Journal of General Virology (1995), 76, 751-758.
751
Printed in Great Britain
cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by r a s oncogene products Mirella Gaboli, Alessandra Angeretti, David Lembo, Marisa Gariglio, Giorgio Gribaudo and Santo Landolfo* Institute of Microbiology, Medical School of Novara, University of Turin, and Immunogenetics and Experimental Oncology Center, CNR, 10126 Torino, Italy
Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfeeted with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras eDNA, together with pAACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of pAACMVCAT were also observed in cell lines carrying stably transfected ras oneogenes. Further support for the role of the CRE sequence in
MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, fl-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21 r~s. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.
Introduction
element for enhancer-dependent expression in uninfected HeLa cells (Boshart et al., 1985; Stinski & Roehr, 1985). Sambucetti et al. (1989) have demonstrated that infection induces interaction of nuclear factors with the repeated elements of the IE enhancer. The most prominent of these are the NF-~:B, AP-1 and CREB/ATF factors. It is therefore conceivable that the MCMV IE gene enhancer is also regulated by the mouse counterpart of these factors and signalling pathways. Products of ras genes, such as p21 ras, play a fundamental role in basic cellular functions (for review see Barbacid, 1987; Downward, 1992; Boguski & McCormick, 1993). In yeast, there is evidence that Ras has a mechanism of activation mediated through protein kinase A (PKA), but in higher eukaryotic cells, examination of some types of mammalian adenylyl cyclase suggests that it is not regulated by Ras proteins. Although a ras-dependent phosphorylation of ATF/CREB proteins has not yet been demonstrated, evidence suggesting ATF/CREB responsiveness to p21 r,s has been presented (Kedar et al., 1990; Galien et al., 1991). ATFs/CREBs proteins are a multigene family of transcriptional transactivators that provide trans-
The murine cytomegalovirus (MCMV), a betaherpesvirus has a large DNA genome of 230 kbp encoding over 100 genes grouped into immediate early (IE), early (E) and late (L) genes (Ebeling et aL, 1983; Keil et al., 1984). The three IE genes (IE1, IE2 and IE3) are expressed in the absence of prior viral protein synthesis, and the products are directly involved in the regulation of gene expression throughout infection (Keil et al., 1987; Messerle et al., 1991, 1992). Transcription of IE genes is regulated by a strong viral enhancer composed of an array of repeated sequence motifs containing several consensus binding sites for cellular transcription factors, including one Sp-1 site, nine NF-xB sites, of which seven partially overlap AP-1 sites, and one CREB/ATF-binding site, TGACGTCA, (DorschH/isler et al., 1985). This motif is repeated five times within the human CMV enhancer and is an essential
* Author for correspondence. Fax
[email protected] 0001-2761 © 1995 SGM
+ 39 11 6636436. e-mail
752
M . Gaboli and others
criptionally productive interactions when bound to the CREs of gene promoters, and whose binding and/or transcriptional activation functions are modulated by p h o s p h o r y l a t i o n s catalysed p r e d o m i n a n t l y by c A M P d e p e n d e n t P K A ( H a b e n e r , 1990; E n g l a n d e r & Wilson, 1992; de G r o o t & Sassone-Corsi, 1993; M a s s o n et al., 1993). We have previously d e m o n s t r a t e d that the expressed v-Ha-ras p r o d u c t stimulates I E e n h a n c e r activity ( L e m b o et al., 1994). However, a n initial a t t e m p t to locate ras responsive elements has indicated that the s t i m u l a t i o n o f the overall e n h a n c e r activity is the result o f the c o o r d i n a t e d i n t e r a c t i o n s o f t r a n s c r i p t i o n factors with each other a n d their cognate b i n d i n g sites. This p a p e r examines the role o f o n e o f these families, n a m e l y the A T F / C R E B factors in the t r a n s a c t i v a t i o n o f M C M V I E gene e n h a n c e r b y ras oncogenes. A t r a n s i e n t transfection assay ~vas used to d e m o n s t r a t e that a C R E sequence, h o m o l o g o u s to the A T F b i n d i n g site a n d c A M P sensitive, does indeed c o n t r i b u t e to this transactivation.
