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E2F target genes: unraveling the biology
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TRENDS in Biochemical Sciences
Vol.29 No.8 August 2004
E2F target genes: unraveling the biology Adrian P. Bracken1, Marco Ciro1, Andrea Cocito1 and Kristian Helin1,2 1 2
Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy Biotech Research & Innovation Centre, DK-2100 Copenhagen, Denmark
The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target genes of E2F, leading to the identification of several hundred such genes that are involved not only in DNA replication and cell cycle progression, but also in DNA damage repair, apoptosis, differentiation and development. These new findings greatly enrich our understanding of how E2F controls transcription and cellular homeostasis. When we say ‘E2F’, we really mean the collective term used for at least seven different transcription factors (E2F1–E2F7), six of which require heterodimerization with DP proteins (DP1 and DP2) to be functional (Figure 1). These factors are well studied owing to their importance in both cell cycle progression and cancer [1,2]. E2F was first described as a cellular activity required by adenovirus E1A proteins for transactivating the adenovirus E2 promoter [3]. Subsequently, E2F-responsive sites, similar to those found in the E2 promoter, were identified in the promoters of two cellular genes, MYC [4] and DHFR [5]. The discovery that the tumor suppressor protein pRB interacted with E2F and blocked its transcriptional activity before the G1/S transition established an early model for cell cycle control [6,7]. In this model (Figure 2a), unphosphorylated pRB binds to E2F in G0/G1, leading to the repression of E2F target genes. The subsequent inactivation of pRB by cyclin-dependent kinase (CDK)mediated phosphorylation in late G1 enables E2F to transactivate target promoters, resulting in the synthesis of proteins required for S-phase entry and cell cycle progression. Analogous to this, the binding and inactivation of pRB and its two family members, p107 and p130, by adenovirus E1A or human papilloma virus E7 result in the transactivation of E2F target genes even in the absence of growth factor stimulation (Figure 2b). This model of ‘deregulation’ of E2F activity is known to contribute to tumor development [1]. Several studies have shown that the repressive capacity of the pRB family members is dependent upon the recruitment of chromatin modifiers to E2F-regulated Corresponding author: Kristian Helin ([email protected]). Available online 10 July 2004
promoters [8,9]. The recruitment of these chromatinmodifying enzymes (histone deacetylases, histone methyltransferases and DNA methyltransferases) is thought to result in the ‘active’ repression of promoters, which presumably prevents the binding of other cis-acting proteins required for transactivation. On the basis of this knowledge and studies of the five E2F factors (E2F1–E2F5) that are currently known to bind to the pRB family members, a more elaborate model of how E2F target genes are controlled has been suggested (Figure 3a). Incorporated into this model are the findings that E2F4 and E2F5 are exported from the nucleus in G1 phase, that levels of E2F1–E2F3 increase at the G1/S boundary, and that E2F4 and E2F5 predominantly occupy the E2F-regulated promoters in G0/G1, whereas E2F1–E2F3 are preferentially bound in S phase [10]. In this model, E2F target genes are actively repressed in G0/G1, whereas they are transactivated by E2F1–E2F3 in late G1 and S. Here, we summarize the efforts made to E2F1 1
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Figure 1. The E2F family. The E2F1–E2F6 proteins contain one DNA-binding (DB) domain and a DIM domain for dimerization with DP. Sequences for transcriptional activation (TA) and pocket protein binding (PB) are present only in E2F1–E2F5, which can be divided on the basis of homology into two distinct subgroups: E2F1, E2F2, E2F3a and E2F3b; and E2F4 and E2F5. Moreover, E2F1, E2F2, E2F3a and E2F3b share a cyclin-A-binding domain (cA) that is absent in E2F4 and E2F5. E2F6 diverges considerably from the other E2F family members, with little homology outside the core DB and DIM domains. E2F6 seems to work as a transcriptional repressor by recruiting polycomb group proteins to its target promoters. The two isoforms of E2F7 (E2F7a and E2F7b) diverge further from E2F1–E2F5 and do not contain a DIM domain; instead, they contain two DB domains, both of which are required for binding to the E2F DNA-binding consensus site. DP1 and DP2 are distantly related to the E2F family, sharing homology in the DB and DIM domains.
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date to identify the target genes of the E2F transcription factors. Recent large-scale approaches have greatly expanded the list of E2F target genes revealing several exciting new aspects of E2F biology, the implications of which are discussed.
