Regulation of the cyclin E gene by transcription factor E2F1.

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12146-12150, December 1995 Genetics

Regulation of the cyclin E gene by transcription factor E2F1 KIYOSHI OHTANI, JAMES DEGREGORI, AND JOSEPH R. NEVINS Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710

Communicated by Gordon G. Hammes, Duke University Medical Center, Durham, NC, July 27, 1995

ABSTRACT A variety of results point to the transcription factor E2F as a critical determinant of the G,/S-phase transition during the cell cycle in mammalian cells, serving to activate the transcription of a group of genes that encode proteins necessary for DNA replication. In addition, E2F activity appears to be directly regulated by the action of retinoblastoma protein (RB) and RB-related proteins and indirectly regulated through the action of G, cyclins and associated kinases. We now show that the accumulation of G, cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin Dl promoters, both promoters are activated by E2F gene products, and at least for cyclin E, the E2F sites contribute to cell cycle-dependent control. Most important, the endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in an autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through Gl.

Through the combined use of genetic and biochemical approaches, the identification of regulatory activities controlling the cell cycle has progressed at a rapid pace. Although yeast systems have provided many of the initial advances (1), more recent contributions have been made from the study of mammalian cell growth control and, in particular, the events that are disrupted during oncogenic transformation. Such work has provided evidence that the cellular transcription factor E2F is a target for the action of the retinoblastoma gene product (RB) as a growth suppressor. Indeed, various studies now suggest that the E2F transcription factor is an integral part of the growth-regulatory network which controls the progression of cells from Go and early GI into the S phase of the cell cycle. In particular, expression of E2F1 can induce quiescent cells to enter S phase (2), and deregulated expression of E2F1 can lead to oncogenic transformation of an established cell line (3) or, in cooperation with an activated ras oncogene, transformation of primary rat embryo cells (4). The control of E2F accumulation during GI appears to be intimately associated with GI cyclin-dependent kinase activity, likely through the phosphorylation of RB and RB family members (5, 6). In view of the ability of E2F to regulate various genes associated with GI/S progression (7), including the autoregulatory control of the E2F1 gene (8-10), we have investigated the possible role of E2F1 in the control of GI cyclin accumulation.*

RNase Protection Assay. The assay was performed as described (8). Gel Mobility-Shift Assays. Mobility shift assays were performed as described (12). Oligonucleotides corresponding to three putative E2F sites in the cyclin E promoter (positions +497, +7, and -16) were used as competitors. E2F site 1

5

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E2F site 2 5 'gatccAAATGTCCCGCTCTG-3'

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E2F-site oligonucleotides 1, 2, and 3 (CycE 1, 2, and 3) and Not I fragments 1 (+680 to +42) and 2 (+41 to -74) (CycE Not 1 and 2) from the cyclin E gene were used at a molar excess of 100 and 500. Not I fragments 1 and 2 contain E2F sites 1 and 2/3, respectively. Construction of Plasmids. A Sac I (-1 195)-BamHI (+ 79) fragmnent and a 5' deletion fragment (-207 to +79) were subcloned into pCAT Basic (Promega) to make pCycECAT and pCycECAT(-207), respectively. The double mutant of putative E2F sites in the pCycECAT(-207) construct, pCycECAT(E2Fx2-), was made by site-directed mutagenesis which changed TGTCCCGC at position +7 to TGTCATGC and changed TTCCGCGC at position -16 to TTCCGATC. Luciferase reporter plasmids pCycELUC(-207) and pCycELUC(E2Fx2-) were made by subcloning of the corresponding fragments from pCycECAT(-207) and pCycECAT(E2Fx2-) into pGL2 Basic (Promega), respectively. Cyclin Dl promoter-driven chloramphenicol acetyltransferase (CAT) plasmid (pCycDlCAT) was made by subcloning a 1.9-kb Pvu II (-1652 to +231) fragment from the 5' flanking sequence of the human cyclin Dl gene (13) into pCAT Basic. pCycDlCAT(E2F-), in which the E2F site was mutated from TTTGGCGC to TTTGATGC, was made by site-directed mutagenesis. The ElA and E2F1, -2, and -3 expression plasmids have been described (8). Transfection Assays. Transfection of REF52 cells (rat embryo fibroblasts) and CAT and luciferase assays were performed as described (2). All assays were done in duplicate. Northern Blot Analysis. Agarose gel electrophoresis, transfer to nitrocellulose membrane, and hybridization were as described (8).