Methods Cell lines. NIH 3T3 (ATCC) 115/14 (a clone derived from NIH 3T3 carrying an amplified human c-Ha-ras, kindly provided by Dr M. Barbacid, National Cancer Institute (NCI), Frederick, MD, USA), and 226-4-1 (a clone derived from NIH 3T3 transfected with a pointmutated human c-Ki-ras kindly provided by Dr Ottolenghi, Milan, Italy) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with I0% heat-inactivated new born calf serum. Oligonucleotide probes. Synthetic double-stranded oligonucleotide, corresponding to a copy of the 19 bp repeat element within the enhancer of CMVs immediate early genes with an internal CREB/ATF-binding site, was synthesized on an Applied Biosystems 308A DNA synthesizer. The oligonucleotide was as follows: 19 bp repeat, 5'-CACCATTGACGTCAATGGGCT-3', with the complementary strand 5'-AGCCCATTGACGTCAATGGTG-Y. When required, the oligonucleotide was labelled at the Y-end with [y-3~P]ATP by T4 polynucleotide kinase, annealed and purified by polyacrylamide gel electrophoresis. Plasmids. pCMVCAT contains an 1.2 kb PstI-NdeI segment from the HindIlI fragment L of MCMV DNA, positioned upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. This segment contains the IE enhancer and the IE1-3 promoter of MCMV (Gribaudo et al., 1993). pAACMVCAT was prepared from pCMVCAT by removing the unique CRE./ATF-binding site, using internal restriction enzyme sites PaeI (-259 nt) and Xhol (-146 nt). The retroviral vector prasZip6 is derived from the pZipNeoSV(x) and contains the v-Ha-ras gene cloned in the BamHI site. Plasmids prasZip6 and pZipNeoSV(x) were kindly provided by G. Dono (Boston, USA) pRSV-ras(Asn-17) and pRSV-ras(Leu-61 ; Ser-186) are eukaryotic expression vectors, in which dominant inhibitory mutants Ha-ras(Asn- 17)gene and Ha-ras(Leu-61 ; Ser-186)-coding region are under the control of the Rous sarcoma virus LTR (Medema et al., 1991). PlasmidpON407, containing the MCMV IE1-3 promoter up to - 146 nt linked to lacZ, and pON407.19R3, in which three copies of the 19bp repeat were inserted upstream of the MCMV sequences
(Cherrington & Mocarski, 1989), were kindly provided by Dr E. Mocarski (Stanford University, CA, USA).
Nuclear extracts. Confluent NIH 3T3, 115/14 and 226-4-1 cells were harvested after being serum deprived for 24 h. Nuclear extracts were prepared according to Dignam et al. (1983). Buffers A and C are those described in the paper, but to minimize proteolysis 0'2 m~-PMSF, 2 Ixg/mlleupeptin, 1 lag/ml pepstatin A, 2 pg/ml aprotinin and 0-1 mMbenzamidine were added. Electrophoretic mobility shift assays (EMSA). Protein-DNA binding reactions were carried out in a final volume of 20 lal of solution containing 15 I~g of nuclear extracts, 1 ~tg poly (dI:dC) and 5 x 104 c.p.m, of labelled probe in the presence of 10 mM-HEPES pH 7"9, 100 mM-NaC1, 1 mM-EDTA and 1 mM-DTT. Reaction mixtures were incubated at 22 °C for 30 min and loaded immediately onto a 6 % nondenaturing polyacrylamide gel. Competitors were added just before the radiolabelled probe. Gels were run in 0.5 x TBE buffer, dried and autoradiographed on X-ray film. Transfections, CA T assays and fl-galactosidase assays. All plasmids were purified twice by caesium chloride centrifugation. For transient gene expression assay, cells were plated the day before transfection in growth medium supplemented with 10% newborn calf serum at a density of 2.5x 105 cells/60mm diameter dish. Four h before transfection, the medium was replaced by fresh DMEM containing 10% newborn calf serum. Cells were transfected by the calcium phosphate procedure and the amount of DNA in each precipitate was normalized to 12 or 13-5pg with carrier DNA. Eighteen h after transfection, cells were washed twice with DMEM, cultured in medium supplemented with 0.5 % newborn calf serum, and harvested 24 h later. Cell extracts were prepared according to Gorman et al. (1982). CAT activity was assayed by incubation of cell extracts, after appropriate dilution, with acetyl coenzymeA (Sigma) and [14C]chloramphenicolfor 30 min. Acetylated products were separated by thin-layer chromatography, visualized by autoradiography, excised from the plates and counted in a liquid scintillation counter. CAT activity is expressed as the percentage of input chlorampbenicol converted into the 1'- and 3'monoacetylated forms, fl-Galactosidase activity was assayed by adding diluted samples to an equal volume of the assay 2 x buffer (200 raMsodium phosphate, pH 7-3, 2 mM-MgClz, 100 mM-2-mercaptoethanol, 1.33 mg/ml o-nitrophenyl-fl-D-galactopyranoside, ONPG). Samples were incubated at 37 °C until a yellow colour was present and the reaction was terminated by adding 1 M-sodium carbonate to a final concentration of 0.625 M. Absorbance at 420 nm was read with a spectrophotometer and compared to a fl-galactosidase standard curve to allow quantification of activity in the samples.