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Identification of E2F target genes The discovery of its connection with pRB, and therefore cancer, greatly increased efforts aimed towards understanding how E2F controls cell proliferation. When the first E2F family members were identified, they were indeed shown to be capable of transactivating E2F-responsive promoters and to push immortalized quiescent cells into the S phase of the cell cycle [11–13]. These results were satisfying, because they showed that the ectopic expression of key interactors of pRB was sufficient to deregulate cell cycle progression, thereby mimicking loss of pRB. These observations have led many laboratories to search for the transcriptional targets of E2F, the hypothesis being that these genes themselves would be key regulators of cell cycle progression. Initially, E2F target genes were identified by searching for E2F-binding sites in the promoters of genes already known to be induced at the G1/S transition. In this way, E2F was found to regulate
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Figure 2. Classical models that link E2F with cell cycle progression and cancer. (a) The G1/S transition and entry into S phase. The retinoblastoma (pRB) family of proteins, also called the pocket proteins (PP), bind and negatively regulate the transcriptional activity of E2F in G0 and early G1. Upon stimulation by growth factors, complexes of cyclin (Cyc) and cyclin-dependent kinase (CDK) accumulate and phosphorylate (P) pocket proteins, resulting in the release of E2F and the transactivation of target genes. This enables cells to progress into S phase. (b) Virally transformed cells enter S phase even in the absence of growth factors, in part, because the viral proteins bind and block the capacity of the pocket proteins to inhibit E2F transcriptional activity. The pocket proteins are bound by adenovirus E1A proteins, the E7 proteins from human papilloma viruses, and the large T protein from SV40. E1A is shown here as an example.
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Figure 3. Models of the regulation of E2F target genes. (a) Model of the E2F-mediated regulation of genes at the G1/S transition. Genes such as CCNE1 and CDC6 are repressed in G0 and early G1 by complexes of E2F4 or E2F5 and a pocket protein (PP) in association with co-repressors (REP). Phosphorylation (P) of the pocket protein in late G1 results in the displacement of this repressor and the subsequent association of E2F1–E2F3, resulting in the transactivation of E2F target genes at the G1/S transition. (b) Model of the E2F-mediated regulation of genes that accumulate subsequent to G1/S. Genes such as CCNA2 and CDC2 are presumably regulated by additional repressors and/or activators (ACT) because accumulation of their mRNA is delayed with respect to the classical E2F target genes shown in (a). (c) Model of the E2F-mediated regulation of genes in early G1. Genes such as MYC and CCND1 are repressed in G0 by complexes of E2F4 or E2F5 and a pocket protein in association with co-repressors. The mechanisms involved in the derepression and the subsequent transactivation of these genes before the accumulation of cyclin (Cyc)/ cyclin-dependent kinase (CDK) activity are not well understood. www.sciencedirect.com
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several additional genes with functions in cell cycle control (e.g. CCNE1 and CDC25A) and DNA replication (e.g. MCM2-7 and CDC6) [1,2]. In the past three years, large-scale systematic approaches have been used to identify many hundreds of E2F target genes. Such approaches include detecting gene expression changes in E2F-overexpressing cells by using DNA oligonucleotide microarrays [14–18], and chromatin immunoprecipitation (ChIP) assays using E2F-specific antibodies [19–21]. Both of these approaches are advantageous in that they are unbiased and can be done on a large scale. Predictably, they have shown that the expression of many additional DNA replication and cell cycle control genes is regulated by E2F (Table 1). Several unexpected findings have, however, also emerged. For example, many newly identified E2F target genes are not induced in late G1, but accumulate in the early G1 or S/G2 phases of the cell cycle [10,22]. Perhaps most surprisingly, E2F also regulates many genes with functions in processes that are unrelated to cell cycle progression such as DNA damage repair, apoptosis, differentiation and development. Furthermore, many negative regulators of cell growth are induced by E2F and numerous genes are repressed upon its overexpression. How does E2F regulate genes outside G1/S? CCND1 and MYC mRNAs accumulate in early G1 upon the stimulation of quiescent cells to enter the cell cycle, whereas CDC2 and CCNA2 mRNAs accumulate later in S/G2 (Table 1), suggesting that different mechanisms of regulation must exist for these genes. The best-studied example of an E2F target gene that accumulates in S phase is CCNA2 [23]. In non-growing cells, the pocket proteins p107 and p130 form complexes with E2F4 or E2F5 on the CCNA2 promoter, which coincides with gene repression [24]. Upon entry into the cell cycle, accumulation of CCNA2 mRNA is delayed with respect to CDC6 or other ‘classical’ E2F target genes [24]. This late accumulation of CCNA2 mRNA could be due to the delayed release of E2F4 or another co-repressor from the CCNA2 promoter, and the subsequent delayed association of, and transactivation by, E2F1–E2F3 [25]. However, E2F1–E2F3 seem to associate with the CCNA2 promoter at the G1/S transition [24,26]. Assuming that this E2F association is