RESULTS Isolation of the Human Cyclin E Promoter. To identify sequences containing the cyclin E promoter, a 50-base oligonucleotide complementary to the 5' end of the human cyclin E cDNA sequence (11, 14) was used to screen a human placenta genomic DNA library. One positive clone was iso-

MATERIALS AND METHODS Isolation of Human Cyclin E Promoter Sequences. A human placenta genomic DNA library (1.4 x 106 clones) was screened with a 50-base oligonucleotide probe complementary to the 5' end of the published human cyclin E cDNA (11) as described (8).

Abbreviations: CAT, chloramphenicol acetyltransferase; RACE, rapid amplification of cDNA ends. *The human cyclin E gene sequence reported in this paper has been deposited in the GenBank data base (accession no. L48996).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Genetics: Ohtani et al. lated from a screen of 1.4 x 106 recombinant phage. A 2.1-kb Sac I fragment that hybridized to this probe was subcloned for further analysis. Sequence analysis confirmed that this fragment contained the 5' sequence of the cyclin E cDNA (Fig. 1A). The sequence matches the cDNA sequence to a point 63 nt 3' of the ATG initiation codon and then diverges in sequence. Surrounding the point of divergence is the sequence CGTTGTGAGT, a close match to a consensus splice donor sequence, suggesting that this is the 3' end of an exon. We used both a RACE procedure (15) and RNase protection to identify the sequences corresponding to the 5' end of the cyclin E mRNA. This analysis identified two additional exons 5' of the sequence found in the cDNA (Fig. 1B). RNase protection assays using an RNA probe spanning the upstream sequence of the cyclin E cDNA yielded protected fragments of -45 nt and 175 nt (Fig. 1C). The 45-nt fragment corresponds to the second exon identified in the RACE products, and the 175-nt fragment corresponds to the first exon defined by the RACE experiment. Use of an additional RNA protection probe that spanned this upstream region revealed several protection products that map to the region, including the exon defined by RACE, and that presumably define the start site for transcription. Multiple potential E2F binding elements as well as Spl recognition sequences are found in the cyclin E 5' flanking region. Gel mobility-shift assays demonstrated that oligonucleotides representing cyclin E promoter E2F sequences competed for E2F binding (Fig. 1D). These results show that the putative E2F binding sites in the cyclin E gene are functional, with site 2 exhibiting the strongest binding. E2F-Dependent Activation of the Cyclin E and DI Promoters. To assess the role of E2F1 in the control of cyclin E and Dl promoter activity, we made use of a recombinant adenovirus, AdE2F1 (17), to overexpress the E2F1 protein in quiescent cells. Plasmids containing the cyclin E or Dl promoter fragment cloned upstream of a CAT reporter gene were transfected into REF52 cells, and the transfectants were starved for serum and then infected with the AdE2F1 virus or a control virus lacking the E2F1 cDNA (AdCMV). Cyclin E promoter activity was stimulated '170-fold and cyclin Dl promoter activity was stimulated 5-fold upon infection with the AdE2F1 virus (Fig. 2A). In contrast, coinfection with the control virus (AdCMV) had no effect on either cyclin E or Dl promoter activity. We also assayed for the ability of additional E2F family members to activate the cyclin E and Dl promoters. Although we have the E2F1 cDNA only in the form of a recombinant adenovirus, we can nevertheless measure activation by cotransfection with appropriate plasmids. Plasmids expressing the E2F2 and E2F3 products were also able to activate transcription from both the cyclin E and Dl promoters, although E2F2 had only a small effect on the cyclin Dl promoter (Fig. 2B). We conclude that the cyclin E and Dl promoters do indeed respond to each of the E2F family members. As additional evidence for a role for E2F in the activation of the cyclin Dl and cyclin E promoters, we have made use of promoter constructs in which the E2F recognition sites have been eliminated by mutation. We initially constructed a series of 5' deletion mutants of the cyclin E promoter to define the essential promoter sequences. Based on these results, we employed a -207 promoter construct for further analysis, since this construct was as active as a promoter containing the full complement of upstream sequence. The two E2F sites which were previously shown to bind E2F were mutated and the construct was then assayed for E2F1-mediated activation. Although these mutations did not completely eliminate E2F1 responsiveness, most likely due to remaining E2F sites in the promoter, there was nevertheless a substantial reduction in the response to E2F1. Likewise, mutation of the E2F site in the -

