognized
by different classes of transcription factors
(10, 11) .
One example is cooperating PEA3/Ets- and AP1-sites, which constitute
RREs identified in the collagenase gene, macrophage scavenger receptor
gene, the polyoma virus enhancer, and the LTR of a member within the
murine VL30 retrotransposon family
(NVL3)
(11, 12, 13) . Here we report the
identification of a novel type of RRE present in the LTR of a VL30
member (B10) expressed in mouse epidermis. This 20-bp sequence mediates
induced transcription in keratinocytes in response to both EGF and an
activated Ha-ras gene. The RRE is distinct from a TPA response
element previously identified within the same LTR, and it does not
mediate induced transcription in response to TPA. These results show
that two different signal pathways that cooperate in mouse skin
carcinogenesis can act through different nuclear targets. A
characterization reveals that the identified RRE is composed of two
cooperating sequence motifs that are target for EGF-induced binding
activity corresponding to specific members within the AP1/ATF family of
transcription factors and a novel 178-kDa nuclear factor, respectively.
Cell Culture Conditions and Transfections
All
chemicals and media were purchased from Sigma, unless stated otherwise.
NIH3T3 cells were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) containing 10% (v/v) fetal calf serum
(Hyclone), 100 IU/ml penicillin, and 100 mg/ml streptomycin. Balb/MK
cells were cultivated in MCDB 153 medium, supplemented with 50
µM CaCl, 0.1 mM ethanolamine, 0.1
mM phosphoethanolamine, 10 ng/ml epidermal growth factor, 5
µg/ml insulin, 0.5% chelex-treated fetal calf serum, 100 IU/ml
penicillin, and 100 mg/ml streptomycin. Preconfluent NIH/3T3 and
Balb/MK cells were transfected by the calcium phosphate precipitation
method and lipofection, respectively
(17) (lipofectin was from
Life Technologies, Inc.). Cells were grown on 25-cm
dishes
and transfected with 3 µg of reporter plasmid DNA and 2 µg of
either a normal or activated c-Ha-ras expression plasmid.
Medium was changed 16 h after transfection, and the cells were cultured
for an additional 24 h. Cells treated with TPA were incubated with 100
ng/ml TPA (Pharmacia Biotech Inc.) for the final 16 h, and EGF-treated
cells were EGF-starved for 30 h prior to EGF treatment (10 ng/ml for 16
h). Cellular homogenates were prepared as described previously,
chloramphenicol acetyltransferase (CAT) and luciferase activities were
normalized to the activity of a cotransfected (1 µg/transfection)
Rous sarcoma virus LTR-driven luciferase (pRSV.Luc)
(18) or a
VL30 B10 promoter-driven CAT (pB10 Sna.CAT) reporter plasmid.
Plasmid Constructs
The construction of B10.U3,
B10.Stu, B10.StuSna, B10.SnaRep, and B10.Sna plasmids, which all
contained different parts of the B10 LTR (including TATA box and cap
site) fused to the bacterial CAT gene has been described elsewhere
(15). The pT109 plasmid contained the -109 to +51 promoter
region of the Herpes simplex thymidine kinase gene fused to a
luciferase reporter gene
(18) . B10 RRE.Luc, M1.Luc, M2.Luc,
M3.Luc, M4.Luc, and NVL3 RRE.Luc. were made by subcloning the
corresponding oligonucleotides into the HindIII-XhoI
site of the pT109 reporter plasmid. The construction of the expression
plasmids for normal (pHO6N1) and Val-12-mutated (pHO6T1) human c-Ha-Ras
protein have been described previously
(19) .
Preparation of Nuclear Extracts and EMSA
Nuclear
cell extracts were prepared according to Struhl et al.(20) in the presence of leupeptin (10 mg/ml), pepstatin (5
mM) (Boehringer Mannheim), and aprotinin (100 KIU/ml) (Bayer
Inc., Germany). EMSA was performed using 8 µg of nuclear protein
extract, 1.0 µg of poly(dIdC), and
P-labeled
oligonucleotide in GS buffer (80 mM KCl, 20 mM HEPES,
pH 7.8, and 4 mM MgCl
). The probe and competitor
were added together, and the binding reactions were incubated for 20
min at room temperature. The resulting protein-DNA complexes were
resolved on a preelectrophoresed 5% polyacrylamide gel (30:0.8) with 45
mM Tris borate and 0.5 mM EDTA as running buffer.
