©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activated Ha-Ras but Not TPA Induces Transcription through Binding Sites for Activating Transcription Factor 3/Jun and a Novel Nuclear Factor (*)

Mats Nilsson (§) , Rune Toftg , Staffan Bohm (¶)

From the (1) Center for Nutrition and Toxicology and Department of Bioscience at Novum, Karolinska Institute, NOVUM, S-141 57, Huddinge, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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),() 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.


MATERIALS AND METHODS

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 Vand 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 = 422SRand 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.

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.

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 Rand 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.

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 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) .

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-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.

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.


FOOTNOTES

*
This work was supported by a grant from the Swedish Cancer Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-8-608-9107; Fax: 46-8-608-1501.

Present address: Harvard Medical School, Dept. of Neurobiology, 220 Longwood Ave., Boston, MA 02115.

The abbreviations used are: EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; LTR, long terminal repeat; CRE, cAMP-responsive element; RRE, ras-responsive element; CREB, CRE binding protein; ATF, activating transcription factor; bp, base pair(s); SRE, serum response element.


ACKNOWLEDGEMENTS

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.


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