Proc. Natl. Acad. Sci. USA 92 (1995)

12147

cyclin D1 promoter virtually abolished activation by E2F1 (Fig. 2C). We conclude that both the cyclin E and Dl promoters can respond to E2F. Cell Cycle Control of Cyclin E Promoter Activity. Northern analyses revealed that cyclin E expression was tightly regulated in REF52 cells, with an accumulation in GI similar to the kinetics of E2F1 (Fig. 3A). In contrast, there was little evidence of control of the cyclin Dl mRNA in the REF52 cells. To assess the role of E2F, plasmids containing the cyclin Dl and E 5' flanking sequences upstream of the luciferase gene were transfected into REF52 cells along with a cytomegalovirus (CMV) promoter-driven f3-galactosidase reporter plasmid as an internal control. Cells were brought to quiescence and then harvested every 4 hr after serum addition. Assays for cyclin Dl promoter activity in the transfected cells revealed only low levels of activity that did not change during the growth response, consistent with the analyses of the endogenous gene (data not shown). Cyclin E promoter activity was detected at a low level in the unstimulated cells and then increased -7-fold following serum stimulation (Fig. 3B), coinciding with the kinetics of accumulation of the endogenous cyclin E mRNA (Fig. 3A). Mutation of the two E2F sites in the cyclin E promoter resulted in a 3.5-fold increase in the unstimulated cells as well as an elevation of the overall level of cyclin E promoter activity during the early part of G1, resulting in an earlier peak of promoter activity. We conclude that the activity of the cyclin E promoter during a -growth response is affected by E2F, primarily a negative control as has been observed in past experiments analyzing both the B-myb promoter (18) and the E2F1 promoter (8-10). The relief of this negative control, leading to an activation of the promoter, could be viewed as the combination of loss of negative-acting E2F complexes found in Go cells as well as the accumulation of E2F activity that would displace the negative-acting complexes. However, given the fact that promoter activity does increase despite the absence of E2F sites, additional factors must contribute to the activation of the promoter. Activation of the Endogenous Cyclin E Gene by E2F1. Since the E2F1 recombinant virus can infect the entire population of quiescent cells, it is also possible to measure the activation of the endogenous cyclin genes in addition to the transfected plasmids, an important assay because the transfection experiments do not measure the control of the gene in its normal context. As shown by Northern analysis (Fig. 4), the cyclin E mRNA was increased -22-fold upon infection with the AdE2F1 virus over that in the control infection. In contrast, we observed no increase in cyclin Dl mRNA under these conditions. We thus conclude that E2F1 not only has the potential to activate the cyclin E gene in transfection assays but also activates the endogenous cyclin E gene. The lack of activation of the cyclin Dl gene may reflect the fact that cyclin Dl expression does not appear to be regulated in REF52 cells.