Oligonucleotides and Antibodies Used in Gel Shift
Analysis
Nuclear extracts and antibodies were incubated for 30
min at 4 °C prior to gel shift analysis. Anti-c-Fos, -CREB1,
-CREB2,-ATF1, -ATF2, and -ATF3 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). These affinity-purified
antibodies were raised against carboxyl-terminal peptides (c-Fos,
CREB2, and ATF3) or recombinant human proteins (CREB1, ATF1, and ATF2).
Anti-CREB, -c-Jun, -JunB, and -JunD were prepared and characterized as
described previously
(21, 22, 23) . The NVL3 RRE
oligonucleotide corresponds to the RRE present in the NVL3 VL30
LTR
(16) ; the PEA3 oligonucleotide corresponds to the
PEA3-binding site found in the polyoma virus enhancer
(24) ; and
the AP-1 oligonucleotide corresponds to the AP-1 binding site found in
the collagenase promoter
(25) . The CRE oligonucleotide
corresponds to the CRE present in the choriogonadotropin
gene
(26) ; the SRE oligonucleotide corresponds to the serum
response element found in the c-Fos promoter
(27) ; and the VLY
oligonucleotide corresponds to a p65/c-Rel-binding
site present in the B10 VL30 LTR (14). The sense strands of the
annealed oligonucleotides were as follows, (lowercase letters denote
nucleotides present in double-stranded oligonucleotides after a fill-in
reaction): B10 RRE, agctTGGACATGACTCCTTAGTTACtcga; M1,
agctTGGCTTTGACTCCTTAGTTACtcga; M2, agctTGGACACTGCTCCTTAGTTACtcga; M3,
agctTGGACATGACTCCTTAGtcga; M4, agctTGGACATGACTCCGCTGTTACtcga; GGmut,
agctTTTACATGACTCCTTAGTTACtcga; NVL3 RRE,
agctTACAGGATATGACTCTGCAGGTTGGCtcga; PEA3, agctTTAAGCAGGAAGTGACCtcga;
AP-1, ctagAGAAGCATGAGTCAGACACctag; CRE,
agctCGAGAAATTGACGTCATGGTTAAagct; SRE, agctTGATGTCCATATTAGGACATCAtcag;
VLY, agctTAAACTTGTACTTTCCCtcga.
Sucrose Density Gradient Centrifugation
200 µl
of a sample containing 1.5 mg of nuclear protein was layered onto a
25-40% (w/v) linear sucrose gradient prepared in GS buffer.
Gradients were centrifuged in a SW60-Ti rotor in a Beckman L8-M
centrifuge at 260,000- 300,000 g for 15 h to a
preset cumulative centrifugal effect (
t) of
1.7
10
rad
/s. Fractions of 150 µl
were collected from the bottom of the tube by gravity flow. Catalase
(11.3 S) and
-globulin (6.6 S) were used as external sedimentation
markers and detected by measuring the optical density of fractions at
280 nm.
Gel Permeation Chromatography
A Superose 12 column
(Pharmacia Biotech Inc.) was equilibrated in GS buffer. The column was
calibrated with albumin, ovalbumin, DNase I, and cytochrome c.
The flow rate was 0.4 ml/min, the sample volumes were less than 2% of
the total column volume, and 0.4-ml fractions were collected. The
calculations of molecular parameters were performed according to Siegel
and Monty
(28) . The Stokes radii
(R) were determined graphically from a
plot of -log Stokes radii versusV
/V
, where
V
and V
are the
elution volume of the factor and the void volume of the column,
respectively. The molecular weight in solution was calculated with the
assumption that the partial specific volume of the macromolecule was
0.725 cm
/g and that the solvation factor was 0.2 g of
solvent/g of solute, both of which are typical values of
proteins
(29) . The molecular weight and frictional ratio were
calculated using the equation M
=
422SR
and
f/f
= 1.393
(R
/M
),
respectively
(28, 30) .