required for CCNA2 transactivation, then its occurrence at the G1/S phase suggests that transcription factors or chromatin remodeling events in addition to E2F1–E2F3 are also required for transactivation (Figure 3b). This suggestion is consistent with the ‘combinatorial control’ model recently proposed by Nevins and colleagues [27] for E2F target gene regulation. In this model, individual E2F-dependent promoters are transactivated by the combination of E2F and additional specific transcription factors. Such a model could explain the delayed upregulation of S/G2 genes such as CCNA2 by E2F (Figure 3b). Less is known about the nature of E2F’s regulation of genes that accumulate in early G1. Recent data have shown that an E2F4/E2F5–p107–SMAD3 protein complex represses the MYC promoter in cells treated with transforming growth factor-b (TGF-b) [28,29]. It would www.sciencedirect.com
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be interesting to know whether this complex is associated with the repressed MYC promoter in quiescent cells and, if so, whether it is subsequently dissociated from the promoter upon serum stimulation. A mechanism can be envisaged in which the cytoplasmic relocalization of SMAD3 is a key event in the derepression of the MYC promoter [30,31]. Transcription factors other than E2F might be also required to transactivate the gene subsequent to derepression (Figure 3c). In summary, E2F family members are required to control the timely activation and repression of numerous genes that are essential for ordered progression through the cell cycle. This control is executed in several phases of the cell cycle and not just at the G1/S transition. Mechanistically, it is likely to be achieved at many levels and involve the action of several E2F members, some of which repress (when associated with pocket proteins and other co-repressors) and others of which transactivate (possibly in combination with additional non-related transcription factors). It is clear, however, that a substantial effort is still required to understand the mechanisms by which E2F exerts its regulation of target genes in different phases of the cell cycle. Can E2F1–E2F3 execute pocket-protein-independent gene repression? Genome-wide expression approaches have pinpointed an unexpectedly large number of genes whose expression is repressed upon overexpression of E2F [16–18,32]. For example, Mu¨ller et al. [16] identified several hundred genes that were repressed upon overexpression of E2F1–E2F3. The regulation of six of these genes (PAI-1, CTGF, EPLIN, TGFB2, BCL3 and INHBA) was subsequently shown to be reduced even in the absence of protein synthesis. This suggests that E2F1–E2F3 can function as direct repressors of transcription independently of pocket proteins. In fact, overexpression of pRB results in an increase in mRNA levels of both PAI-1 and CTGF [16]. It is therefore likely that E2F1–E2F3 negatively regulate the transcription of numerous target genes by mechanisms that remain to be defined. One possibility is that E2F1–E2F3 recruit co-repressors to block transcription (Figure 4a) in a manner similar to the capacity of MYC to repress the expression of some target genes directly [33]. An even more intriguing possibility has emerged from recent observations that suggest that antisense regulation of the human genome is a widespread event. Using a set of computational tools, one study has identified about 1600 antisense RNA transcripts and mapped them to the gene locus of their target mRNA [34]. In addition, Cawley et al. [35] have shown that many transcription factors frequently bind at the start site of antisense mRNA sequences located within or at the end of coding gene loci. It is therefore possible that E2F1–E2F3 bind to the promoters of antisense transcripts and activate their transcription. These antisense transcripts could subsequently bind to the corresponding target mRNA and downregulate its expression in the absence of protein synthesis (Figure 4b). Although these hypotheses await experimental validation, it is already clear that E2F transcription factors very probably
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Table 1. E2F target genesa
exert part of their biological effect via the downregulation of target genes. Does E2F control negative growth regulators to finetune cell cycle progression? Of the newly identified E2F target genes, several encode negative regulators of cell proliferation (Table 1). This www.sciencedirect.com
might seem surprising, because E2F is best known for its role in regulating the transcription of genes that positively affect cell cycle progression. CDKN2C (encoding p18), CDKN2D (p19), CDKN1C (p57), E2F7, RB1 (pRB) and RBL1 (p107) are upregulated by E2F during S phase, yet these genes are known to regulate cell growth negatively when overexpressed [36–39]. Their induction could,
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Table 1. E2F target genesa (continued)
however, function to ensure orderly progression through the cell cycle. For example, the induction of CDK inhibitors (p18, p19 and p57) would be predicted to decrease CDK activity in S phase, which in turn would result in both the firing of fewer replication origins and a decrease in E2F activity during the latter stages of S phase. Recently, the E2F repressor E2F7 has been identified as an E2F target gene that is induced in S phase [38,40]. Notably, E2F7 represses only a subset of E2F target genes, such as CCNE1 and CDC6, and does not repress latertranscribed genes such as CCNA2 and CDC2 [38]. This potentially provides another way in which the E2F transcription factors can control orderly progression through the cell cycle, because the downregulation of CCNE1 and CDC6 expression in late S phase is important for preventing the refiring of origins and genomic instability [41]. www.sciencedirect.com