DISCUSSION The progression of cells through GI and into S phase coincides with the temporal expression of sets of genes whose products are required for the next phase of the cell cycle (19, 20). Various experiments suggest a role for E2F in the control of genes encoding proteins involved in DNA synthesis such as dihydrofolate reductase, thymidine kinase, and DNA polymerase a (7), as well as regulatory genes such as c-myc, N-myc, PRAD1/Bcll (cyclin D1), erb-B (epidermal growth factor receptor), c-myb, and B-myb (7, 13, 16, 18, 21-23), and cyclin A (24). Moreover, E2F sites have been shown to be important for cell cycle-regulated transcription in several instances (25, 26) and, like the results we present here, many of these genes are indeed activated when E2F1 is expressed in quiescent cells (27). Given the role of these genes in normal cell proliferation, we speculate that one or more of these gene products may be

12148

Proc. Natl. Acad. Sci. USA 92 (1995)

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FIG. 1. Human cyclin E promoter. (A) DNA sequence of the human cyclin E gene that includes the 5' end of the cyclin E cDNA. The ATG translation initiation codon identified in the second exon defined by a rapid amplification of cDNA ends (RACE) procedure is doubly underlined. The ATG initiation codon previously identified in the cDNA clone is underlined by a dashed line. The regions of exons identified by the RACE procedure and RNase protection are underlined. The 5' ends of the cyclin E mRNA, as determined by RNase protection, are indicated by arrows and the 5' end is numbered as + 1. Three putative E2F sites analyzed in this study are boxed and numbered 1, 2 and 3. (B) Mapping the 5' end of the cyclin E mRNA by RACE analysis. The RACE procedure was carried out (15) and six clones were isolated. These were sequenced and their structures relative to the genomic sequence are depicted. Dashed lines indicate introns. The ATG translation initiation codon previously identified in the cDNA clone is underlined and the newly identified ATG translation initiation codon in the second exon is boxed. (C) RNase protection analysis of the cyclin E mRNA 5' end. Probe A (Left) begins at position +715 in the second intron and ends at -159. Probe B (Right) begins at +79 in the first exon and ends at -159. Protected fragments were electrophoresed in a 7% polyacrylamide sequencing gel next to products of a sequencing reaction of the cyclin E genomic DNA as size markers. Lanes 1, no RNA; 2, 20 ,ug of yeast tRNA; 3, 20 ,tg of total RNA from human embryonic kidney cell line 293; 4, Poly(A)+ RNA derived from 1 mg of 293-cell total RNA. (D) E2F binding to the cyclin E promoter. A gel mobility-retardation assay was performed (12) with c-myc E2F binding-site oligonucleotide as probe (16). The first lane contained probe only, and the second lane contained probe and HeLa cell nuclear extract as a source of E2F. Competitions were performed with c-myc E2F binding site, wild-type E2 promoter oligonucleotide (E2WT), a mutant form of the E2 promoter oligonucleotide (E2mut) at a molar excess of 100, and 100or 500-fold molar excess of the indicated oligonucleotides and fragments from the cyclin E promoter (see Materials and Methods).

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FIG. 2. Cyclin E and Dl promoters are activated by E2F. (A) Activation of the cyclin Eand Dl promoters by E2F1. REF52 cells were transfected with 5 ,ug of pCycECAT or pCycD1CAT plasmid along with 3 jig of pRSV-f3-gal plasmid and 12 ,ug of carrier DNA. Twelve hours after transfection, cells were washed and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 0.1% fetal bovine serum. After 24 hr of serum starvation, cells were infected with the AdE2F1 virus or AdCMV control virus at a multiplicity of 500 plaque-forming units per cell and further cultured for 21 hr. Cells were harvested and assayed for CAT activity (2). CAT activities are represented relative to ,B-galactosidase activity as an internal control. (B) E2F family members activate the cyclin E and Dl promoters. REF52 cells were transfected with 5 ,ug of pCycECAT or pCycD1CAT plasmid, 3 pug of pRSV-,3-gal, 100 ng of the indicated E2F or E1A plasmid, and 12 ,ug of carrier DNA. After transfection, cells were washed and placed in DMEM with 0.1% serum for 48 hr. CAT activity was assayed as inA. (C) Activation of cyclin E and D1 promoters by E2F1 is dependent on E2F binding sites. REF52 cells were transfected with 5 ,ug of pCycECAT(-207) or pCycDlCAT (wild type, WT) or their mutants pCycECAT(E2Fx2-) or pCycDlCAT(E2F-), 3 ,tg of pRSV-13-gal, 100 ng of E2F1 expression plasmid where indicated (+), and 12 ,tg of carrier DNA. Cells were serum starved for 48 hr and assayed for CAT activity as in A.