Results Role o f C R E element in transactivation o f the M C M V I E gene enhancer by v-Ha-ras or activated c-Ha-ras products To determine the c o n t r i b u t i o n o f the u n i q u e C R E B / A T F - b i n d i n g site, (from - 231 n t to - 223 nt) to MCMV I E gene e n h a n c e r t r a n s a c t i v a t i o n , the p A A C M V C A T derived from p C M V C A T p l a s m i d by deleting the 113 nt c o n t a i n i n g the C R E B / A T F - b i n d i n g site (from p o s i t i o n - 2 5 9 n t to - 1 4 6 n t ) was cotransfected in N I H 3T3 with the expression vector prasZip6, at the m o l a r ratios o f 1 : 4 ( r e p o r t e r / e x p r e s s i o n vector). As a control, the p A A C M V C A T was co-
MCMV
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I E gene C R E transactivation by p 2 F a`
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T
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-259 ,~l'
[
146
[[-~C E
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]
AT
TATA pON407.19R3
LACZ ] TATA _,4° I
pON407
AC Z
I
Fig. 1. Plasmid constructs used in these experiments. In pCMVCAT MCMV IE gene enhancer and IE1-3 promoter are linked to the chloramphenicol acetyltransferase gene (open box). Transcription start site and TATA box are indicated. Filled boxes represent the unique 19 bp element with the internal CRE sequence between position -231 nt and position -223 nt. In pAACMVCAT the deletion of 113 nt, containing the CRE element, is indicated. In pON407.19R3 MCMV IE1-3 gene promoter (sequence up to - 146 nt) is linked to the lacZ gene and three tandem copies of the 19 bp element, each of which brings an internal CRE sequence (filled box). pON407 contains MCMV IEI-3 gene promoter fused to lacZ gene.
~1 ~
o 8
v
pCMVcat
pCMVcat+ prasZip6
pAACMVcat
pAACMVcat+ prasZip6
100 90 80 70 60 50 40 30 20 10 0
pCMVcat pAACMVcat 115/14 Clone
pCMVcat pAACMVcat 226-4-1 Clone
Fig. 2. Effect of activated p2V a~on pCMVCAT and pAACMVCAT. (a) NIH 3T3 cells were transfected either with pCMVCAT (1.5 lag) or pAACMVCAT (1.5 lag). Where indicated, cells were cotransfected with prasZip6 (6 lag). In controls, the amount of DNA was normalized with parental vector, which lacks the v-Ha-ras gene. Eighteen h after transfection cells were serum-deprived for 24 h, harvested and CAT activities were assayed for each sample (see Methods). The diagram represents acetylation value means and standard deviations. (b) 226-4-1 and 115/14 cells, stably transfected with a point-mutated human c-Ki-ras or an amplified human cHa-ras respectively, were transfected with 1.5 lag of the indicated plasmids. Eighteen h after transfection cells were serum-deprived for 24 h, harvested and CAT activities were assayed for each sample. Result means and standard deviations are represented in the diagram.
t r a n s f e c t e d with the p Z i p N e o S V ( x ) , which lacks the vHa-ras c D N A , at the s a m e m o l a r ratios. A s s h o w n in Fig. 1, c o t r a n s f e c t i o n o f p C M V C A T c o n s t r u c t w i t h p r a s Z i p 6 resulted in a sevenfold increase o f C A T activity, w h e r e a s c o t r a n s f e c t i o n o f p A A C M V C A T resulted in a 4-4-fold increase. T h e b a s a l activities o f the t w o c o n structs were a l m o s t c o m p a r a b l e . Since C A T activity decreases when the C R E is r e m o v e d f r o m the I E enhancer, it m a y be s u p p o s e d to p l a y a p a r t in the response to v - H a - r a s . T o d e t e r m i n e w h e t h e r M C M V I E e n h a n c e r transa c t i v a t i o n t h r o u g h o u t the C R E B / A T F e l e m e n t c o u l d also be o b s e r v e d in cells stably t r a n s f e c t e d with a c t i v a t e d
ras p r o t o - o n c o g e n e s , 115/14 a n d 226-4-1 cell lines, w h i c h stably express the amplified c-Ha-ras o r the p o i n t m u t a t e d c-Ki-ras, were t r a n s f e c t e d with p C M V C A T o r p A A C M V C A T . A s s h o w n in Fig. 2, C A T activities f r o m cells with p A A C M V C A T were lower t h a n those f r o m cells transfected w i t h p C M V C A T . A l t h o u g h these decreases are n o t very high, they a r e significant (as s h o w n b y s t a n d a r d deviations) a n d r e p r o d u c i b l e . A l t o g e t h e r these results suggest t h a t the A T F f a m i l y m a y p a r t l y c o n t r i b u t e to R a s t r a n s a c t i v a t i o n o f M C M V I E e n h a n c e r in ras-transformed cells. F i n a l l y , it s h o u l d be n o t e d t h a t the efficiency o f transfection, e v a l u a t e d by m o l e c u l a r h y b r i d i z a t i o n as
M. Gaboli and others
754
~'~" .~.= "~ ~ ~'= ~ 8
20 18 16 14 12 10 4 2 0
galactosidase activity o f p O N 4 0 7 . 1 9 R 3 a b o u t fivefold, whereas it only slightly modifies the reporter activity o f pON407. It should be noted that p O N 4 0 7 contains a C R E B CS site ( A C G T C A , f r o m position - 1 3 1 nt to position - 136 nt), which m a y be responsible for the l'9fold increase o f p-galactosidase activity observed when an activated p 2 F as is expressed. pON407
pON407+ pON407.19R3 pON407.19R3 prasZip6 +prasZip6
Fig. 3. Effect ofv-Ha-ras expression on MCMV IE gene enhancer CRE sequence. NIH 3T3 cells were transfected with either pON407 (2 lag) or pON407.19R3 (2 lag), in presence or absence of prasZip6 (8 lag). In controls, the amount of DNA was normalized with the parental vector, which does not contain the v-Ha-ras gene. Cells were serum-deprived for 24 h before assaying ~-galactosidase activities (see Methods). Fold activation reflects the level of p-galactosidase activity measured from transfection of the indicated plasmids divided by the level measured from mock-transfected cells. Means and standard deviations are represented in the diagram.