A role for E2F in the DNA damage response? The discovery that DNA damage results in ATM-mediated phosphorylation of E2F1 and its subsequent stabilization has suggested that E2F might have a role in mediating the DNA damage response [42]. Such a role is supported by the presence of many checkpoint (CHK1, TP53, ATM, BRCA1 and BRCA2) and DNA damage repair (RPA1–RPA3, RAD51, RAD54, MSH2–MSH6 and MLH1) genes among E2F targets (Table 1). But checkpoint and DNA repair proteins have been also proposed to function in non-stressed cells to suppress genomic rearrangements resulting from DNA replication or chromosome segregation errors during the normal cell cycle [43]; therefore, their upregulation could be simply another facet of the role of E2F in regulating normal progression through the cell cycle. This point is well illustrated by the E2F-mediated upregulation of CHK1, which encodes an essential
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Nuclear retention RNAi machinery + gene silencing
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Figure 4. Models of pocket-protein-independent repression by E2F1–E2F3. (a) Direct transcriptional repression of E2F target genes is achieved independently of retinoblastoma protein (pRB) through the recruitment of co-repressors (REP) by E2F1–E2F3, in a manner analogous to how the transactivator MYC represses some target genes by the recruitment of co-repressors to promoters (reviewed in Ref. [33]). (b) E2F1–E2F3 bind within a gene locus and activate the expression of a noncoding antisense transcript (red). Subsequent binding of the antisense transcript to the sense transcript (black) negatively affects gene expression by any of several possible mechanisms, including activation of the RNA interference (RNAi) machinery, nuclear retention of the mRNA, and heterochromatization of the corresponding gene loci (reviewed in Ref. [73]).
component of the DNA damage signaling pathway. In addition to its role in the DNA damage response, CHK1 accumulates in S phase in non-stressed cycling cells and ensures that DNA replication has been successfully completed before entry into M phase [44]. Mechanistically, this function is partly achieved by regulating the levels of CDC25A phosphatase, which is required for activating the CDC2/cyclin B complex and entry into mitosis. Thus, regulation of CHK1 expression might be another way in which E2F facilitates orderly progression through the cell cycle. In Table 1 we have separated the genes encoding DNA damage repair and checkpoint proteins from those encoding replication proteins. There are, however, several examples of functional overlap between these two groups, and thus this separation could be inaccurate or misleading. For example, the RPA1–RPA3 family of replication proteins is also involved in the DNA damage response [45,46], whereas the RFC1–RFC4 and POLE2 DNA damage repair proteins might be essential for replication [47]. This overlap significantly blurs the definition of E2F as a regulator of DNA damage checkpoint and DNA damage repair. What is clear is that E2F regulates the expression of a host of factors that function during S phase in a DNA synthesis or a DNA repair context, or both. It is not clear, however, whether the E2F transcription factors regulate DNA repair and DNA damage genes because these genes have a role in ‘normal’ DNA synthesis or because they are important for the cellular response to DNA damage and are involved in DNA repair.
physiological role for E2F in apoptosis is suggested by the observation that mice deficient in E2f1 have defects in thymocyte apoptosis [50]. Many key apoptotic genes, including APAF1, CASP3, CASP7 and TP73, are induced by E2F1 [16,51–53] (Table 1). Furthermore, several reports have highlighted the importance of TP73 in E2F1-mediated apoptosis [52–54], and ChIP analysis has recently shown that E2F1 is recruited to the TP73 promoter in apoptotic cells [55]. A possible link between the E2F-dependent regulation of apoptosis genes and cell cycle progression has been recently demonstrated [56]. Consistent with their genes being ‘classical’ E2F targets, both caspase-3 and caspase-7 proteins are upregulated at the G1/S transition [56]. Furthermore, E2F1 binds to the CASP7 promoter in nonapoptotic cells [56] and, despite the fact that caspase-3 and caspase-7 accumulate in S phase, they do so in an inactive state because additional posttranslational modifications are required for their activation [57]. It is therefore possible that endogenous E2F1 does not induce apoptosis under normal physiological conditions, but instead might function to ‘prime’ growing cells for apoptotic signals. In a model analogous to that of the E2F-mediated regulation of DNA repair and checkpoint proteins, the transcriptional regulation of apoptotic genes could constitute part of the cell’s capability to maintain an intact genome. In this model, growing cells that have undergone serious DNA damage could activate the already highly expressed, but inactive caspases, independently of E2F1 and subsequently undergo apoptosis.
Is E2F involved in regulating apoptosis, development and differentiation? Many newly identified E2F target genes have been found in diverse functional groups such as apoptosis, development and differentiation (Table 1). Below, we discuss the potential role of E2F in some of these biological processes.
