JOURNAL OF VIROLOGY, Dec. 1991, p. 6397-6406

Vol. 65, No. 12

0022-538X/91/126397-10$02.00/0 Copyright C) 1991, American Society for Microbiology

The Adenovirus ElA Transforming Protein Activates the Proliferating Cell Nuclear Antigen Promoter via an Activating Transcription Factor Site GILBERT F. MORRIS* AND MICHAEL B. MATHEWS Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724

Received 24 June 1991/Accepted 22 August 1991

The transforming region of adenovirus (El) stimulates expression of a reporter construct linked to the gene in a cotransfection assay (G. F. Morris and M. B. Mathews, J. Biol. Chem. 264:13856-13864, 1989). The major products of the El region were assessed individually for their contribution to transactivation of the PCNA promoter. The ElA 13S and 12S products and the E1B 19-kDa product elevated expression from the PCNA promoter, whereas the E1B 55-kDa product did not. Induction of the PCNA promoter by ElA differed from transcriptional activation of the adenovirus E3 promoter in that the PCNA promoter is activated by the ElA 12S product whereas the E3 promoter is repressed; furthermore, the PCNA promoter is activated upon ElA overexpression, whereas the E3 promoter responds less well to high amounts of ElA. A site for the activating transcription factor ATF located approximately 50 nucleotides upstream from the transcription initiation site in the PCNA promoter mediates a positive response to the ElA 12S and 13S products. promoter for the human proliferating cell nuclear antigen (PCNA)

The proliferating cell nuclear antigen (PCNA) functions in DNA replication (59) as a processivity factor for DNA polymerase 8 (10, 60, 69; see reference 45 for a review). This function appears to be essential for growth of mammalian (34, 43) and yeast (6) cells. As suggested by its dual designation as PCNA (50) and cyclin (9, 46), mRNA levels for the protein increase in response to stimulation of cell growth (2, 35, 47) and fluctuate during the cell cycle (43, 52). From this pattern of synthesis and its critical role in DNA replication, it seems likely that activation of PCNA gene expression is a necessary component of the transition from the quiescent to the proliferating state. Adenovirus infection can cause a quiescent cell to enter the cell cycle by a mechanism that requires expression of the viral ElA gene (see references 8, 51, and 65 for reviews). Alternative splicing generates five different mRNAs from the primary ElA transcript, with the two larger mRNAs, 13S and 12S (8, 18, 51), as the major products. Translation of the 13S mRNA produces a 289-residue protein (289R) containing an internal stretch of 46 amino acids that is conserved in ElA from different adenovirus serotypes and absent in the 243R product of the ElA 12S mRNA (8, 18, 51, 56). The transcriptional activation functions of ElA have generally been attributed to the properties of this 46-amino-acid region (8, 18, 56), designated conserved region 3 (CR3). Other functions ascribed to ElA, such as transcriptional repression (8, 57, 61, 65), induction of DNA synthesis (8, 32, 51, 62, 65), and cooperation with other oncogenes to transform cells (51, 62, 65, 81), map to sequences common to both the 13S and 12S products. Two additional conserved regions of ElA (CR1 and CR2) within these shared sequences are involved in binding a number of cellular proteins that appear to mediate the aforementioned pleiotropic effects of ElA (26, 79). Although the 12S ElA transcript is usually associated with transcriptional repression, mutant viruses that express the *

EIA 12S product without the 13S product can activate expression of the PCNA gene (36, 82) as well as other cellular genes (15, 17, 39, 67). This induction of cellular genes by the ElA 12S product is clearly distinct from CR3-mediated transactivation and could be related to the transformation properties of the protein. However, in the context of a viral infection, it is difficult to determine whether the effect on the cellular PCNA gene is directly attributable to the 12S product or is mediated through effects of 12S on other viral genes. A mechanism for activation of transcription via the cellular E2F transcription factor by the ElA 12S product, acting alone or in concert with the viral E4 product, has recently been established (4, 25, 33). We show here that the PCNA promoter, in contrast to the adenovirus E3 and simian virus (SV40) early promoters, is activated by the 12S product expressed in the absence of other viral genes. A site for the activating transcription factor (ATF) (41) located approximately 50 nucleotides upstream from the cap site in the PCNA promoter mediates a positive response to both the ElA 12S and 13S products. MATERIALS AND METHODS Plasmids. pSV2CAT was obtained from C. Gorman (23), and pE3CAT was obtained from N. Jones (74). pEl contains adenovirus type 5 sequences from nucleotides 1 to 2045 and adenovirus type 2 sequences from nucleotides 2045 to 4110 (14). pElAwt (81) and pBACAT (14) were described previ-