RESULTS
Identification of a 20-bp Sequence within the B10
LTR-mediating Ha-Ras-induced Transcription
We have previously
used the LTR of the VL30 clone B10 to identify sequences and nuclear
factors mediating induced transcription in keratinocytes in response to
the skin tumor promoter TPA
(14) . To study if this LTR in
addition could mediate induced transcription in response to an event
mimicking tumor initiation, we employed transient transfection
experiments using a B10 LTR-driven reporter gene plasmid together with
expression vectors coding for either the normal or the activated form
(Ras-Val-12) of the human c-Ha-Ras oncoprotein, respectively. The
reporter gene plasmid (B10.U3) contained the U3-region of the B10 LTR
fused to a CAT reporter gene. Fig. 1shows that the VL30 LTR
present in B10.U3 mediated a 10-fold increased CAT activity when a
Balb/MK mouse keratinocyte cell line was co-transfected with a plasmid
expressing the oncogenic Ha-ras gene
(ras). No enhanced CAT activity was detected in
cells transfected with a plasmid expressing the normal form of the
c-Ha-ras gene (ras) or a control plasmid (pUC9, data
not shown).
Figure 1:
Mapping of a
ras-responsive region within the B10 LTR. Shown is a schematic
representation of the different B10 LTR-driven CAT plasmids and an
autoradiograph of a CAT analysis performed with the different plasmids.
The B10.U3 contains a full-length (375 bp) U3 region (hatchedarea) and the first open reading frame in the R region
cloned in frame with the CAT gene in a promoterless vector.
Transcription start site is indicated by +1. The direct repeats in
the LTR are indicated with arrows. Deletions in the LTR were
made by digestion with restriction enzymes indicated as in the figure
(Xb, XbaI, -301; St, StuI,
-215; Sn, SnaB1, -122). The different CAT
plasmids were transfected together with an expression vector encoding
either the normal (ras) or the activated form
(ras) of c-Ha-Ras. Protein aliquots were
taken for CAT assay with the amounts adjusted with respect to the
relative transfection efficiencies, as determined by luciferase
activity of co-transfected pRSVLuc vector. CM,
chloramphenicol; AcCM, acetylated chloramphenicol. -fold
induction (F.I.) and percent CAT conversion (%) are indicated.
The same pattern of expression was obtained in three independent
experiments.
To determine the minimal sequence required for
ras-induced VL30 transcription, we performed CAT assays using
a number of plasmid constructs (Fig. 1), which contained
different deletions in the U3 region of the B10 LTR. Fig. 1shows
that activated Ha-ras gene expression in Balb/MK cells
resulted in a 10-50-fold stimulation of CAT activity from the
B10.U3, B10.Stu, and B10.SnaRep plasmids. The relative low -fold
induction obtained with the B10.U3 plasmid (9.7 fold) compared with
B10.Stu (25.4-fold) and B10.SnaRep (48.6-fold) was reproducible and
indicated that the sequence 5` to the StuI site repressed
inducibility. The B10 LTR contains a direct repeat of two 35-bp
sequences (indicated with arrows in Fig. 1)
(15) .
No ras-induced transcription was obtained with plasmids that
lacked both repeat units (B10.StuSna and B10.Sna). This observation led
us to construct a plasmid that contained one repeat unit by ligating a
20-bp oligonucleotide corresponding to the 5` end of one repeat unit to
the SnaBI site of B10.Sna. Interestingly, the 20-bp sequence
restored the ras responsiveness (compare B10.Sna with
B10.SnaRep). To determine if the 20-base pair sequence alone was
sufficient to mediate ras-induced transcription, we cloned an
oligonucleotide corresponding to this sequence 5` to the herpes simplex
virus thymidine kinase promoter in a luciferase reporter plasmid
(pT109). As shown in Fig. 2A, this plasmid, termed B10
RRE.Luc, mediated a 4-fold induction when cotransfected together with
the vector expressing an activated Ha-ras gene as compared
with cells transfected with a plasmid expressing the normal
c-Ha-ras gene. This result demonstrated that the 20-bp
sequence functioned as a RRE.