Development Several genes that are essential for normal development are induced by E2F1–E2F3 [16,18], consistent with the observation that E2f1 and E2f3 knockout mice show developmental defects [58]. In mammalian embryogenesis, the correct spatial expression of homeobox (Hox) genes is crucial for proper development. The polycomb group (PcG) and trithorax group proteins provide a transcriptional memory mechanism that functions to ensure maintenance of the correct transcription patterns of the Hox genes, thereby guaranteeing the proper execution of developmental programs [59–61]. Notably, several of these Hox and PcG genes are among the developmental genes upregulated in E2F1–E2F3
Apoptosis As in the case of the DNA damage repair genes, the E2F-mediated upregulation of pro-apoptotic genes might be an aspect of the role of E2F in normal cell cycle progression. Contrary to this hypothesis is the wellestablished fact that overexpression of E2F1 results in the induction of apoptosis [48,49]. In addition, a www.sciencedirect.com
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expression arrays [16,18], suggesting that E2F might be also involved in the transcriptional control of embryonic development. In support of this notion is the finding in Xenopus that xE2F is required both for the correct expression of some Hox genes and for proper development [62]. It is not clear, however, whether the developmental defects observed in E2F-depleted organisms are actually related to specific faults in proliferation. For example, many Hox genes have been shown to have important roles in cellular proliferation [63–65] and the PcG proteins EZH2 and EED are required for normal cell proliferation [66]. Thus, although E2F regulates several genes involved in development, these genes are also required for normal cell proliferation. Development, differentiation and cell proliferation are very closely linked, and E2F family members might be the regulators that connect these three processes. To determine whether E2F transcriptional control is involved directly in the regulation of development, we need to design systems in which development can be discriminated from cell proliferation. Differentiation E2F is also thought to have a role in differentiation; indeed, mouse mutant strains lacking functional E2f4, E2f5, p107, p130 and Rb all show defects in the differentiation of several cell lineages [8]. An obvious explanation for these differentiation defects is that many E2F target genes such as CCNE1 and CCNA2 remain highly expressed in the absence of the E2F repressors and thus prevent exit from the cell cycle. The recent demonstration that the activating E2F members E2F1–E2F3 upregulate the expression of several differentiation factors suggests, however, that they might have a more central role in differentiation (Table 1). For example, the TEAD4 transcription factor is a regulator of muscle-specific genes [67] and its gene is a transcription target of E2F1–E2F3 [16]. Notably, its expression is known to increase during mitogenic stimulation of quiescent fibroblasts and also during myogenic differentiation, suggesting that TEAD4 functions in both cell cycle and differentiation [68]. A possible explanation for this dual upregulation is that cells that have been stimulated to differentiate undergo several additional cell cycles before ultimately stopping and terminally differentiating [69]. This process, which is sometimes referred to as ‘clonal expansion’, has been recently shown in differentiating adipocyte cells to involve the upregulation of E2F target genes such as CCNA2 and the concomitant upregulation of tissue-specific factors [70]. One of these factors, peroxisome proliferator-activated receptor-g (PPARg), is crucial for the control of terminal adipocyte differentiation and is regulated in an E2F-dependent manner. Significantly, the PPARg gene is not expressed with other E2F-responsive genes in cycling cells of other cell types. It is therefore possible that unknown mechanisms function to block the ability of E2F to transactivate the PPARg gene in non-adipocyte cells. Alternatively, E2F might require the combined action of other coactivators to activate the PPARg gene in cycling adipocyte cells. This latter hypothesis expands on the www.sciencedirect.com
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combinatorial model (Figure 3c) discussed above and predicts that E2F could regulate many different target genes in specific tissues types during the process of clonal expansion before terminal differentiation. As such, it will be interesting to determine whether E2F also regulates the expression of other tissue-specific factors essential for the differentiation of various tissues. In conclusion, the identification of differentiation genes as E2F targets indicates that E2F might have a specific role in promoting differentiation.
Perspectives and future directions The E2F transcription factors are crucial for regulating cell cycle progression and tumorigenesis. Therefore, to understand these biological processes, we need to unravel the mechanisms by which these factors exert control. The nature and function of the several hundred E2F target genes identified by genome-wide approaches have already disclosed, and will continue to open up, many directions in the field of E2F biology. Several potential pitfalls in the recently developed genome-wide approaches remain, however, to be overcome. It is not clear whether all of the reported E2F target genes are directly regulated by E2F and, if so, whether this regulation is relevant in vivo for the basic cell processes in which E2F is involved. It is also not clear whether the genomic fragments that have been shown to bind to E2F by ChIP correspond to real E2F targets, and whether E2F binding to these sites is ratelimiting for target gene expression. To resolve these issues, it will be useful to combine ChIP experiments with expression arrays in the same cellular systems. Nevertheless, the genome-wide screens published so far have identified many diverse types of E2F target gene, which provide significant insights into how the E2Fs control cellular homeostasis. Moreover, on the basis of the presence of several E2F target genes involved in tumorigenesis, we anticipate the addition of several novel oncogenes and tumor suppressors to the list of E2F target genes. Several years of research will be required to address some of the issues in this field. For example, the involvement of E2F in processes not linked to the cell cycle needs further investigation. Little is known about the biological functions of E2F6 and E2F7, and additional family members are likely to be identified. The emerging concepts of both combinatorial control and E2F1–E2F3 as repressors still require experimental validation. The E2F story looks set to continue to provide crucial links for understanding cellular homeostasis and the development of cancer.
