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The role of E2F in the mammalian cell cycle
125
Biochimica et Biophysica Acta, 1155 (1993) 125-131 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00
BBACAN 87265
The role of E2F in the mammalian cell cycle P e g g y J. F a r n h a m , Jill E. S l a n s k y a n d R i c h a r d K o l l m a r McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI (USA) (Received 10 December 1992)
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
II.
The E2F protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
III. The role of E2F sites in gene activation at the G1/S-phase boundary . . . . . . . . . . . . . . . . . . .
126
IV. Regulation of E2F activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
127
V.
129
Is E2F a central regulator of the entrance into S phase? . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
I. Introduction
The proliferative cell cycle is divided into 4 phases (Fig. 1). During the two gap phases, G1 and G2, the cell accumulates the necessary products for replication of the genome (S phase) and cell division (M phase), respectively. Protein synthesis is required for a cell to pass through a restriction point in G1 phase [1,2] and commit to DNA synthesis. Although the proteins that control the entrance into S phase are unknown, several genes that encode products involved in nucleotide synthesis and DNA replication are activated at the G1/S-phase boundary. The promoters of many of these genes contain binding sites for the transcription factor E2F. Thus, a common control mechanism involving E2F may be responsible for the cell cycle regulation of these genes. E2F has also been implicated in cell cycle control through its association with the retinoblastoma (Rb) tumor suppressor protein and with the cyclins E and A. The purpose of this review is to summarize
Correspondence to: P.J. Farnham, McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Avenue, Madison, WI 53706, USA. Abbreviations: Rb, Retinoblastoma; DHFR, dihydrofolate reductase.
what is known about the role of E2F in gene regulation and to discuss the possibility that E2F is a central regulator of the entrance into S phase. II. The E2F protein
E2F is a cellular DNA-binding protein with an apparent molecular mass of about 60 kDa. It was first identified in HeLa cells as a transcriptional activator of the adenovirus E2 promoter [3,4]. A similar activity from F9 cells has been called DRTF1 [5-7]. E2F binds to DNA with the consensus 5'-TITSSCGC-3' (S = C or G) [8,9]. Variations at the first, second, and last position of the consensus do not abolish E2F binding, but reduce the affinity of the site for E2F. E2F binds not only to DNA, but also to the Rb protein. E 2 F / R b interactions were used to isolate the human E2F-1 cDNA. Several clones were obtained when expression libraries were probed with the region of the Rb protein that binds E2F [10-13]. One clone was human E2F-1 (also called RBP3 [12], RBAP-1 [11], and Apl2 [13]) as defined by its sequence-specific DNA binding and transcriptional activation properties. The DNA-binding domain of E2F-1 (Fig. 2) resembles helix-loop-helix DNA-binding domains and is located near the N-terminus of the protein, adjacent to a
126 basic region. A transactivation region at the C-terminus of the protein overlaps with the region of E2F-1 that is required for binding to the Rb protein. A proline-rich region of unknown function is located N-terminal to the DNA binding domain and two potential phosphorylation sites for cdk kinase are found at amino acids 159 and 346. Cloned E2F-1 binds to and activates transcription from the adenovirus E2 and dihydrofolate reductase (DHFR) promoters [11,12,14]. The mouse DHFR promoter contains two inverted, overlapping E2F sites between - 8 and + 1 which bind cloned E2F-1 [11], and three other similar sites at -266, -244 and -213 which have not been tested in binding assays (Fig. 3A). DHFR promoter constructs containing different numbers of these putative E2F sites were transfected with a human E2F-1 expression construct and assayed in serum-starved cells (Fig. 3B). Decreasing the number of E2F sites in the various constructs resulted in decreased transactivation by E2F-1. A murine E2F-1 cDNA homologous to human E2F-1 (Y. Li and P.J. Farnham, unpublished data; J. Lees and E. Harlow, personal communication) and two other human cDNAs that are similar to E2F-1 in the DNA binding domain (J. Lees and E. Harlow, unpublished data) have also been cloned. In addition, a cDNA has been cloned for an unrelated mouse protein called DP-1 that can also bind specifically to an E2F site [15]. Throughout this review, E2F refers to the uncharacterized proteins that bind to the recognition site, whereas E2F-1, E2F-2, or E2F-3 refers to the cloned E2F cDNAs. IlL The role of E2F sites in gene activation at the G1 / S-phase boundary
The promoters of several genes that are activated in mid or late G1 phase bind cellular proteins at E2F sites. These genes include DHFR, thymidine kinase, DNA polymerase alpha, cdc2, thymidylate synthase, and c-myc (see Ref. 9 for a list of these sites). This observation suggests that E2F may mediate growth regulation of these genes. In this section, we will discuss what is known about the role of the E2F binding sites in the transcriptional regulation of these promoters. DHFR catalyzes the conversion of dihydrofolate to tetrahydrofolate, which is required for the biosynthesis of purines and thymidylate. DHFR mRNA levels increase at the G1/S-phase boundary in mouse, human, and hamster cells [16-20]. Mouse DHFR promoter sequences from -270 to + 20 are sufficient to confer an increase in transcription at the G1/S-phase boundary on a reporter gene in 3T3 cells synchronized by serum starvation. Mutational analysis of the DHFR promoter indicates that sequences spanning the transcription initiation site are required for the transcrip-