ously. PCNA-CAT constructs. PCNA-CAT plasmids containing human PCNA promoter sequences of various lengths fused at +60 to the bacterial chloramphenicol acetyltransferase (CAT) gene in pBACAT were described previously (53). The exception, the -560 to -2 construct, contains CAT coding sequences fused at a position two nucleotides upstream from the transcription initiation site in the PCNA gene (53). Deletions of seven nucleotides in the ATF homologous sequence in the -1265 and -87 versions of the PCNA promoter were prepared by site-directed mutagenesis to

Corresponding author. 6397

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generate the two ATFA-CAT plasmids. Plasmids -1265 dl-66/-47CAT and -1265 dl-112/-47CAT have PCNA promoter sequences from -66 to -47 or -112 to -47 in the -1265-+60CAT plasmid replaced by a 19-nucleotide sequence derived from pBACAT (boxed in Fig. 6A). A BamHI restriction site was introduced by changing four nucleotides in the ATF site by site-directed mutagenesis to create plasmid -87 ATF-Bam. Site-directed mutagenesis was also used to change the sequence ATTAAA at the transcription initiation site to AGATCT (-87 - +60 INTR-Bgl). The first A (underlined) in the wild-type sequence is the initiating nucleotide. El expression constructs. Plasmids pCMVElB, pCMV19K, and pCMV55K were provided by E. White (76). ElA expression constructs were prepared by substituting inserts from the constructs of Zerler et al. (81) in place of the E1B 19-kDa protein (19K) sequences in pCMV19K. Briefly, pCMVElA and pCMV12S were generated by subcloning the ElA sequences from pMTEIA and pMTEB12S with HindlIl linkers into pCMV19K from which the 19K insert had been removed by digestion with EcoRI and Hindlll. pCMV13S was prepared by subcloning the XmaI-to-XbaI fragment of pMT13S into the pCMVElA construct. Cell culture. Monolayer cultures of HeLa cells (ATCC CCL 2) were grown under 5% CO2 in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and 100 ,ug of penicillin and streptomycin per ml and transfected at 50%

chimeric genes to study the mechanism of induction of PCNA synthesis by adenovirus ElA products. One chimera consists of the human PCNA promoter (nucleotides -1265 to +60 relative to the transcription initiation site) fused directly upstream of a reporter gene encoding bacterial CAT. Previous work showed that in transient assays, plasmid pEl, encoding both of the adenovirus early region 1 genes, ElA and E1B, stimulates the synthesis of CAT directed by the PCNA promoter in HeLa cells (53). Cotransfection of a plasmid that expressed the ElA gene alone (pElAwt) did not induce PCNA-CAT expression to a significant extent (Fig. 1A, left). We thought that this result might be due to a difference in the level of ElA expression from the two constructs as a result of positive interactions between the El genes. Thus, ElA activates E1B expression (14, 38, 56, 58), and E1B can increase the levels of ElA gene expression via both cis-acting (37) and trans-acting (28, 80) effects. In turn, the level of the ElA protein can affect its transactivation efficiency (11) and its ability to transform cells (1, 64). Because the ElA promoter is autoregulated by the ElA protein (14, 27, 38, 56), we placed the adenovirus type 5 ElA protein coding sequences under the control of the human cytomegalovirus (CMV) immediate-early promoter (CMVE1A; see Fig. 2). The CMV promoter with its enhancer can provide high levels of EIA expression, as shown by the RNA analysis of Fig. 1B, and it is relatively insensitive to the effects of ElA in HeLa cells (22). pCMVEIA

confluence.