previously described ( L e m b o et al., 1994), is c o m p a r a b l e for the three cell lines (data not shown).
ras transactivation o f three tandem copies of the M C M V CREB/ATF-binding site T o confirm further that activated p 2 F a` could transactivate the M C M V IE enhancer t h r o u g h the C R E B / A T F - b i n d i n g site, plasmid pON407.19R3, which contains three t a n d e m copies o f the 19 bp C R E , was cotransfected in N I H 3T3 cells with either the prasZip6 or its c o u n t e r p a r t without the v-Ha-ras, pZipNeoSV(x). As shown in Fig. 3, activated p 2 F as increases the fl-
o 20 -= 18 "~ 16 14 12 10 t~ 8 •~ 6 °4 ~ 2 ~ 0 ~k
pON407.19R3
pON407.19R3+ pRSVras(Asn-17)
pON407.19R3+ prasZip6
pON407A9R3+ prasZip6+ pRSVras(Asn-17)
Effect of dominant inhibitory Ha-ras mutations on C R E transactivation by v-Ha ras product To evaluate the effects o f p21 r"s inhibitors on p O N 4 0 7 . 1 9 R 3 basal and v-Ha-ras-induced expression, in N I H 3T3 cells, two d o m i n a n t inhibitory ras m u t a n t proteins, which selectively inhibit the activity o f either cellular or oncogenic ras, have been studied (Stacey et al., 1991 ; M e d e m a et al., 1991). The first, ras(Asn-17), has a single amino acid substitution at position 17, where serine is changed to asparagine, causing a preferential affinity for G D P , resulting in an inactive f o r m o f ras which interferes with the endogenous c-ras signalling system (Feig & Cooper, 1988; Bortner et al., 1993; Quilliam et al., 1994). The second d o m i n a n t inhibitory ras mutant, ras(Leu-61 ;Ser-186) has a serine at position 186 replacing a cysteine, and inhibits the function o f p21TM by competition for its cellular target (Gibbs et aL, 1989; Michaeli et al., 1989). As shown in Fig. 4(a), the fl-galactosidase activity was u n c h a n g e d when p O N 4 0 7 . 1 9 R 3 was cotransfected with prasZip6 and pRSV-ras(Asn-17), c o m p a r e d with the activity observed with the activated ras only. A lack o f function o f o u r pRSV(Asn-17) was ruled out by testing the activity o f this vector on quiescent N I H 3T3 cells
20 18 16 14 12 10 8 6 4 2 0
pON407.19R3
pON407.19R3+ pRSVras (Leu-61; Ser-186)
pON407A9R3+ prasZip6
pON407,19R3+ prasZip6+ pRSVras (Leu-61; Set-186)
Fig. 4. Effect of dominant inhibitory Ha-ras mutants on v-Ha-ras transactivation of MCMV IE gene enhancer CRE sequence. (a) NIH 3T3 cells were transfected with pON407.19R3 (1.5 lag) and, where indicated were cotransfected with prasZip6 (6 ~tg), with pRSVras(Asn-17) (6 lag) or with both plasmids. Cellular extracts prepared from 24 h serum-deprived ceils were assayed as described in Methods. fl-Galactosidase activity is represented as fold activation with respect to the fl-galactosidase activity of mock-transfected cells. The experiment was repeated three times and the average results and standard deviations are shown. (b) NIH 3T3 cells were transfected with pON407.19R3 (1.5 lag) and, where indicated were cotransfected with prasZip6 (6 lag), with pRSV-ras(Leu-61 ;Ser-186) (6 lag) or with both plasmids. Experimental conditions were those described in (a). fl-Galactosidase activity is represented as fold activation with respect to the fl-galactosidase activity of mock-transfected cells. Means and standard deviations are represented.