tional increase [21]. These sequences consist of two overlapping, inverted binding sites ( - 8 to + 1) for E2F [22] and an initiator element ( - 3 to +5) which is involved in start site recognition [23,24]. The 29 base pairs containing the E2F sites and the DHFR initiator element were cloned upstream of a reporter gene without any other promoter elements. A 35-fold increase in activity of this promoter was observed during S phase [14]. Thus, the E2F sites at the DHFR initiation site are both necessary and sufficient for G1/Sphase regulation. Thymidine kinase catalyzes the phosphorylation of thymidine to thymidylate. The levels of murine, human, and hamster thymidine kinase mRNA increase at the G1/S-phase boundary after mitogen stimulation of quiescent cells [25-31]. In extracts prepared from Sphase cells, protein binds to similar 20-25 bp elements containing consensus E2F sites in the mouse [32] and human [33] promoters. Competition with an E2F site from the E2 promoter suggests that the protein (called Yi2) binding to the mouse thymidine kinase promoter is related to E2F [9,32]. However, a probe that contains an E2F site does not compete for binding of the S-phase protein on the human thymidine kinase element [33]. This could be due to different affinities of these sites, or the DNA-binding protein could be different from, but related to, E2F. Mutations in the E2F site abolish the S-phase-specific binding and result in loss of serum regulation of the human thymidine kinase promoter [33]. No point mutants have been made to determine if the E2F site is required for regulation of the mouse thymidine kinase promoter. DNA polymerase alpha synthesizes the lagging strand of DNA at the replication fork. Levels of DNA polymerase alpha mRNA increase as cells enter S phase [34], and the promoter region of this gene can Late response genes: DHFR TS TK Delayed early response genes: c-myc Early response l genes: c-fos
GO
CAD DNA
POL a CDCZ Cycln A EZF
,,
G1
S
GZ
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Fig. 1. The cell cycle. Addition of mitogens to quiescent cells initiates a series of events that lead to activation of growth-regulated genes at different times. Some genes are regulated differently in the first cell cycle after quiescent cells are stimulated with mitogens. Therefore, this cycle is shown as distinct from the subsequent (proliferative) cell cycles. Most of the regulation of the late response genes discussed in this review has been analyzed in the first cycle. The restriction point (R) is indicated. The abbreviations for genes are dihydrofolate reductase (DHFR), thymidine kinase (TK), thymidylate synthase (TS), carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (CAD), DNA polymerase alpha (DNA pol a).
127 confer G1/S-phase regulation on a reporter gene [35]. A consensus site that binds E2F is present at -128 [9,35] and sequences similar to the E2F consensus site are present throughout the promoter. One such sequence, GTTGGCGC, is located at + 18 and is present in all constructs tested for growth regulation. A DNA polymerase alpha promoter construct that extends from - 4 0 2 to +45 increases 6-fold in activity at the G1/Sphase boundary; successive 5' deletions gradually reduce the transcriptional increase. These results are similar to those obtained with the 5' deletion constructs of the DHFR promoter (Fig. 3C). Although each construct contains the E2F sites located between - 8 and + 1 that are required for regulation, deletion of the upstream putative E2F sites reduces the maximal increase in transcription at the G1/S-phase boundary. It is possible that multiple E2F sites contribute to the overall regulation of both the DHFR and DNA polymerase alpha promoters. The large S-phasespecific increase in transcription from the synthetic DHFR promoter (composed only of the E2F sites and the initiator element) as compared to the increases observed using longer DHFR promoter constructs could be due to the very low basal activity of the synthetic promoter. Perhaps the E2F sites are not sufficient to confer a large increase in transcription at the G1/S-phase boundary if strong constitutive activators (such as SP1) are present. Alternatively, there may be a repressor site in the longer DHFR promoter that limits the activation seen at the G1/S-phase boundary. Cdc2 is a kinase that associates with various cyclins and is required for progression through the proliferative cell cycle. Cdc2 transcription and mRNA levels increase at the G1/S-phase boundary [36,37]. Deletion of an E2F consensus site in the cdc2 promoter results in equal transcription rates in cycling and serum-starved cells. Thus, E2F also appears to be important in the regulation of the cdc2 gene. Other genes, such as thymidylate synthase [38-42], ribonucleotide reductase [43-45], carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydrooro-
proline-rich region 43 86
I
I
KSP I 120
IV. Regulation of E2F activity If E2F is responsible for activation of genes at the G1/S-phase boundary, then E2F activity must change
SPGK I 307
I I
tase [46-48] (R.J. Miltenberger and P.J. Farnham, unpublished data), and cyclin A [49-53], are also activated at the G1/S-phase boundary. Although less is known about the DNA sequences and transcription factors involved in their regulation, each of these promoters contains at least one putative E2F site. The E2F site in the thymidylate sythase promoter binds a cellular protein which is competed by a consensus E2F site (Y. Li and P.J. Farnham, unpublished data). A 7/8 match with a consensus E2F site is present in a 60base-pair region that is required for growth regulation of the carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase promoter (R.J. Miltenberger, R. Kollmar, and P.J. Farnham, unpublished data). E2F may also regulate the delayed-early c-myc gene [54,55]. A consensus site which binds E2F is located between - 58 and - 65, upstream of the P2 promoter. A 3-fold increase in c-myc transcription 4 h after serum stimulation of quiescent cells is dependent on the E2F site [8]. Perhaps, the 3-fold increase in mid G1 is the beginning of a larger increase that peaks at the G1/Sphase boundary. Alternatively, the E2F elements might mediate a transcriptional increase at different points in the cell cycle by binding to different members of the E2F gene family. In summary, the promoters of several genes that are activated in mid or late G1 phase contain sequences that resemble E2F sites. Mutagenesis of these sequences in the DHFR, thymidine kinase, and cdc2 promoters indicates that protein binding to these sites is important for their growth regulation. The analysis of the role of the E2F sites in the other promoters is not yet complete. However, the available evidence is consistent with the hypothesis that E2F is involved in the regulation of the genes that are activated at the G1/S-phase boundary.