Acknowledgements We thank Claire Attwooll, Emmanuelle Trinh, Eros Lazzerini Denchi and Luisa Di Stefano for critically reading the manuscript and helpful discussions. This work was supported by grants from the Association for International Cancer Research, Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Italiana per la Ricerca sul Cancro (FIRC), The European Union Fifth Framework Program, The Italian Health Ministry, and the Danish Ministry of Research.
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References 1 Nevins, J.R. (2001) The Rb/E2F pathway and cancer. Hum. Mol. Genet. 10, 699–703 2 Muller, H. and Helin, K. (2000) The E2F transcription factors: key regulators of cell proliferation. Biochim. Biophys. Acta 1470, M1–12 3 Kovesdi, I. et al. (1986) Identification of a cellular transcription factor involved in E1A trans-activation. Cell 45, 219–228 4 Thalmeier, K. et al. (1989) Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter. Genes Dev. 3, 527–536 5 Blake, M.C. and Azizkhan, J.C. (1989) Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo. Mol. Cell. Biol. 9, 4994–5002 6 Goodrich, D.W. et al. (1991) The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell 67, 293–302 7 Chellappan, S.P. et al. (1991) The E2F transcription factor is a cellular target for the RB protein. Cell 65, 1053–1061 8 Trimarchi, J.M. and Lees, J.A. (2002) Sibling rivalry in the E2F family. Nat. Rev. Mol. Cell Biol. 3, 11–20 9 Ferreira, R. et al. (2001) The Rb/chromatin connection and epigenetic control: opinion. Oncogene 20, 3128–3133 10 Cam, H. and Dynlacht, B.D. (2003) Emerging roles for E2F: beyond the G1/S transition and DNA replication. Cancer Cell 3, 311–316 11 Johnson, D.G. et al. (1993) Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365, 349–352 12 Lukas, J. et al. (1996) Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression. Mol. Cell. Biol. 16, 1047–1057 13 Qin, X.Q. et al. (1994) Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc. Natl. Acad. Sci. U. S. A. 91, 10918–10922 14 Polager, S. et al. (2002) E2Fs up-regulate expression of genes involved in DNA replication, DNA repair and mitosis. Oncogene 21, 437–446 15 Stanelle, J. et al. (2002) Gene expression changes in response to E2F1 activation. Nucleic Acids Res. 30, 1859–1867 16 Mu¨ller, H. et al. (2001) E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev. 15, 267–285 17 Ishida, S. et al. (2001) Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis. Mol. Cell. Biol. 21, 4684–4699 18 Young, A.P. et al. (2003) Mechanisms of transcriptional regulation by Rb–E2F segregate by biological pathway. Oncogene 22, 7209–7217 19 Weinmann, A.S. et al. (2001) Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol. Cell. Biol. 21, 6820–6832 20 Ren, B. et al. (2002) E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints. Genes Dev. 16, 245–256 21 Weinmann, A.S. et al. (2002) Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis. Genes Dev. 16, 235–244 22 Stevaux, O. and Dyson, N.J. (2002) A revised picture of the E2F transcriptional network and RB function. Curr. Opin. Cell Biol. 14, 684–691 23 Schulze, A. et al. (1995) Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site. Proc. Natl. Acad. Sci. U. S. A. 92, 11264–11268 24 Takahashi, Y. et al. (2000) Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression. Genes Dev. 14, 804–816 25 Ghosh, M.K. and Harter, M.L. (2003) A viral mechanism for remodeling chromatin structure in G0 cells. Mol. Cell 12, 255–260 26 Rayman, J.B. et al. (2002) E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/ mSin3B corepressor complex. Genes Dev. 16, 933–947 27 Schlisio, S. et al. (2002) Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function. EMBO J. 21, 5775–5786 28 Chen, C.R. et al. (2002) E2F4/5 and p107 as Smad cofactors linking the TGFb receptor to c-myc repression. Cell 110, 19–32 29 Kowalik, T.F. (2002) Smad about E2F. TGFb repression of c-Myc via a Smad3/E2F/p107 complex. Mol. Cell 10, 7–8 www.sciencedirect.com