Figure 2:
Identification and functional analysis of
B10 RRE. A, the B10 RRE.Luc is a luciferase reporter gene
plasmid containing an oligonucleotide corresponding to the 20-bp
sequence involved in ras-induced B10 transcription, cloned
upstream (-109) to the herpes simplex thymidine kinase promoter.
Shown is the -fold induction of lucifer-ase activity in transiently
transfected Balb/MK cells treated with EGF (10 ng/ml for 16 h) and in
cells transfected with an activated c-Ha-ras gene. EGF
treatments were performed using cells cultured in absence of EGF for 30
h. The -fold induction obtained in response to EGF and activated
c-Ha-ras-expression is shown relative to the activity obtained
in untreated cells and cells transfected with an expression plasmid
coding for the normal c-Ha-ras gene, respectively. The -fold
induction was normalized relative to cells transfected in parallel with
the pT109 plasmid, containing the thymidine kinase promoter only.
Protein aliquots were taken for luciferase assays with the amounts
adjusted with respect to the relative transfection efficiencies, as
determined by CAT activity of co-transfected pB10.Sna.CAT. The bars in the figure represent the mean values of the relative induced
luciferase activity from five independent experiments. The standard
deviations of the mean values are indicated. B, VLTRE.Luc is a
luciferase reporter gene plasmid containing an oligonucleotide
corresponding to a TPA responsive element, previously identified in the
B10 LTR, cloned upstream (-109) to the herpes simplex thymidine
kinase promoter. Shown is the -fold induction of luciferase activity 16
h after TPA-treatment (100 ng/ml) of Balb/MK cells transiently
transfected with B10 RRE.Luc or VLTRE.Luc. The -fold induction was
calculated as described for A. Protein aliquots were taken for
luciferase assays with the amounts adjusted with respect to the
relative transfection efficiencies, as determined by CAT activity of
co-transfected pRSVCAT. The bars in the figure represent the
mean values of the relative induced luciferase activity from four
independent experiments. C, the NVL3 RRE.Luc is a luciferase
reporter gene plasmid containing an oligonucleotide corresponding to
the RRE previously identified in the NVL3 VL30 LTR, cloned upstream
(-109) of the herpes simplex thymidine kinase promoter.
Balb/MK and NIH/3T3 cells were transfected with an expression plasmid
for a normal or an activated Ha-ras gene, and the B10 RRE.Luc,
NVL3 RRE.Luc, or pT109.Luc as reporter plasmids. Shown is the -fold
induction obtained in response to an activated Ha-ras gene
calculated relative to the activity obtained in cells transfected with
the pT109.Luc, as described for A. The bars represent
the mean values of the relative -fold induction obtained in four
independent experiments. The standard deviation of the mean is also
indicated. The lowerpanel shows a sequence alignment
of the B10 RRE and NVL3 RRE.
Functional Characterization of B10 RRE
Next we
investigated if the identified RRE was a nuclear target for a signal
pathway stimulated through the EGF receptor. We analyzed the capacity
of the B10 RRE.Luc in mediating EGF-induced transcription. The result
shown in Fig. 2A indicated that this assumption was
correct in that the B10 RRE.Luc responded with a 4-fold induction of
luciferase activity after readdition of EGF to cells cultivated in the
absence of EGF for 30 h. We have previously identified a nonconsensus
CRE/ATF-site that binds CREB- and Jun-related proteins and cooperates
with a juxtaposed p65/c-Rel-binding site in
mediating TPA-induced transcription in
keratinocytes
(15, 14) . This sequence (VLTRE) is
situated 27 base pairs 3` to the B10 RRE and is unresponsive to
EGF
(15) . We wanted to rule out that the RRE was responsive to
TPA. Fig. 2B shows that VLTRE inserted 5` to the
thymidine kinase promoter in the pT109 plasmid (VLTRE.Luc) responded to
TPA treatment with a 4-fold increase in luciferase activity, whereas
B10 RRE.Luc was unresponsive to the same treatment. Taken together,
these results suggested that the B10 RRE was responsive to an
EGF/Ha-ras-signal pathway that is not induced by the protein
kinase C-activator and tumor promoter TPA.