RB-binding r e g i o n 409 426
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109 128 181 b a s i c r e g i o n HLH r e g i o n DNA-binding domain
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437
trans-activation domain
Fig. 2. The human E2F-1 protein. The DNA-binding,trans-activation,and Rb-bindingdomainshave been defined by mutagenesis[11-13]. A proline-rich regionand two putativephosphorylationsites (KSPand SPGK)havebeen foundby sequenceanalysis[13].
128 A -270
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Fig. 3. (A) The mouse D H F R promoter. The position of transcription factor binding sites is shown and the 5' endpoints of the different promoter deletions are represented by vertical bars. Open box, putative E2F site; filled box, E2F site; oval, Spl site. (B) 3T3 cells were transfected with the D H F R promoter luciferase constructs and a plasmid with or without the human E2F-1 cDNA expressed from the CMV promoter. The cells were incubated in medium containing 0.5% bovine calf serum for 48 h and then harvested for luciferase assays. The fold induction by E2F was calculated for each promoter as the ratio of luciferase activity in the presence of the E2F-1 expression plasmid to that in the presence of the vector control. (C) 3T3 cells were transfected with the D H F R promoter luciferase constructs. The cells were incubated in medium containing 0.5% bovine calf serum for 48 h, followed by incubation in medium containing 10% bovine calf serum. Cells were harvested at the indicated times for luciferase assays to determine promoter activity and for flow cytometry to define the time at which the cells entered S phase. Luciferase values are reported as a ratio of the activity of each promoter at different times in the cell cycle relative to the activity of the same promoter in starved cells,
through the cell cycle. The levels of E2F-1 mRNA do fluctuate through the cell cycle. Little E2F-1 mRNA is present in quiescent 3T3 cells, in resting T cells, or in mouse liver. However, the levels of E2F-1 mRNA increase at the G1/S-phase boundary after serum stimulation of 3T3 cells [14], mitogen stimulation of resting human T cells [11], and partial hepatectomy of C 3 H / H e J mice (M. Bennett, N. Drinkwater, and P.J. Farnham, unpublished data). The levels of E2F-2 and E2F-3 mRNAs and of the different E2F proteins in specific stages of the cell cycle have not yet been examined. Protein binding to E2F sites can be detected in all stages of the cell cycle [8]. This detection of E2F DNA binding activity in GO and G1 cells initially led to the conclusion that the D H F R promoter was not regulated by E2F [21]. However, the low level of E2F-1 mRNA in GO and G1 cells suggests that perhaps E2F-2 or E2F-3 binds to DNA during these stages. Also, a 180-kDa protein from proliferating HeLa cells has been purified on a DNA affinity column containing the E2F sites located between - 8 and + 1 in the D H F R promoter [21]. This protein has not been further characterized because only small amounts are obtained during purification (Y. Li and P.J. Farnham, unpublished data); in particular, it is not known whether it corresponds to E2F-2 or E2F-3. E2F activity may also be regulated by association with different cellular proteins in different stages of the cell cycle. In G1 phase, E2F binds to the Rb protein [56-60]; this association continues through S phase [61]. The existence of the E 2 F / R b complex in S phase is not understood. Rb undergoes cell cycle-dependent phosphorylation, with underphosphorylated Rb present in GO and G1 phase cells and phosphorylated Rb present in S and G 2 / M phase cells [62]. Since E2F has been shown to bind underphosphorylated Rb [11,59], it is not clear if the S-phase E 2 F / R b complex is due to a novel E2F interaction with phosphorylated Rb or if some unphosphorylated Rb exists in S phase. E2F also forms a complex with p107, the kinase cdk2, and cyclin E (in mid- to late-G1) or cyclin A (in S phase) [5,61,63-67]. One study detected an E 2 F / p l 0 7 complex in early G1 [9]. Other combinations of E2F, Rb, p107, cdk2, and various cyclins may be important in different growth states or in different cell types. All complexes appear to dissociate in G 2 / M phase, resulting in free E2F. How might changes in E2F abundance or formation of different E2F protein complexes control transcriptional activation at different stages in the cell cycle? The low levels of E2F-1 mRNA in the first G1 phase after stimulation of resting cells probably cause the low level of transcription of genes such as DHFR. The increase in E2F-1 mRNA at the G1/S-phase boundary would then result in increased transcription of these genes. Thus, E2F-1 activity in the G1 phase immediately following GO is controlled mainly by limiting
129 amounts of E2F-1. In addition, E2F activity in G1 phase may be controlled by interaction with Rb. Transfection of a plasmid expressing Rb protein results in repression of transcription from promoters containing E2F sites [68-70]. This repression could be simply a consequence of the growth-suppressing ability of Rb, and would then be unrelated to E 2 F / R b interactions. However, there is a correlation between the ability of Rb to bind to E2F and to suppress growth, suggesting that the direct interaction of Rb and E2F is critical for growth suppression [71]. E2F-Rb interactions can be disrupted by the E1A protein of adenovirus 2, the large T antigen of simian virus 40, and the E7 protein of papilloma virus [5,11,12,72,73]. In the presence of these viral proteins, E2F sites confer activation on the mouse D H F R [14], hamster D H F R [74], n-myc [74], c-myc [55], and cdc2 [37] promoters. Rb may interfer with E2F activity by masking the activation domain of E2F [11] or the E 2 F / R b complex bound to a promoter might hinder binding of other transcription factors [58,69,75] The function of the different E 2 F / p l 0 7 / c y c l i n / cdk2 complexes in different stages of the cell cycle is open to speculation. One reason to bring the cdk2 kinase to a promoter could be to stimulate transcription by phosphorylation of a factor in the initiation complex. The target of phosphorylation could be E2F itself since the DNA binding activity