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30 Reguly, T. and Wrana, J.L. (2003) In or out? The dynamics of Smad nucleocytoplasmic shuttling. Trends Cell Biol. 13, 216–220 31 Shi, Y. and Massague, J. (2003) Mechanisms of TGF-b signaling from cell membrane to the nucleus. Cell 113, 685–700 32 Vernell, R. et al. (2003) Identification of target genes of the p16INK4A– pRB–E2F pathway. J. Biol. Chem. 278, 46124–46137 33 Wanzel, M. et al. (2003) Transcriptional repression by Myc. Trends Cell Biol. 13, 146–150 34 Yelin, R. et al. (2003) Widespread occurrence of antisense transcription in the human genome. Nat. Biotechnol. 21, 379–386 35 Cawley, S. et al. (2004) Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116, 499–509 36 Ortega, S. et al. (2002) Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim. Biophys. Acta 1602, 73–87 37 Sherr, C.J. and Roberts, J.M. (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501–1512 38 Di Stefano, L. et al. (2003) E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes. EMBO J. 22, 6289–6298 39 Stott, F.J. et al. (1998) The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 17, 5001–5014 40 de Bruin, A. et al. (2003) Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation. J. Biol. Chem. 278, 42041–42049 41 Spruck, C.H. et al. (1999) Deregulated cyclin E induces chromosome instability. Nature 401, 297–300 42 Lin, W.C. et al. (2001) Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation. Genes Dev. 15, 1833–1844 43 Myung, K. et al. (2001) Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell 104, 397–408 44 Bartek, J. and Lukas, J. (2003) Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3, 421–429 45 Zou, L. and Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548 46 Iftode, C. et al. (1999) Replication protein A (RPA): the eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol. 34, 141–180 47 Ellison, V. and Stillman, B. (2003) Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5 0 recessed DNA. PLoS Biol. 1, E33 48 Ginsberg, D. (2002) E2F1 pathways to apoptosis. FEBS Lett. 529, 122–125 49 Matsumura, I. et al. (2003) E2F1 and c-Myc in cell growth and death. Cell Cycle 2, 333–338 50 Field, S.J. et al. (1996) E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85, 549–561 51 Moroni, M.C. et al. (2001) Apaf-1 is a transcriptional target for E2F and p53. Nat. Cell Biol. 3, 552–558 52 Irwin, M. et al. (2000) Role for the p53 homologue p73 in E2F-1induced apoptosis. Nature 407, 645–648 53 Stiewe, T. and Putzer, B.M. (2000) Role of the p53-homologue p73 in E2F1-induced apoptosis. Nat. Genet. 26, 464–469 54 Rodicker, F. et al. (2001) Therapeutic efficacy of E2F1 in pancreatic cancer correlates with TP73 induction. Cancer Res. 61, 7052–7055 55 Pediconi, N. et al. (2003) Differential regulation of E2F1 apoptotic target genes in response to DNA damage. Nat. Cell Biol. 5, 552–558 56 Nahle, Z. et al. (2002) Direct coupling of the cell cycle and cell death machinery by E2F. Nat. Cell Biol. 4, 859–864 57 Boatright, K.M. and Salvesen, G.S. (2003) Mechanisms of caspase activation. Curr. Opin. Cell Biol. 15, 725–731 58 Cloud, J.E. et al. (2002) Mutant mouse models reveal the relative roles of E2F1 and E2F3 in vivo. Mol. Cell. Biol. 22, 2663–2672 59 van Lohuizen, M. (1998) Functional analysis of mouse Polycomb group genes. Cell. Mol. Life Sci. 54, 71–79 60 Orlando, V. (2003) Polycomb, epigenomes, and control of cell identity. Cell 112, 599–606 61 Deschamps, J. et al. (1999) Initiation, establishment and maintenance of Hox gene expression patterns in the mouse. Int. J. Dev. Biol. 43, 635–650
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62 Suzuki, A. and Hemmati-Brivanlou, A. (2000) Xenopus embryonic E2F is required for the formation of ventral and posterior cell fates during early embryogenesis. Mol. Cell 5, 217–229 63 Chen, H. and Sukumar, S. (2003) HOX genes: emerging stars in cancer. Cancer Biol. Ther. 2, 524–525 64 van Oostveen, J. et al. (1999) The role of homeobox genes in normal hematopoiesis and hematological malignancies. Leukemia 13, 1675–1690 65 Cillo, C. et al. (2001) Homeobox genes in normal and malignant cells. J. Cell. Physiol. 188, 161–169 66 Bracken, A.P. et al. (2003) EZH2 is downstream of the pRB–E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 22, 5323–5335 67 Jacquemin, P. et al. (1996) A novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS
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DNA binding domain. J. Biol. Chem. 271, 21775–21785 68 Hsu, D.K. et al. (1996) Identification of a murine TEF-1-related gene expressed after mitogenic stimulation of quiescent fibroblasts and during myogenic differentiation. J. Biol. Chem. 271, 13786–13795 69 Brown, G. et al. (2003) Cell differentiation and proliferation – simultaneous but independent? Exp. Cell Res. 291, 282–288 70 Fajas, L. et al. (2002) E2Fs regulate adipocyte differentiation. Dev. Cell 3, 39–49 71 Iyer, V.R. et al. (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283, 83–87 72 Whitfield, M.L. et al. (2002) Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell 13, 1977–2000 73 Carmichael, G.G. (2003) Antisense starts making more sense. Nat. Biotechnol. 21, 371–372