Determination of the Sequence Requirement for ras-induced
Transcription by Mutational Analysis of the B10 RRE
The 20-base
pair B10 RRE sequence is shown in Fig. 2C. It contained
a motif, TGACTCC, which deviates from a canonical AP1-site, TGACTCA,
with one nucleotide. To further characterize the sequence requirement
of Ha-ras-induced transcription, oligonucleotides containing
substitutions within the AP1-like site and its flanking sequences were
synthesized (Fig. 3, M1-M4). Plasmids
containing these oligonucleotides upstream of the thymidine kinase
promoter in the pT109 luciferase reporter gene plasmid were
cotransfected with Ha-ras expression vectors.
Fig. 3
shows the -fold induction mediated by these plasmids in
response to an activated Ha-ras gene relative to the activity
obtained in cells transfected with the normal Ha-ras gene. The
result from this experiment demonstrated that the nucleotide
substitutions in the AP1-like site (M2.Luc) abolished
ras-responsiveness. Interestingly, mutations both 5` (M1.Luc)
and 3` (M3- and M4.Luc) to the AP1-like site also inhibited the
capacity of the B10 RRE to mediate induced transcription. This result
indicated that the AP1-like site alone was not sufficient to mediate
transcription induced by a mutated ras gene.
Figure 3:
Mutational analysis of the B10 RRE. Shown
are the sequences of oligonucleotides corresponding to B10 RRE.Luc and
mutated variants of B10 RRE.Luc (M1-M4) cloned
upstream (-109) to the herpes simplex thymidine kinase promoter
in a luciferase reporter gene plasmid (pT109). The bars in the
figure represent mean values of the relative -fold induction of
luciferase activity from five independent experiments calculated as
described in the legend to Fig. 2A. The standard deviations of
the mean values are indicated.
Identification of Nuclear Proteins Showing EGF-induced
Binding to the B10 RRE
In order to identify possible qualitative
and/or quantitative differences in DNA-protein complex formation, we
performed EMSA. In the experiment shown in Fig. 4A, we
used an oligonucleotide corresponding to B10 RRE as probe and nuclear
extracts prepared prior to and after readdition of EGF (10 ng/ml) to
cells cultivated without EGF for 48 h. Interestingly, several complexes
were identified that showed induced formation 1-2 h after
addition of EGF. Three complexes (designated 1, 2, and 3) were formed
in a sequence-specific manner as judged from the ability of a 100-fold
molar excess of unlabeled B10 RRE to compete for complex formation,
whereas a p65/c-REL-binding site (VLY) in the same
molar excess was without effect (Fig. 4B). In accordance
with the functional results, no induced binding activity corresponding
to complex 1, 2, and 3 was detected in nuclear extracts prepared from
EGF-starved cells treated for 1 and 2 h with TPA (data not shown).
TPA-induced binding activity could, however, be detected in the same
nuclear extracts using the VLY probe (14, and data not shown).
Figure 4:
EMSA of B10 RRE binding activity in
Balb/MK nuclear extracts. A, EMSA using nuclear extracts
prepared prior to (Cont), 1, and 2 h after readdition of EGF
to Balb/MK cells cultivated without EGF for 48 h. Equal amounts of
proteins (8 µg) from the different extracts were incubated with
P-labeled oligonucleotides corresponding to the B10 RRE
(upperpanel) or an Ets-binding site present in the
polyoma virus enhancer (PEA3) (lowerpanel).
Protein-DNA complexes were resolved by 5% nondenaturating
polyacrylamide gel electrophoresis and autoradiography of the fixed and
dried gels. Complexes formed in a sequence-specific manner are
indicated with 1, 2, and 3. B, EMSA
using the B10 RRE probe and Balb/MK nuclear extract prepared from cells
grown in the presence of EGF. An 100-fold molar excess of unlabeled
oligonucleotides were used as competitors (GGmut.,
M1-M4, and VLY sequences are given
under ``Material and Methods''). GGmut contained nucleotide
substitutions in a sequence similar to the PEA3/Ets-binding site within
the NVL3 RRE. 0 indicates that no competitor was added in this
incubation. C, EMSA using oligonucleotides corresponding to
M2, M3, and B10RRE as probes and
Balb/MK nuclear extract prepared from cells grown in the presence of
EGF.