increases after phosphorylation of a dephosphorylated E2F fraction [76]. Other potential targets in the transcription complex are the CCAAT enhancer factor that binds next to the E2F site in the human TK promoter [33] and the C-terminal domain of RNA polymerase II, which is required for D H F R transcription [77] and is phosphorylated prior to initiation [78]. The presence of different cyclins in the E2F/pl07/cyclin/cdk2 complexes could result from specific interactions between particular members of the E2F family and particular cyclins. The different E2F/cyclin complexes may activate different promoters that are expressed in different stages of the cell cycle. For example, the E2F binding sites in the c-myc and the D H F R promoter are necessary for transcriptional activation in mid G1 phase and at the G1/S-phase boundary, respectively. However, cotransfection of E2F-1 increases transcription from the DHFR, but not from the c-myc, promoter in serumstarved cells (Ref. 14 and J.E. Slansky, unpublished data). Alternatively, other proteins in addition to E2F-1 may be required to activate the c-myc promoter. To date, the D H F R promoter is the only cellular promoter that has been shown to be activated by E2F-1, although proteins bind to E2F sites in the EGFR [8], the D H F R [11,21,22], thymidine kinase [32,33], c-myc [54,55] n-myc [74], E2 [4,79], E1A [79], thymidylate synthase (Y. Li and P. J. Farnham, unpublished data) and Rb (P.D. Robbins, personal communication) promoters. Other
promoters contain consensus E2F sites that have not yet been tested for binding or activation (see above and Ref. 56). Transfection of E2F-1, E2F-2, and E2F-3 with reporter constructs driven by these promoters is required before it is known if the different E2Fs regulate different promoters. Preliminary evidence suggests that E2F-1, like many transcription factors, may function as a heterodimer (D. Heimbrook, personal communication). Affinitypurified E2F from HeLa cells displays multiple protein bands on a denaturing polyacrylamide gel. Little E2F DNA-binding activity is obtained after renaturation of individual bands. However, when the separated proteins are mixed together, a dramatic increase in DNAbinding activity is seen. Western blot analysis shows that E2F-1 is related to some, but not all, of these proteins. The presence of multiple E2Fs and the possibility of different hetero- and homodimer pairs would add another level of complexity to E2F regulation. However, cloned E2F-1 can bind to DNA without a partner [11,12]. Understanding the relevance of potential heterodimer formation to E2F function must await cloning of this putative partner. In summary, the activity of E2F appears to be regulated by changes in mRNA levels and through association with other cellular proteins. Cloning of the E2F promoter and analysis of E2F mRNA stability will help to understand how E2F expression is regulated. Determining whether the different E2F/pl07/cyclin/cdk2 complexes are positive or negative regulators of E2F activity requires further experiments. The promoters of two genes that encode putative positive (cyclin A) and negative (Rb) regulators of E2F activity contain E2F sites, suggesting that E2F may regulate the levels of its regulators. It is not yet known if E2F increases its activity by repressing the production of Rb protein and increasing the production of cyclin A, or if E2F represses its activity by increasing the amounts of Rb and decreasing cyclin A levels. V. Is E2F a central regulator of the entrance into S phase?
A regulator of the G1 to S-phase transition may function by activation of genes required for S phase and/or repression of genes that prevent entrance into S phase. Genes whose promoters have E2F sites include those required for specific S-phase functions, such as D H F R and thymidine kinase, and those critical for controlling the entrance into S phase, such as Rb [80], and cyclin A [49,50]. E2F as a central regulator could coordinate the expression of these genes and ensure that products needed for S phase are synthesized concurrently with the decision to enter S phase. We do not know yet whether E2F is the sole activator of transcription at the G1/S-phase boundary or
130 whether E2F regulates only a subset of G 1 / S phaseactivated genes. In yeast, genes involved in DNA synthesis, such as thymidylate synthase and DNA polymerase, are activated at the G1/S-phase boundary by SWI6 and a 120-kDa protein. A second group of genes that are homologous to mammalian cyclins are activated at the same time by SWI6 and SWI4. DNA binding specificity is controlled by SWI4 or the 120-kDa protein, whereas regulation is mediated by SWI6 [81,82]. If other classes of mammalian G1/S-phaseactivated promoters exist that are regulated by proteins different from E2F, perhaps the expression of these classes of genes is kept synchronous by a common activator, such as a cyclin. In conclusion, members of the E2F gene family are important for activation of genes required for and involved in S-phase functions. Ongoing analyses will reveal the roles of E2F-1, E2F-2, and E2F-3 in the regulation of individual growth-regulated genes. It is not yet known if E2F activity is required for entrance into S phase. Cell lines and transgenic animals expressing mutant E2F proteins will aid in understanding how the E2F gene family participates in cell growth control. For example, overexpression of a mutant E2F that does not bind Rb may lead to neoplastic growth. Expression of a mutant E2F which can form an inactive complex with cyclin A may prevent the entrance into S phase and cause growth arrest. If expression of different mutant E2Fs can either enhance or repress cell proliferation, an important regulator of the cell cycle has indeed been identified.
Acknowledgments We thank members of the Farnham laboratory for critical reading of the manuscript and the investigators that allowed us to refer to unpublished data. We apologize to those colleagues whose work we did not cite. This work was supported by Public Health Service grants CA45240, CA07175, and CA23076 from the National Institutes of Health. J.E.S. was supported, in part, by Public Health Service training grant CA09135 and as a Cremer Scholar.