well as the potential for control of cyclin Dl, suggesting that the action of the E2F1 transcription factor may extend beyond the activation of a group of genes encoding proteins essential fqr S phase. In particular, these findings suggest that the action of E2F1 in the GI/S transition may not be a simple linear pathway leading to the activation of S-phase

genes. Rather, the results suggest a feedback loop whereby the accumulation of E2F1 would lead to an increased accumulation of cyclin E, as the result of loss of negative control, which would then further amplify the accumulation of E2F1 (Fig. 5). As such, E2F1 takes on a central role in mediating the control of cell cycle progression through GI

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FIG. 3. E2F-dependent, cell cycle-regulated expression of cyclin E. (A) Northern blot analysis of cyclin E, E2F1, and cyclin Dl mRNAs during induction. REF52 cells were serum starved for 24 hr and then stimulated with 20% fetal bovine serum. Cells were harvested every 4 hr after stimulation and RNA was isolated. Twenty micrograms of total RNA (for cyclin D1) and poly(A)+ RNA isolated from 180 ,ug of total RNA (for cyclin E and E2F1) were electrophoresed in a 1% agarose gel, transferred to nitrocellulose membrane and separate membranes were probed with cyclin E or E2F1 cDNA and cyclin Dl cDNA. Both filters were reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. [293], 293-cell RNA. (B) Serum induction of cyclin E promoter activity. REF52 cells were transfected with 5 jig of pCycELUC(-207) (wild type, WT) or its mutant pCycELUC(E2Fx2-), 1 ,ug of pCMV-,B-gal, and 14 ,ug of carrier DNA. Twelve hours after transfection, cells were washed and placed in DMEM with 0.1% fetal bovine serum for 24-28 hr. Serum was then added to a final concentration of 20%, and the cells were harvested at indicated time points for assay of luciferase and ,B-galactosidase activities. Luciferase activity was normalized to P-galactosidase activity as an internal control. serum serum

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Genetics: Ohtani et aL

Proc. Natl. Acad. Sci. USA 92 (1995) enter S phase, clearly implicates E2F as a critical regulator

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and into S phase. The realization that E2F1 transcription is upder autoregulatory control, the fact that G1 cyclin kinase activity controls the activity of E2F1 (8), and now the finding that E2F1 controls the transcription of the cyclin E gene, all ppint to a process whereby the accumulation of E2F activity may well be a critical event in the decision to enter S phase. This realization, together with previous experiments that demonstrate an ability of E2F1 to induce quiescent cells to Growth Stimulatory Signals

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FIG. 5. A central role for E2F1 in GI/S-phase control. Growthstimulatory signals would include various growth factors that lead to the activation of GI cyclin accumulation. One target for the action of G, cyclins, in conjunction with cyclin-dependent kinases (cdk), is the RB protein and possibly RB family members ("RB"). Phosphorylation of RB abolishes its capacity to interact with E2F, thus leading to the accumulation of active E2F. E2F1 has the capacity to activate transcription of its own gene and, as shown here, cyclin E expression as well.