M C M V I E gene C R E transactivation by p21 ras
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20
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Table 1. Regulation of the C R E sequence o f M C M V I E gene enhancer by v-ras and forskolin treatment is agonistic*
~~ lO
~
Ratio
8
4 2
0
pON407
pON407+ pON407.19R3 pON407.19R3 forskolin +forskolin
Fig. 5. Effect of forskolin treatment on pON407 and pON407.19R3. NIH 3T3 cells were transfected as indicated, serum-deprived and treated for 24 h with 8 laN-forskolinin DMSO or with 0'2 % DMSO only. fl-Galactosidaselevelswere measured and expressedas a ratio to that in mock-transfected cells. Means and standard deviations are shown in the diagram.
transfected with p C M V C A T and treated with plateletderived growth factor (PDGF), which is known to activate cellular p21TM by increasing the ratios of p 2 W S G T P / p 2 W s G D P (Satoh et al., 1990a, b; Gibbs et al., 1990). A strong inhibition of CAT activity following P D G F stimulation upon cotransfection of pRSV(Asn17) was indeed observed (data not shown). By contrast, when an expression vector for Ha-ras(Leu-61 ;Ser-186) was cotransfected with prasZip6, we observed inhibition of pON407.19R3 transactivation by oncogenic ras (Fig. 4b). This inhibition is dose-dependent since increasing concentrations o f pRSVras(Leu-61 ; Ser- 186) were able to completely antagonize the v-Ha-ras transactivation of pON407.19R3. Moreover, the pRSV vector itself, containing the RSV promoter alone, used as control in transfection experiments did not repress the v-Ha-ras transactivation of pON407.19R3 at the molar ratios employed (data not shown). Taken as a whole, these data suggest that C R E B / A T F o binding site activation is restricted to an activated form of ras, and that the normal ras product plays a role in the regulation of basal expression of this enhancer sequence. Agonistie effect of c A M P and v-Ha-ras product on the M C M V C R E element Stamminger et al. (1990) have demonstrated that the H C M V IE gene enhancer basal activity can be considerably augmented by elevated levels of intraceUular cAMP in a cell type-specific manner and that the 19 bp repeat is responsible for this effect. To confirm that the CRE sequence of MCMV IE gene enhancer is responsive to cAMP in N I H 3T3 cells we next tested the effect of forskolin on pON407 and pON407.19R3 constructs. Eighteen h after transfection, cells were treated with 8 ~M-forskolin and fl-galactosidase activities were assayed 24 h later. As shown in Fig. 5, pON407.19R3 flgalactosidase activity was enhanced, whereas no stimu-
755
Treatment Control Forskolin Ratio (+ forskolin/- forskolin)
(- )
(+ )
prasZip6
prasZip6
1-00 4-04 4-04
4.82 6.84 1.41
( + v-Ha-ras/ - v-Ha-ras)
4-82 1.69
* NIH 3T3 cells were transfected with 2 lag of pON407.19R3, and cotransfected with prasZip6 (8 lag) ( + ) or with the parental vector ( - ) . Thus total DNA was kept constant at 10 lag in these studies. Forskolin treatment (8 laM)is indicated, whereas no indication stands for DMSO treatment. Results represent the average of three experiments, these deviated from the mean by less then 10%. To obtain fold induction of MCMV CRE sequence resulting from cotransfection with prasZip6, the pON407.19R3 activity in presence of v - H a - r a s was divided by that obtained in the absence of v - H a - r a s for each condition.
lation was observed with pON407 construct. These results establish that the same motif is responsive to the activated p21 ras, as well as to an increase of cAMP levels. To examine the role that P K A plays in ras activation of CRE, the ras effect on pON407.19R3 activity was determined under two conditions of cellular P K A activity. The first condition served as a control and the cells were left untreated; in the second condition, P K A activity was activated by treating the cells with 8 pMforskolin 18 h after transfection and 24 h prior to harvesting. In the untreated controls, ras stimulates basal pON407.19R3 activity by about fivefold. In the forskolin-treated group, cells transfected with prasZip6 expression vector and treated with 8 laM-forskolin showed a pON407.19R3 fl-galactosidase activity sevenfold higher than control cells (see Table 1). As forskolin treatment alone results in a reproducible fourfold increase over basal pON407.19R3 activity, we conclude that P K A and Ras signalling pathways are agonistic in the activation of the M C M V C R E / A T F element in N I H 3T3 cells. These results are further confirmed by the observation that stably transfected N I H 3T3 cells, which express the amplified c-Ha-ras or point mutated c-Ki-ras, present both a basal and forskolin-inducible pON407.19R3 activity higher than those observed in normal N I H 3T3 fibroblasts (data not shown).
ras oncogenes modulate ATFs in fibroblast nuclear extracts To investigate the factors which bind to the CRE sequence of the MCMV, N I H 3T3, 115/14 and 226-4-1 cell lines were initially grown in 10% calf serum. Thereafter the cultures were serum deprived for 24 h and
756
M . Gaboli and others
Competitor +
1
NIH
3T3
-
+
115/14
+
226-4-1
Fig. 6. Mobility-shift analysis of nuclear extracts binding to M C M V IE gene enhancer CRE. Nuclear proteins extracted from NIH 3T3 cells, or from ras-transformed 115/14 and 226-4-1 clones were assayed for binding to a synthetic oligonucleotide corresponding to M C M V IE gene enhancer sequence from position - 2 3 6 nt to position - 2 1 6 nt. A 200-fold excess of homologous competitor was added where indicated. Lane 1 contains free probe only. The experiment was repeated at least four times with different nuclear extract preparations and one representative is reported.
nuclear extracts prepared. As shown in Fig. 6, when a radiolabelled oligonucleotide corresponding to the CRE was mixed with the nuclear extracts and analysed in EMSA, a strongly retarded complex was observed with 115/14 and 226-4-1 cell lines. By contrast, only a faint band was generated with extracts from NIH 3T3 cells. Moreover, nuclear extracts from NIH 3T3 cells transfected with prasZip6 contain a CRE-binding activity identical to that observed in nuclear extracts from rastransformed cells (data not shown). These results suggest that high activity of MCMV IE enhancer in 115/14 and 226-4-1 cell lines is associated to an higher availability of ATF/CREB factors than in NIH 3T3 cells.