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Biochimica et Biophysica Acta, 1155 (1993) 125-131 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00
BBACAN 87265
The role of E2F in the mammalian cell cycle P e g g y J. F a r n h a m , Jill E. S l a n s k y a n d R i c h a r d K o l l m a r McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI (USA) (Received 10 December 1992)
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
II.
The E2F protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
III. The role of E2F sites in gene activation at the G1/S-phase boundary . . . . . . . . . . . . . . . . . . .
126
IV. Regulation of E2F activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
127
V.
129
Is E2F a central regulator of the entrance into S phase? . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
I. Introduction
The proliferative cell cycle is divided into 4 phases (Fig. 1). During the two gap phases, G1 and G2, the cell accumulates the necessary products for replication of the genome (S phase) and cell division (M phase), respectively. Protein synthesis is required for a cell to pass through a restriction point in G1 phase [1,2] and commit to DNA synthesis. Although the proteins that control the entrance into S phase are unknown, several genes that encode products involved in nucleotide synthesis and DNA replication are activated at the G1/S-phase boundary. The promoters of many of these genes contain binding sites for the transcription factor E2F. Thus, a common control mechanism involving E2F may be responsible for the cell cycle regulation of these genes. E2F has also been implicated in cell cycle control through its association with the retinoblastoma (Rb) tumor suppressor protein and with the cyclins E and A. The purpose of this review is to summarize
Correspondence to: P.J. Farnham, McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Avenue, Madison, WI 53706, USA. Abbreviations: Rb, Retinoblastoma; DHFR, dihydrofolate reductase.
what is known about the role of E2F in gene regulation and to discuss the possibility that E2F is a central regulator of the entrance into S phase. II. The E2F protein
E2F is a cellular DNA-binding protein with an apparent molecular mass of about 60 kDa. It was first identified in HeLa cells as a transcriptional activator of the adenovirus E2 promoter [3,4]. A similar activity from F9 cells has been called DRTF1 [5-7]. E2F binds to DNA with the consensus 5'-TITSSCGC-3' (S = C or G) [8,9]. Variations at the first, second, and last position of the consensus do not abolish E2F binding, but reduce the affinity of the site for E2F. E2F binds not only to DNA, but also to the Rb protein. E 2 F / R b interactions were used to isolate the human E2F-1 cDNA. Several clones were obtained when expression libraries were probed with the region of the Rb protein that binds E2F [10-13]. One clone was human E2F-1 (also called RBP3 [12], RBAP-1 [11], and Apl2 [13]) as defined by its sequence-specific DNA binding and transcriptional activation properties. The DNA-binding domain of E2F-1 (Fig. 2) resembles helix-loop-helix DNA-binding domains and is located near the N-terminus of the protein, adjacent to a
126 basic region. A transactivation region at the C-terminus of the protein overlaps with the region of E2F-1 that is required for binding to the Rb protein. A proline-rich region of unknown function is located N-terminal to the DNA binding domain and two potential phosphorylation sites for cdk kinase are found at amino acids 159 and 346. Cloned E2F-1 binds to and activates transcription from the adenovirus E2 and dihydrofolate reductase (DHFR) promoters [11,12,14]. The mouse DHFR promoter contains two inverted, overlapping E2F sites between - 8 and + 1 which bind cloned E2F-1 [11], and three other similar sites at -266, -244 and -213 which have not been tested in binding assays (Fig. 3A). DHFR promoter constructs containing different numbers of these putative E2F sites were transfected with a human E2F-1 expression construct and assayed in serum-starved cells (Fig. 3B). Decreasing the number of E2F sites in the various constructs resulted in decreased transactivation by E2F-1. A murine E2F-1 cDNA homologous to human E2F-1 (Y. Li and P.J. Farnham, unpublished data; J. Lees and E. Harlow, personal communication) and two other human cDNAs that are similar to E2F-1 in the DNA binding domain (J. Lees and E. Harlow, unpublished data) have also been cloned. In addition, a cDNA has been cloned for an unrelated mouse protein called DP-1 that can also bind specifically to an E2F site [15]. Throughout this review, E2F refers to the uncharacterized proteins that bind to the recognition site, whereas E2F-1, E2F-2, or E2F-3 refers to the cloned E2F cDNAs. IlL The role of E2F sites in gene activation at the G1 / S-phase boundary
The promoters of several genes that are activated in mid or late G1 phase bind cellular proteins at E2F sites. These genes include DHFR, thymidine kinase, DNA polymerase alpha, cdc2, thymidylate synthase, and c-myc (see Ref. 9 for a list of these sites). This observation suggests that E2F may mediate growth regulation of these genes. In this section, we will discuss what is known about the role of the E2F binding sites in the transcriptional regulation of these promoters. DHFR catalyzes the conversion of dihydrofolate to tetrahydrofolate, which is required for the biosynthesis of purines and thymidylate. DHFR mRNA levels increase at the G1/S-phase boundary in mouse, human, and hamster cells [16-20]. Mouse DHFR promoter sequences from -270 to + 20 are sufficient to confer an increase in transcription at the G1/S-phase boundary on a reporter gene in 3T3 cells synchronized by serum starvation. Mutational analysis of the DHFR promoter indicates that sequences spanning the transcription initiation site are required for the transcrip-