To
analyze if the sequences required for complex formation correlated with
those required for functionality, we used unlabeled oligonucleotides
corresponding to the different mutated variants shown in
Fig. 3
in competition experiments. The oligonucleotide that
contained a mutated AP1-like site (M2) specifically competed for the
formation of complex 1 and 2 while leaving complex 3 unaffected. The
same pattern of competition was observed using the
oligonucleotide-containing mutations 5` to the AP1-like site (M1). An
opposite result was obtained using an excess of unlabeled
oligonucleotides corresponding to mutations 3` to the AP1-like site.
These oligonucleotides (M3 and M4) specifically competed for the
formation of complex 3. The reciprocal capacity of M2 and M3 to form
complexes with nuclear extract was also evident using these two
oligonucleotides as probes (Fig. 4C). These results,
together with the results from the functional analysis, thus indicated
that the B10 RRE was composed of two binding sites that cooperated in
function. One of the sites was positioned immediately 5` and within the
AP1-like site while the other was located 3` to the AP1-like site.
The B10 RRE Sequence Is a Target for ATF3/Jun-binding
Activity
The binding characteristics of the B10 RRE probe were
further analyzed by competition experiments using nuclear Balb/MK
extract and a 100-fold molar excess of oligonucleotides corresponding
to binding sites for transcription factors known to be involved in
mediating ras-induced transcription. Fig. 5A shows that an oligonucleotide (AP1), which contained the consensus
AP1-site present in the collagenase gene, specifically competed for the
formation of complex 3, suggesting that the AP1-like site indeed was a
target for AP1-binding activity. However, in the reverse experiment,
using a consensus AP-1-binding site as a probe and an excess of B10 RRE
as competitor only a partial inhibition of AP1-binding was observed
(data not shown). This result suggested that the B10 RRE, displayed a
lower affinity and/or a restricted binding specificity for AP1-binding
activity. No inhibition of B10 RRE complex formation was detected using
an oligonucleotide corresponding to the SRE present in the c-fos gene. Interestingly, the formation of complex 3 was inhibited when
competing with an oligonucleotide corresponding to a consensus CRE
present in the choriogonadotropin gene (Fig. 5A), which
suggested the presence of CREB/ATF-proteins in this complex. An
oligonucleotide corresponding to a NVL3 RRE also competed for complex 3
formation due to the presence of an AP1-site. This result indicated
that the PEA3/Ets-binding site in the NVL3 RRE was without effect. In
agreement with this result, no competition was detected using an
oligonucleotide (PEA3) that corresponded to the Ets1- and Ets2-binding
sites present in the polyoma virus enhancer
(10) . Furthermore,
in a reverse experiment using the PEA3/Ets binding site as a probe and
a 100-fold molar excess of a B10 RRE as competitor, no competition was
observed to PEA3/Ets binding activity (data not shown). These results
indicated that the B10 RRE-binding factors were unrelated to PEA3/Ets.
Figure 5:
Characterization and identification of
proteins forming complexes with B10RRE. A, EMSA was performed
as described in legend to Fig. 4. Competition were performed using a
100-fold molar excess of unlabeled oligonucleotides corresponding to
response elements known to be involved in Ras-induced transcription.
The sequences of AP1, PEA3, SRE, CRE, NVL3 RRE, and B10 RRE are given
under ``Material and Methods''. B, EMSA showing
immunological detection of proteins binding to B10 RRE using antibodies
specifically recognizing c-Jun (c-Jun), JunB
(
JunB), JunD (
JunD), c-Fos
(
c-Fos), and an antibody recognizing members within the
CREB/ATF family of transcription factors (
CREB). A
supershift formed with the
Jun B antibody is indicated with an
arrow. C, EMSA showing immunological detection of
proteins binding to B10 RRE using antibodies specifically recognizing,
CREB-1 (
CREB-1), CREB-2 (
CREB-2), ATF-1
(
ATF-1), ATF-2 (
ATF-2), and ATF-3
(
ATF-3).