Discussion The expression of murine cytomegalovirus IE genes is under the control of a strong enhancer containing repeated sequences that act in an additive manner (Gribaudo et al., 1993; Lembo et al., 1994). In the HCMV IE gene enhancer, the 19 bp repeat, containing a sequence similar to the CRE, is essential for enhancerdependent expression of the major immediate early gene in HeLa cells (Boshart et al., 1985; Stinski & Roehr,
1985; Hunninghake et aL, 1989; Stamminger et aL, 1990). The data presented in this paper demonstrate that also in the MCMV enhancer, the unique CRE contributes at least in part to the enhancer activation by activated ras. In this regard, we show by transient transfection assays that deletion of 113 nt, including the CRE, from MCMV IE gene enhancer reduces transactivation by ras oncogene products, but does not impair basal activity. In addition, the product of the v - H a - r a s gene activates three tandem CRE/ATF-binding sites linked to a reporter gene controlled by MCMV IE gene promoter (sequence up to - 1 4 6 nt from transcription start). Finally, two dominant negative mutants of ras, Ha-ras Asn-17, which interferes with the endogenous c-ras signalling system, but not with activated ras proteins, and Ha-ras(Leu61;Ser-186), which has been proposed to inhibit the function of p21 ra~ by competition for its cellular target, have opposite effects on MCMV IE gene CRE sequence activation by v-Ha-ras. In fact, Ha-ras(Asn-17) mutant cannot prevent pON407.19R3 transactivation by v-Haras, whereas Ha-ras(Leu-61 ;Ser-186) inhibits the effect of this activated Ras. The sum of these results demonstrates that oncogenic, rather than normal p21r% is involved in MCMV IE gene enhancer CREB/ATFbinding site activation by ras. In line with our results, Kedar et al. (1990) found that in NIH 3T3 cells the flpolymerase gene promoter stimulation by ras products was dependent on the presence of a CRE, i.e. GTGACGTCACC, the cognate binding site of CREB/ATF family members. Galien et al. (1991) showed that ras oncogene activates the intracisternal A particle long terminal repeat (IAP LTR) promoter through a CRE. Moreover, by using forskolin, an activator of adenylyl cyclase leading to cellular cAMP level elevation, we established that in NIH 3T3 fibroblasts transfected with pON407.19R3 the same motif is responsive to the v - H a - r a s gene product and to cAMP. In addition, ras oncogenic proteins and PKA pathways appear to be agonistic in regulating MCMV IE gene C R E / A T F element enhancer activity, since forskolin treatment significantly increased pON407.19R3 transactivation by v-Ha-Ras protein and pON407.19R3 expression in cells stably transfected with amplified c - H a - r a s or point mutated c-Ki-ras. A relationship between ras and PKA signalling pathways is still controversial, ras oncogene proteins, but not normal counterparts, induce terminal differentiation of PC12 cells into neuron-like cells by a mechanism independent of the cAMP/PKA pathway. Moreover, microinjection of anti-p21TM antibodies did not inhibit neurite formation by cAMP (Barbacid, 1987). By contrast, exposure of rat thyroid ceils to oncogenic p21ras results in dedifferentiation due to down-regu-
M C M V I E gene C R E transactivation by p 2 F ~
lation of nuclear cAMP-dependent PKA (Avvedimento et al., 1991; Gallo et al., 1992). The ras and PKA
pathways are also mutually antagonistic in regulating rat prolactin promoter activity (Conrad & GutierrezHartmann, 1992). However, none of these authors has identified either the Ras response element (RRE) or the CRE, and this may explain their observation. According to Avvedimento et al. (1991) and Conrad & GutierrezHartman (1992), in fact, their promoter sequences do not contain any previously defined RREs, nor classical CRE or AP-1 sites. Our results, by contrast, focus on the identity between RRE and CRE sequences, suggesting that CREB/ATF proteins are the nuclear end-point of both oncogenic p21~* and cAMP pathways, at least with respect to MCMV IE gene enhancer CRE. In support to this conclusion, we demonstrated by EMSA that nuclear extracts from ras-transformed NIH 3T3 cells contain a much higher CRE-binding activity than normal NIH 3T3 fibroblasts, suggesting that the expression of oncogenic p21 ra, increases nuclear activated CREB/ATF proteins. This higher binding activity with extracts from ras-transformed NIH 3T3 cells seems to be generated by a post-translational modification as suggested by Northern blot analysis and resistance to cycloheximide treatment. In the former case the amount of CREB/ATF mRNA from ras-transformed cells was comparable to that found in NIH 3T3 cells. In the latter case treatment of the ras-transformed cells with cycloheximide for at least 18 h did not impair the amount of proteins binding to the CRE-binding site (data not shown). Taken as a whole, these findings underscore the likelihood that MCMV IE gene enhancer expression is modulated by oncogenic Ras proteins through the CREB/ATB-binding sites. However, because of the artificiality of the system any conclusion about the contribution of ATF proteins to CMV replication in vivo is still premature. We thank GianPaolo Dotto, Boston, MA, USA for the prasZip6 and pZipNeoSV plasmids, Dr Johannes Bos, Utrecht, The Netherlands for the pRSVrasAsn-17 and pRSVras(Leu-61;Ser-186) plasmids, Dr Edward S. Mocarski, Stanford, CA, for pON407 and pON407.19R3 plasmids. This work was supported by grants from the Italian National Research Council (P.F. 'A.C.R.O.' and 'F.A.T.M.A.') and from the Associazione Italiana per la Ricerca sul Cancro. D.L. was supported by a fellowship from the Italian A.I.D.S. Project.