tional increase [21]. These sequences consist of two overlapping, inverted binding sites ( - 8 to + 1) for E2F [22] and an initiator element ( - 3 to +5) which is involved in start site recognition [23,24]. The 29 base pairs containing the E2F sites and the DHFR initiator element were cloned upstream of a reporter gene without any other promoter elements. A 35-fold increase in activity of this promoter was observed during S phase [14]. Thus, the E2F sites at the DHFR initiation site are both necessary and sufficient for G1/Sphase regulation. Thymidine kinase catalyzes the phosphorylation of thymidine to thymidylate. The levels of murine, human, and hamster thymidine kinase mRNA increase at the G1/S-phase boundary after mitogen stimulation of quiescent cells [25-31]. In extracts prepared from Sphase cells, protein binds to similar 20-25 bp elements containing consensus E2F sites in the mouse [32] and human [33] promoters. Competition with an E2F site from the E2 promoter suggests that the protein (called Yi2) binding to the mouse thymidine kinase promoter is related to E2F [9,32]. However, a probe that contains an E2F site does not compete for binding of the S-phase protein on the human thymidine kinase element [33]. This could be due to different affinities of these sites, or the DNA-binding protein could be different from, but related to, E2F. Mutations in the E2F site abolish the S-phase-specific binding and result in loss of serum regulation of the human thymidine kinase promoter [33]. No point mutants have been made to determine if the E2F site is required for regulation of the mouse thymidine kinase promoter. DNA polymerase alpha synthesizes the lagging strand of DNA at the replication fork. Levels of DNA polymerase alpha mRNA increase as cells enter S phase [34], and the promoter region of this gene can Late response genes: DHFR TS TK Delayed early response genes: c-myc Early response l genes: c-fos
GO
CAD DNA
POL a CDCZ Cycln A EZF
,,
G1
S
GZ
R
Fig. 1. The cell cycle. Addition of mitogens to quiescent cells initiates a series of events that lead to activation of growth-regulated genes at different times. Some genes are regulated differently in the first cell cycle after quiescent cells are stimulated with mitogens. Therefore, this cycle is shown as distinct from the subsequent (proliferative) cell cycles. Most of the regulation of the late response genes discussed in this review has been analyzed in the first cycle. The restriction point (R) is indicated. The abbreviations for genes are dihydrofolate reductase (DHFR), thymidine kinase (TK), thymidylate synthase (TS), carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (CAD), DNA polymerase alpha (DNA pol a).
127 confer G1/S-phase regulation on a reporter gene [35]. A consensus site that binds E2F is present at -128 [9,35] and sequences similar to the E2F consensus site are present throughout the promoter. One such sequence, GTTGGCGC, is located at + 18 and is present in all constructs tested for growth regulation. A DNA polymerase alpha promoter construct that extends from - 4 0 2 to +45 increases 6-fold in activity at the G1/Sphase boundary; successive 5' deletions gradually reduce the transcriptional increase. These results are similar to those obtained with the 5' deletion constructs of the DHFR promoter (Fig. 3C). Although each construct contains the E2F sites located between - 8 and + 1 that are required for regulation, deletion of the upstream putative E2F sites reduces the maximal increase in transcription at the G1/S-phase boundary. It is possible that multiple E2F sites contribute to the overall regulation of both the DHFR and DNA polymerase alpha promoters. The large S-phasespecific increase in transcription from the synthetic DHFR promoter (composed only of the E2F sites and the initiator element) as compared to the increases observed using longer DHFR promoter constructs could be due to the very low basal activity of the synthetic promoter. Perhaps the E2F sites are not sufficient to confer a large increase in transcription at the G1/S-phase boundary if strong constitutive activators (such as SP1) are present. Alternatively, there may be a repressor site in the longer DHFR promoter that limits the activation seen at the G1/S-phase boundary. Cdc2 is a kinase that associates with various cyclins and is required for progression through the proliferative cell cycle. Cdc2 transcription and mRNA levels increase at the G1/S-phase boundary [36,37]. Deletion of an E2F consensus site in the cdc2 promoter results in equal transcription rates in cycling and serum-starved cells. Thus, E2F also appears to be important in the regulation of the cdc2 gene. Other genes, such as thymidylate synthase [38-42], ribonucleotide reductase [43-45], carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydrooro-
proline-rich region 43 86
I
I
KSP I 120
IV. Regulation of E2F activity If E2F is responsible for activation of genes at the G1/S-phase boundary, then E2F activity must change
SPGK I 307
I I
tase [46-48] (R.J. Miltenberger and P.J. Farnham, unpublished data), and cyclin A [49-53], are also activated at the G1/S-phase boundary. Although less is known about the DNA sequences and transcription factors involved in their regulation, each of these promoters contains at least one putative E2F site. The E2F site in the thymidylate sythase promoter binds a cellular protein which is competed by a consensus E2F site (Y. Li and P.J. Farnham, unpublished data). A 7/8 match with a consensus E2F site is present in a 60base-pair region that is required for growth regulation of the carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase promoter (R.J. Miltenberger, R. Kollmar, and P.J. Farnham, unpublished data). E2F may also regulate the delayed-early c-myc gene [54,55]. A consensus site which binds E2F is located between - 58 and - 65, upstream of the P2 promoter. A 3-fold increase in c-myc transcription 4 h after serum stimulation of quiescent cells is dependent on the E2F site [8]. Perhaps, the 3-fold increase in mid G1 is the beginning of a larger increase that peaks at the G1/Sphase boundary. Alternatively, the E2F elements might mediate a transcriptional increase at different points in the cell cycle by binding to different members of the E2F gene family. In summary, the promoters of several genes that are activated in mid or late G1 phase contain sequences that resemble E2F sites. Mutagenesis of these sequences in the DHFR, thymidine kinase, and cdc2 promoters indicates that protein binding to these sites is important for their growth regulation. The analysis of the role of the E2F sites in the other promoters is not yet complete. However, the available evidence is consistent with the hypothesis that E2F is involved in the regulation of the genes that are activated at the G1/S-phase boundary.