Next, we wanted to identify the proteins present in the different
DNA-protein complexes. Given the finding that both AP1-sites and a CRE
competed for binding to the B10 RRE, we performed gel shift analyses
using antisera specific for proteins known to bind to these sequences.
Fig. 5B shows that the anti-c-Jun and anti-JunD
antibodies specifically disrupted complex 3 as did a polyclonal
antiserum raised against CREB. Moreover, a supershift was generated
using anti-JunB antibodies (indicated with an arrow in
Fig. 5B), while an anti-c-Fos antibody had no effect on
complex formation. The anti-c-Fos and all anti-Jun antibodies generated
supershifts when using the collagenase AP1-site as a probe (data not
shown). This result was in agreement with the oligonucleotide
competition experiments and indicated that complex 3 contained Jun- and
CREB-related factors, whereas complex 1 and 2 contained proteins
unrelated to these transcription factors. In order to identify the
specific CREB/ATF-proteins that bound to the B10 RRE probe, we used
antibodies specific for different CREB/ATF members. As shown in
Fig. 5C, an antibody that specifically recognize ATF3
disrupted formation of complex 3, whereas anti-ATF1, -ATF2, -CREB1, and
-CREB2 had no effect. This suggested that complex 3 was composed of
several complexes with similar mobility and that these contained homo-
and/or heterodimers of ATF3 and different Jun isotypes.
Initial Purification of the Major Factor with Affinity
for the 3` Part of the RRE
The results indicated that the
sequence, TTAGTTAC, immediately 3` to the AP1-like site was both
essential for functionality and bound protein(s) unrelated to
transcription factors known to be involved in ras-induced
transcription (complex 1 and 2 in Fig. 3). A computer search
revealed no significant similarity between the sequence TTAGTTAC and
previously published transcription factor binding sites. We therefore
established conditions for an initial purification and characterization
of the physiochemical nature of the factor(s) with affinity to this
sequence. Balb/MK nuclear extract was run on 25-40% (w/v) linear
sucrose gradients. The DNA binding activity in collected fractions were
determined by EMSA using a P-labeled B10 RRE as probe
(Fig. 6A). Fig. 6A shows that binding
activity corresponding to complex 2 (indicated with an arrow)
was observed in fractions 9-14 with maximal activity in fraction
13. The binding activity specific to the AP1-like site eluted in
fraction 15-16, whereas the factor responsible for the formation
of the relative faint complex 1 did not withstand the purification
conditions. Due to the identical binding specificity between the
factors in complex 1 and 2, we cannot rule out the possibility that
complex 1 is composed of complex 2 with an additional factor. By using
catalase (11.3 S) and
-globulin (6.6 S) as marker proteins, we
calculated the sedimentation coefficient for the major factor with
affinity for the TTAGTTAC sequence (complex 2) to be 9.7 S.
Fig. 6B shows the partial purification of the same
factor by gel permeation chromatography on a Superose 12 column. EMSA
using the RRE as probe and protein aliquots of the obtained fractions
showed that this factor eluted in fraction 24-28. Calibration of
the column with standard proteins gave a Stokes radius for the
protein(s) present in complex 2 of 43.6 Å. A calculation using
the obtained R
and the sedimentation
coefficient revealed that this factor had an apparent molecular mass of
178,000 Da in solution. The frictional ratio (f/f
)
was calculated to 1.08 consistent with a globular molecule.
Figure 6:
Partial purification and physiochemical
characterization of the factor in complex 2. A, sucrose
gradient centrifugation. Nuclear extract was layered onto linear
25-40% (w/v) sucrose density gradients, centrifuged, and
fractionated as described under ``Materials and Methods.''
Aliquots (20 µl) from each fraction were incubated with
P-labeled B10 RRE and analyzed by EMSA on a native
polyacrylamide gel. The arrows indicate the position of
complex 2 formation. The relative levels of complex 2 formation in each
fraction were determined by densitrometric scanning of EMSA
autoradiograms. Separate gradients were run with the standard proteins:
1, catalase (11.3 S) and 2,
-globulin (6.6 S).