References AVVEDIMENTO,V. E., MUSTI, A. M., UEFFING,M., OBICI, S., GALLO, A., SANCHEZ, M., DEBRASI, D. & GOTTESMAN, M.E. (1991). Reversible inhibition of a thyroid-specific trans-acting factor by Ras. Genes and Development 5, 22 28. BARBACID, M. (1987). ras genes. Annual Review of Biochemistry 56, 779 827. BOGUSKI,M. S. & McCORMICK,F. (1993). Proteins regulating ras and its relatives. Nature 366, 643-654. BORTNER, D.M., LANGER, S.J. & OSTROWSKI, M. C. (1993). Nonnuclear oncogenes and the regulation of gene expression in transformed cells. Critical Reviews in Oncogenesis 4, 137-160.
757
BOSHART, M.F., WEBER, F., JAHN, G., DORSCH-HASLER, K., FLECKENSTEIN,B. & SCHAFFNER,W. (1985). A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Celt 41, 521 530. CHERRINGTON, J.M. & MOCARSKI, E.S. (1989). Human cytomegalovirus iel transactivates the ~ promoter-enhancer via an 18base-pair repeat element. Journal of Virology 63, 1435-1440. CONRAD, K.E. & GUTIERREZ-HARTMANN,A. (1992). The ras and protein kinase A pathways are mutually antagonistic in regulating rat prolactin promoter activity. Oncogene 7, 1279-1286. DE GROOT, R.P. & SASSO~-CORSl,P. (1993). Hormonal control of gene expression; multiplicity and versatility of cyclic adenosine 3',5'monophosphate-responsive nuclear regulators. Molecular Endocrinology 7, 145-153. DIGNAM, J. D., LEBOVITZ, R. M. & ROEDER, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Research 11, 1475-1489. DORSCH-H~,SLER,K., KEIL, G. M., WEBER, F., JASIN, M., SCHAFFNER, W. & KOSZlNOWSKI,U. H. (1985). A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus. Proceedings of the National Academy of Sciences, USA 82, 8325-8329. DOWNWARD, J. (1992). Regulatory mechanisms for ras proteins. BioEssays 14, 177-184. EBELING, A., KEIL, G. i . , KNUST, E. & KOSZINOWSKI,U. H. (1983). Molecular cloning and physical mapping of routine cytomegalovirus DNA. Journal of Virology 47, 421-433. ENGLANDER,E. W. & WILSON,S. H. (1992). DNA damage response of cloned DNA fl-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A. Nucleic Acids Research 20, 5527-5531. EEIG, L.A. & COOPER, G.M. (1988). Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Molecular and Cellular Biology 8, 3235-3243. GALIEN, R., MERCIER, G., GARCETTE, M. & EMANOIL-RAVIER,R. (1991). RAS oncogene activates the intracisternal A particle long terminal repeat promoter through a c-AMP response element. Oncogene 6, 849-855. GALLO,A., BENUSIGLIO,E., BONAPACE,I. M., FELICIELLO,A., CASSANO, S., GARBI, C., MUSTL A. M., GOTTESMAN,M. E. & AVVEDIMENTO, E. V. (1992). v-ras and protein kinase C dedifferentiate thyroid cells by down-regulating nuclear cAMP-dependent protein kinase A. Genes and Development 6, 1621-1623. GIBBS, J.B., SCHABER, T. L., SCHOFIELD,T.L. & SCOLNICK,E.M. (1989). Xenopus oocyte germinal vesicle break down induced by [Va113] ras is inhibited by cytosoMocalized ras mutant. Proceedings of the National Academy of Sciences, USA 86, 6630-6634. GIBBS, J. B., MARSHALL,M. S., SCOLNICK,E. M., DIXON, R. A. F. & VOGEL, U. S. (1990). Modulation of guanine nncleotides bound to ras in NIH3T3 cells by oncogenes growth factors, and the GTPase activating. Journal of Biological Chemistry 265, 20437-20442. GORMAN, C.M., MOFFAT, L.F. 86 HOWARD, B.H. (1982). Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells. Molecular and Cellular Biology 2, 1044-1051. GRIBAUDO,G., RAVAGLIA,S., CALIENDO,A., CAVALLO,R., GARIGLIO, M., MARTINOTTI,m. G. & LANDOLFO,S. (1993). Interferons inhibit onset of murine cytomegalovirns immediate-early gene transcription. Virology 197, 303-311. HABENER,J. F. (1990). Cyclic AMP response element binding proteins: a cornucopia of transcription factors. Molecular Endocrinology 4, 1087-1094. HUNNINGHAKE, G.W., MONICK, i . M . , LIU, B. & STINSKI, i . F . (1989). The promoter-regulatory region of the major immediateearly gene of human cytomegalovirus responds to T-lymphocyte stimulation and contains functional cyclic AMP-response elements. Journal of Virology 63, 3026-3033. KEDAR, P. S., LowY, D. R., WIDEN, S. G. & WILSON, S. H. (1990). Transfected human fl-polymerase promoter contains a ras-responsive element. Molecular and Cellular Biology 10, 3852-3856. KEIL, G.M., EBELING-KEIL, A. & KOSZlNOWSKI, U.H. (1984). Temporal regulation of murine cytomegalovirns transcription and
758
M . Gaboli and others
mapping of viral RNA synthesized at immediate early times after infection. Journal of Virology 50, 784-795. KEIL, G.M., EBELrNG-KEIL, A. & KOSZINOWSKI, U.H. (1987). Sequence and structural organization of murine cytomegalovirus immediate-early gene 1. Journal of Virology 61, 1901-1908. LEMBO,D., ANGERETTI,A., FORESTA,P., GRIBAUDO,G., GARIGLIO,M. & LANDOLFO, S. (1994). ras oncogenes transactivate the mouse cytomegalovirus immediate early gene enhancer. Journal of General Virology 75, 1685-1692. MASSON,N., JOHN, J. & LEE, K. A. W. (1993). In vitro phosphorylation studies of a conserved region of the transcription factor ATF1. Nucleic Acids Research 21, 4166-4173. MEDEMA, R.H., WUBBOLTS, R. & BOS, J. (1991). Two dominant inhibitory mutants of p21 ras interfere with insulin-induced gene expression. Molecular and Cellular Biology 11, 5963 5967. MESSERLE, M., KEIL, G. M. & KOSZINOWSKI,U. H. (1991). Structure and expression of murine cytomegalovirus immediate-early gene 2. Journal of Virology 65, 1638-1643. MESSERLE, M., BUEHLER, B., KEIL, G . M . & KOSZINOWSKI, U.H. (1992). Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3. Journal of Virology 66, 27-36. MICHAELI,T., FIELD, J., BALLESTER,R., O'NEIL, K. O. & WIGLER, M. (1989). Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae. EMBO Journal 8, 303%3044. QUILLIAM, L.A., KATO, K., RABUN, K. M., HISAKA, M. M., HUFF, S.Y., CAMPBELL-BURK, S. & DER, C- (1994). Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for
ras interaction with guanine nucleotide exchange factors. Molecular and Cellular Biology 14, 1113-1121. SAMBUCETTI,L.C., CHERRINGTON,J.M., WILKINSON,G . W . G . & MOCARSKI, E. S. (1989). NF-KB activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO Journal 8, 4251-4258. SATOH, T., ENDO, M., NAKAFUKU,M., NAKAMURA,S. & KAZIRO, Y. (1990a). Platelet-derived growth factor stimulates formation of active p21 r~s GTP complex in Swiss mouse 3T3 cells. Proceedings of the National Academy of Sciences, USA 87, 5993 5997. SATOH,T., ENDO, M., NAKAFUKU,M., AKIYAMA,T., YAMAMOTO,T. & KAZlRO, Y. (1990b). Accumulation of p21 ra~ GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proceedings of the National Academy of Sciences, USA 87, 7926-7929. STACEY,D. W., FEIG, L. A. & GIBBS,J. B. (1991). Dominant inhibitory ras mutants selectively inhibit the activity of either cellular or oncogenic ras. Molecular and Cellular Biology 11, 4053-4064. STAMMINGER,T., F1CKENSCHER,H. t~z FLECKENSTEIN,B. (1990). A cell type-specific induction of the major immediate early enhancer of human cytomegalovirus by cyclic AMP. Journal of General Virology 71, 105-113. STINSKI, M. F. • ROEHR, T. J. (1985). Activation of the immediateearly gene of human cytomegalovirus by cis-elements in the promoter-regulatory sequence and by trans-acting components. Journal of Virology 55, 431-441.
(Received 6 July 1994; Accepted 21 November 1994)