RB-binding r e g i o n 409 426
i I
I
109 128 181 b a s i c r e g i o n HLH r e g i o n DNA-binding domain
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I
I
I
368
437
trans-activation domain
Fig. 2. The human E2F-1 protein. The DNA-binding,trans-activation,and Rb-bindingdomainshave been defined by mutagenesis[11-13]. A proline-rich regionand two putativephosphorylationsites (KSPand SPGK)havebeen foundby sequenceanalysis[13].
128 A -270
-117 -90 IA ~fll' j , ,e,
-233 0
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I
luciferase cDNA
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'
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pu~tive E2Fsites
E2F sites
25
20
15
10
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0
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_
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i
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8
1'0 lr2 1'4 1'6 Hoursalter stimulation
lr8
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24
Fig. 3. (A) The mouse D H F R promoter. The position of transcription factor binding sites is shown and the 5' endpoints of the different promoter deletions are represented by vertical bars. Open box, putative E2F site; filled box, E2F site; oval, Spl site. (B) 3T3 cells were transfected with the D H F R promoter luciferase constructs and a plasmid with or without the human E2F-1 cDNA expressed from the CMV promoter. The cells were incubated in medium containing 0.5% bovine calf serum for 48 h and then harvested for luciferase assays. The fold induction by E2F was calculated for each promoter as the ratio of luciferase activity in the presence of the E2F-1 expression plasmid to that in the presence of the vector control. (C) 3T3 cells were transfected with the D H F R promoter luciferase constructs. The cells were incubated in medium containing 0.5% bovine calf serum for 48 h, followed by incubation in medium containing 10% bovine calf serum. Cells were harvested at the indicated times for luciferase assays to determine promoter activity and for flow cytometry to define the time at which the cells entered S phase. Luciferase values are reported as a ratio of the activity of each promoter at different times in the cell cycle relative to the activity of the same promoter in starved cells,
through the cell cycle. The levels of E2F-1 mRNA do fluctuate through the cell cycle. Little E2F-1 mRNA is present in quiescent 3T3 cells, in resting T cells, or in mouse liver. However, the levels of E2F-1 mRNA increase at the G1/S-phase boundary after serum stimulation of 3T3 cells [14], mitogen stimulation of resting human T cells [11], and partial hepatectomy of C 3 H / H e J mice (M. Bennett, N. Drinkwater, and P.J. Farnham, unpublished data). The levels of E2F-2 and E2F-3 mRNAs and of the different E2F proteins in specific stages of the cell cycle have not yet been examined. Protein binding to E2F sites can be detected in all stages of the cell cycle [8]. This detection of E2F DNA binding activity in GO and G1 cells initially led to the conclusion that the D H F R promoter was not regulated by E2F [21]. However, the low level of E2F-1 mRNA in GO and G1 cells suggests that perhaps E2F-2 or E2F-3 binds to DNA during these stages. Also, a 180-kDa protein from proliferating HeLa cells has been purified on a DNA affinity column containing the E2F sites located between - 8 and + 1 in the D H F R promoter [21]. This protein has not been further characterized because only small amounts are obtained during purification (Y. Li and P.J. Farnham, unpublished data); in particular, it is not known whether it corresponds to E2F-2 or E2F-3. E2F activity may also be regulated by association with different cellular proteins in different stages of the cell cycle. In G1 phase, E2F binds to the Rb protein [56-60]; this association continues through S phase [61]. The existence of the E 2 F / R b complex in S phase is not understood. Rb undergoes cell cycle-dependent phosphorylation, with underphosphorylated Rb present in GO and G1 phase cells and phosphorylated Rb present in S and G 2 / M phase cells [62]. Since E2F has been shown to bind underphosphorylated Rb [11,59], it is not clear if the S-phase E 2 F / R b complex is due to a novel E2F interaction with phosphorylated Rb or if some unphosphorylated Rb exists in S phase. E2F also forms a complex with p107, the kinase cdk2, and cyclin E (in mid- to late-G1) or cyclin A (in S phase) [5,61,63-67]. One study detected an E 2 F / p l 0 7 complex in early G1 [9]. Other combinations of E2F, Rb, p107, cdk2, and various cyclins may be important in different growth states or in different cell types. All complexes appear to dissociate in G 2 / M phase, resulting in free E2F. How might changes in E2F abundance or formation of different E2F protein complexes control transcriptional activation at different stages in the cell cycle? The low levels of E2F-1 mRNA in the first G1 phase after stimulation of resting cells probably cause the low level of transcription of genes such as DHFR. The increase in E2F-1 mRNA at the G1/S-phase boundary would then result in increased transcription of these genes. Thus, E2F-1 activity in the G1 phase immediately following GO is controlled mainly by limiting
129 amounts of E2F-1. In addition, E2F activity in G1 phase may be controlled by interaction with Rb. Transfection of a plasmid expressing Rb protein results in repression of transcription from promoters containing E2F sites [68-70]. This repression could be simply a consequence of the growth-suppressing ability of Rb, and would then be unrelated to E 2 F / R b interactions. However, there is a correlation between the ability of Rb to bind to E2F and to suppress growth, suggesting that the direct interaction of Rb and E2F is critical for growth suppression [71]. E2F-Rb interactions can be disrupted by the E1A protein of adenovirus 2, the large T antigen of simian virus 40, and the E7 protein of papilloma virus [5,11,12,72,73]. In the presence of these viral proteins, E2F sites confer activation on the mouse D H F R [14], hamster D H F R [74], n-myc [74], c-myc [55], and cdc2 [37] promoters. Rb may interfer with E2F activity by masking the activation domain of E2F [11] or the E 2 F / R b complex bound to a promoter might hinder binding of other transcription factors [58,69,75] The function of the different E 2 F / p l 0 7 / c y c l i n / cdk2 complexes in different stages of the cell cycle is open to speculation. One reason