We thank Kaye Culler for assistance in the preparation of the manuscript and Tim Kowalik for critical reading of the manuscript. We thank Drs. Yan Geng and Robert Weinberg for sharing unpublished information regarding the structure of the cyclin E gene. J.D. is supported by a National Institutes of Health postdoctoral fellowship. 1. Hartwell, L. H. & Weinert, T. A. (1989) Science 246, 629-633. 2. Johnson, D. G., Schwarz, J. K., Cress, W. D. & Nevins, J. R. (1993) Nature (London) 365, 349-352. 3. Singh, P., Wong, S. W. & Hong, W. (1994) EMBO J. 13, 33293338. 4. Johnson, D. G., Cress, W. D., Jakoi, L. & Nevins, J. R. (1994) Proc. Nati. Acad. Sci. USA 91, 12823-12827. 5. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J. & Livingston, D. M. (1993) Cell 73, 487-497. 6. Kato, J., Matsushime, H., Hiebert, S. W., Ewen, M. E. & Sherr, C. J. (1993) Genes Dev. 7, 331-342. 7. Nevins, J. R. (1992) Science 258, 424-429. 8. Johnson, D. G., Ohtani, K. & Nevins, J. R. (1994) Genes Dev. 8, 1514-1525. 9. Hsiao, K., McMahon, S. L. & Farnham, P. J. (1994) Genes Dev. 8, 1526-1537. 10. Neuman, E., Flemington, E. K., Sellers, W. R. & Kaelin, W. G., Jr. (1994) Mol. Cell. Biol. 14, 6607-6615. 11. Koff, A., Cross, F., Fisher, A., Schumacher, J., Leguellec, K., Philippe, M. & Roberts, J. M; (1991) Cell 66, 1217-1228. 12. Yee, A. S., Raychaudhuri, P., Jakoi, L. & Nevins, J. R. (1989) Mol. Cell. Biol. 9, 578-585. 13. Motokura, T. & Arnold, A. (1993) Genes Chromosomes Cancer 7, 89-95. 14. Lew, D. J., Dulic, V. & Reed, S. I. (1991) Cell 66, 1197-1206. 15. Frohman, M. A. (1990) in PCR Protocols:A Guide to Methods and Applications, eds. Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J. (Academic, San Diego), pp. 28-38. 16. Hiebert, S. W., Lipp, M. & Nevins, J. R. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598. 17. Schwarz, J. K., Bassing, C. H., Kovesdi, I., Datto, M. B., Blazing, M., George, S., Wang, X. & Nevins, J. R. (1995) Proc. Natl. Acad. Sci. USA 92, 483-487. 18. Lam, E. W. & Watson, R. J. (1993) EMBO J. 12, 2705-2713. 19. Johnston, L. H. (1990) Curr. Opin. Cell Biol. 2, 274-279. 20. McKinney, J. D. & Heintz, N. (1991) Trends Biochem. Sci. 16, 430-435. 21. Mudryj, M., Hiebert, S. W. & Nevins, J. R. (1990) EMBO J. 9, 2179-2184. 22. Hara, E., Okamoto, S., Nakada, S., Taya, Y., Sekiya, S. & Oda, K. (1993) Oncogene 8, 1023-1032. 23. Muller, H., Lukas, J., Schneider, A., Warthoe, P., Bartek, J., Eilers, M. & Strauss, M. (1994) Proc. Natl. Acad. Sci. USA 91, 2945-2949. 24. Henglein, B., Chenivesse, X., Wang, J., Eick, D. & Brechot, C. (1994) Proc. Natl. Acad. Sci. USA 91, 5490-5494. 25. Means, A. L., Slansky, J. E., McMahon, S. L., Knuth, M. W. & Farnham, P. J. (1992) Mol. Cell. Biol. 12, 1054-1063. 26. Blake, M. C. & Azizkhan, J. C. (1989) Mol. Cell. Biol. 9,4994-5002. 27. DeGregori, J., Kowalik, T. & Nevins, J. R. (1995) Mol. Cell. Biol. 15, 4215-4224.