B, Gel filtration chromatography. Nuclear extract (200 µl)
was chromatographed on a Superose 12 gel filtration column as described
under ``Materials and Methods.'' The relative levels of
complex 2 formation in each fraction were determined and visualized as
described in A. The column was calibrated with the following
standard proteins: albumin (32.5 Å); ovalbumin (28.6 Å);
DNase I (24.6 Å); and cytochrome C (17.9
Å).
DISCUSSION
There is substantial evidence that EGF functions as a mitogen
for keratinocytes and that the mutational activation of the Ha-ras gene in combination with TPA treatments cause tumor formation in
mouse epidermis. Although TPA treatments and the activation of the
Ha-ras have been shown to result in the stimulation of
identical intracellular signaling events such as the activation of
Raf-1 kinase and mitogen-activated protein kinases as well as certain
transcription factors, they represent two distinct events (initiation
and promotion, respectively) in the mouse skin model for multistage
carcinogenesis
(31) . We have previously reported the
identification of a TPA response element within the LTR of a VL30
retrotransposon expressed in epidermis. This response element is
unresponsive to EGF in keratinocytes. Here we report the identification
of a 20-base pair sequence (B10 RRE) present within the same LTR, which
is unresponsive to TPA while mediating induced transcription in
response to both EGF and an activated Ha-ras gene in
keratinocytes. It is known that forced expression of transforming
growth factor- in keratinocytes of transgenic mice obviates the
need for an activated ras in the development of skin tumors
(papillomas)
(32) . This finding indicates that a constitutively
active EGF-receptor bypasses the need for Ha-ras mutations in
mouse skin tumorigenesis. The feature of conferring elevated
transcription in response to both EGF and activated Ha-ras but
not to the tumor promoter TPA thus suggest that the B10 RRE is a target
for a signaling pathway activated during the initiation phase of
multistage carcinogenesis.
to G
transition in
hepatic cells, and ATF3 in combination with Jun proteins mediate
promoter-specific transactivation distinctly different from that of Jun
proteins in combination with c-Fos
(34) . A growth factor-induced
Ras-dependent kinase has recently been described that phosphorylates
and activates CREB
(35) . It will therefore be interesting to
determine whether the mechanism of ras-induced ATF3 activation
involves post-translational modifications similar to that described for
CREB
(35) .
B sites or the SRE present in the c-fos promoter. Antisera raised against c-Fos, c-Jun, JunB, JunD, CREB1,
CREB2, ATF1, ATF2, and ATF3 do not recognize this factor. Moreover, a
sequence homology analysis does not reveal any obvious match between
the 3` sequence (TTAGTTAC) in the B10 RRE and previously identified
transcription factor binding sites. The B10 RRE 3` sequence does not
contain an E-box that constitutes a target for bHLH proteins such as
Myc
(40) . In addition, this sequence is unrelated to known
binding sites for the signal transducers and activators of
transcription (STAT) family of transcription factors (41). These
findings prompted us to analyze the factor in closer detail. An initial
purification of the factor by sucrose density gradient centrifugation
and gel permeation chromatography shows that the factor has a Stokes
radius of 43.6 Å, sediments at 9.7 S, and has a calculated
molecular mass and frictional ratio (f/f
) of
178,000 Da and 1.08, respectively. These results suggest that the major
EGF-inducible nuclear factor with binding specificity to the site
juxtaposed to ATF3/Jun in the B10 RRE represents a single molecular
complex with a spherical shape in solution. The binding site for this
factor consists of two TTAG/C motifs arranged as a direct repeat. The
functional analysis indicates that mutations in either of the two
TTAG/C motifs abolish binding. The relatively large size of the factor
and the repeated nature of its binding sequence suggest that the factor
might be an oligomer that binds to one side of the DNA helix.
Southwestern blotting, cross-linking, methylation interference
experiments, and sequence-specific affinity purification are in
progress to define the stoichiometry and binding characteristics of the
protein(s) involved. Results from these studies will be important in
facilitating the purification and/or cloning of the factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.