consistently stimulated PCNA promoter-directed CAT expression (Fig. 1), although the degree of transactivation varied from experiment to experiment. We conclude that the ElA products are sufficient to transactivate the PCNA promoter. However, pCMVElA was not always as effective as pEl (Fig. 1), raising the possibility that E1B also exerts an effect (see Fig. 3). To compare these findings with previously described functions of ElA, we tested the three ElA expression vectors pEl, pElAwt, and pCMVElA for their ability to repress the SV40 early promoter (SV2CAT) and to activate the adenovirus E3 promoter (E3CAT) in parallel assays. As expected, ElA repressed expression of SV2CAT and increased expression of E3CAT. The degree of change varied among experiments, but we consistently observed pCMVElA > pElAwt > pEl for the repression function (Fig. 1A, middle). Thus, the degree of repression by a particular ElA expression vector appears to correlate with the amount of EIA expressed. On the other hand, for the activation function, we found pEl > pElAwt > pCMVElA (Fig. 1A, right), implying that the amount of EIA synthesized by an ElA expression construct does not correlate

Transfections and CAT assays. Transfection experiments were performed by the calcium phosphate precipitation technique as described previously (29, 53). The reporter CAT construct was transfected at 5 ,ug/6-cm dish at 50% confluence. The amount of transactivator plasmid was varied as indicated, and salmon sperm DNA was added to bring the total to 20 jig of DNA per 6-cm plate. Cell extracts were prepared and assayed for CAT enzyme activity at 48 h posttransfection (23, 29, 53). RNA analysis. Cytoplasmic RNA was isolated at 48 h posttransfection by phenol-chloroform extraction after cell lysis by Nonidet P-40 (63). RNase protection assays were performed as described previously (63), with some modifications. The cytoplasmic RNA was treated with DNase 1 (3). The probe and cytoplasmic RNA were dried together under vacuum and resuspended in 5 ,l of hybridization solution. After denaturation, the mixture was incubated at 42°C overnight. The hybrids were digested at 30°C for 1 h with 2 to 12 pLg of RNase T1 per ml in 100 ,ul. The RNase protection products were analyzed in 8% polyacrylamide-7 M urea denaturing gels, which were then fixed, dried, and exposed to X-ray film. The protection products were quantified by scanning the dried gel on a Molecular Dynamics phosphoimage analyzer. The PCNA-CAT riboprobe, from -172 of the PCNA promoter to +203 in the CAT gene, flanked with polylinker sequence, was described previously (53). The ElA riboprobe was synthesized by SP6 polymerase transcription of a PvuII fragment (nucleotides 451 to 622) from pElAwt subcloned into the HinclI site of pGEM-1. RNA probes were purified by gel electrophoresis. RESULTS The experiments described here address two questions: which El gene products are able to transactivate expression from the PCNA promoter, and what PCNA promoter sequences constitute the cis-acting responsive elements. Transcriptional effects of El gene products. We have used

with its effectiveness at activation of the adenovirus E3 promoter. The effect of the concentration of ElA on its transcriptional repression and activation functions will be addressed directly below (see Fig. 4). The data of Fig. 1 show that PCNA-CAT expression can be stimulated by high levels of ElA alone or by low levels of ElA in combination with E1B. To determine the effects of each of the gene products of the El region on PCNA expression, the four major products of the region (EIA 12S, ElA 13S, E1B 19K, and E1B 55K) were expressed independently from CMV-based expression vectors diagrammed in Fig. 2. The constructs shown in Fig. 2 were cotransfected into HeLa cells with the PCNA-CAT construct or the E3CAT plasmid. The ElA and the 13S expression vectors (pCMVElA and pCMV13S, respectively) stimulated CAT expression from both the E3 and PCNA promoters (Fig. 3). The ElA 12S product repressed the E3 promoter and acti-

VOL. 65, 1991

12S ElA ACTIVATES PCNA EXPRESSION

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