to bring the cdk2 kinase to a promoter could be to stimulate transcription by phosphorylation of a factor in the initiation complex. The target of phosphorylation could be E2F itself since the DNA binding activity increases after phosphorylation of a dephosphorylated E2F fraction [76]. Other potential targets in the transcription complex are the CCAAT enhancer factor that binds next to the E2F site in the human TK promoter [33] and the C-terminal domain of RNA polymerase II, which is required for D H F R transcription [77] and is phosphorylated prior to initiation [78]. The presence of different cyclins in the E2F/pl07/cyclin/cdk2 complexes could result from specific interactions between particular members of the E2F family and particular cyclins. The different E2F/cyclin complexes may activate different promoters that are expressed in different stages of the cell cycle. For example, the E2F binding sites in the c-myc and the D H F R promoter are necessary for transcriptional activation in mid G1 phase and at the G1/S-phase boundary, respectively. However, cotransfection of E2F-1 increases transcription from the DHFR, but not from the c-myc, promoter in serumstarved cells (Ref. 14 and J.E. Slansky, unpublished data). Alternatively, other proteins in addition to E2F-1 may be required to activate the c-myc promoter. To date, the D H F R promoter is the only cellular promoter that has been shown to be activated by E2F-1, although proteins bind to E2F sites in the EGFR [8], the D H F R [11,21,22], thymidine kinase [32,33], c-myc [54,55] n-myc [74], E2 [4,79], E1A [79], thymidylate synthase (Y. Li and P. J. Farnham, unpublished data) and Rb (P.D. Robbins, personal communication) promoters. Other
promoters contain consensus E2F sites that have not yet been tested for binding or activation (see above and Ref. 56). Transfection of E2F-1, E2F-2, and E2F-3 with reporter constructs driven by these promoters is required before it is known if the different E2Fs regulate different promoters. Preliminary evidence suggests that E2F-1, like many transcription factors, may function as a heterodimer (D. Heimbrook, personal communication). Affinitypurified E2F from HeLa cells displays multiple protein bands on a denaturing polyacrylamide gel. Little E2F DNA-binding activity is obtained after renaturation of individual bands. However, when the separated proteins are mixed together, a dramatic increase in DNAbinding activity is seen. Western blot analysis shows that E2F-1 is related to some, but not all, of these proteins. The presence of multiple E2Fs and the possibility of different hetero- and homodimer pairs would add another level of complexity to E2F regulation. However, cloned E2F-1 can bind to DNA without a partner [11,12]. Understanding the relevance of potential heterodimer formation to E2F function must await cloning of this putative partner. In summary, the activity of E2F appears to be regulated by changes in mRNA levels and through association with other cellular proteins. Cloning of the E2F promoter and analysis of E2F mRNA stability will help to understand how E2F expression is regulated. Determining whether the different E2F/pl07/cyclin/cdk2 complexes are positive or negative regulators of E2F activity requires further experiments. The promoters of two genes that encode putative positive (cyclin A) and negative (Rb) regulators of E2F activity contain E2F sites, suggesting that E2F may regulate the levels of its regulators. It is not yet known if E2F increases its activity by repressing the production of Rb protein and increasing the production of cyclin A, or if E2F represses its activity by increasing the amounts of Rb and decreasing cyclin A levels. V. Is E2F a central regulator of the entrance into S phase?
A regulator of the G1 to S-phase transition may function by activation of genes required for S phase and/or repression of genes that prevent entrance into S phase. Genes whose promoters have E2F sites include those required for specific S-phase functions, such as D H F R and thymidine kinase, and those critical for controlling the entrance into S phase, such as Rb [80], and cyclin A [49,50]. E2F as a central regulator could coordinate the expression of these genes and ensure that products needed for S phase are synthesized concurrently with the decision to enter S phase. We do not know yet whether E2F is the sole activator of transcription at the G1/S-phase boundary or
130 whether E2F regulates only a subset of G 1 / S phaseactivated genes. In yeast, genes involved in DNA synthesis, such as thymidylate synthase and DNA polymerase, are activated at the G1/S-phase boundary by SWI6 and a 120-kDa protein. A second group of genes that are homologous to mammalian cyclins are activated at the same time by SWI6 and SWI4. DNA binding specificity is controlled by SWI4 or the 120-kDa protein, whereas regulation is mediated by SWI6 [81,82]. If other classes of mammalian G1/S-phaseactivated promoters exist that are regulated by proteins different from E2F, perhaps the expression of these classes of genes is kept synchronous by a common activator, such as a cyclin. In conclusion, members of the E2F gene family are important for activation of genes required for and involved in S-phase functions. Ongoing analyses will reveal the roles of E2F-1, E2F-2, and E2F-3 in the regulation of individual growth-regulated genes. It is not yet known if E2F activity is required for entrance into S phase. Cell lines and transgenic animals expressing mutant E2F proteins will aid in understanding how the E2F gene family participates in cell growth control. For example, overexpression of a mutant E2F that does not bind Rb may lead to neoplastic growth. Expression of a mutant E2F which can form an inactive complex with cyclin A may prevent the entrance into S phase and cause growth arrest. If expression of different mutant E2Fs can either enhance or repress cell proliferation, an important regulator of the cell cycle has indeed been identified.
Acknowledgments We thank members of the Farnham laboratory for critical reading of the manuscript and the investigators that allowed us to refer to unpublished data. We apologize to those colleagues whose work we did not cite. This work was supported by Public Health Service grants CA45240, CA07175, and CA23076 from the National Institutes of Health. J.E.S. was supported, in part, by Public Health Service training grant CA09135 and as a Cremer Scholar.
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