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Vol.29 No.8 August 2004
E2F target genes: unraveling the biology Adrian P. Bracken1, Marco Ciro1, Andrea Cocito1 and Kristian Helin1,2 1 2
Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy Biotech Research & Innovation Centre, DK-2100 Copenhagen, Denmark
The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target genes of E2F, leading to the identification of several hundred such genes that are involved not only in DNA replication and cell cycle progression, but also in DNA damage repair, apoptosis, differentiation and development. These new findings greatly enrich our understanding of how E2F controls transcription and cellular homeostasis. When we say ‘E2F’, we really mean the collective term used for at least seven different transcription factors (E2F1–E2F7), six of which require heterodimerization with DP proteins (DP1 and DP2) to be functional (Figure 1). These factors are well studied owing to their importance in both cell cycle progression and cancer [1,2]. E2F was first described as a cellular activity required by adenovirus E1A proteins for transactivating the adenovirus E2 promoter [3]. Subsequently, E2F-responsive sites, similar to those found in the E2 promoter, were identified in the promoters of two cellular genes, MYC [4] and DHFR [5]. The discovery that the tumor suppressor protein pRB interacted with E2F and blocked its transcriptional activity before the G1/S transition established an early model for cell cycle control [6,7]. In this model (Figure 2a), unphosphorylated pRB binds to E2F in G0/G1, leading to the repression of E2F target genes. The subsequent inactivation of pRB by cyclin-dependent kinase (CDK)mediated phosphorylation in late G1 enables E2F to transactivate target promoters, resulting in the synthesis of proteins required for S-phase entry and cell cycle progression. Analogous to this, the binding and inactivation of pRB and its two family members, p107 and p130, by adenovirus E1A or human papilloma virus E7 result in the transactivation of E2F target genes even in the absence of growth factor stimulation (Figure 2b). This model of ‘deregulation’ of E2F activity is known to contribute to tumor development [1]. Several studies have shown that the repressive capacity of the pRB family members is dependent upon the recruitment of chromatin modifiers to E2F-regulated Corresponding author: Kristian Helin ([email protected]). Available online 10 July 2004
promoters [8,9]. The recruitment of these chromatinmodifying enzymes (histone deacetylases, histone methyltransferases and DNA methyltransferases) is thought to result in the ‘active’ repression of promoters, which presumably prevents the binding of other cis-acting proteins required for transactivation. On the basis of this knowledge and studies of the five E2F factors (E2F1–E2F5) that are currently known to bind to the pRB family members, a more elaborate model of how E2F target genes are controlled has been suggested (Figure 3a). Incorporated into this model are the findings that E2F4 and E2F5 are exported from the nucleus in G1 phase, that levels of E2F1–E2F3 increase at the G1/S boundary, and that E2F4 and E2F5 predominantly occupy the E2F-regulated promoters in G0/G1, whereas E2F1–E2F3 are preferentially bound in S phase [10]. In this model, E2F target genes are actively repressed in G0/G1, whereas they are transactivated by E2F1–E2F3 in late G1 and S. Here, we summarize the efforts made to E2F1 1
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Figure 1. The E2F family. The E2F1–E2F6 proteins contain one DNA-binding (DB) domain and a DIM domain for dimerization with DP. Sequences for transcriptional activation (TA) and pocket protein binding (PB) are present only in E2F1–E2F5, which can be divided on the basis of homology into two distinct subgroups: E2F1, E2F2, E2F3a and E2F3b; and E2F4 and E2F5. Moreover, E2F1, E2F2, E2F3a and E2F3b share a cyclin-A-binding domain (cA) that is absent in E2F4 and E2F5. E2F6 diverges considerably from the other E2F family members, with little homology outside the core DB and DIM domains. E2F6 seems to work as a transcriptional repressor by recruiting polycomb group proteins to its target promoters. The two isoforms of E2F7 (E2F7a and E2F7b) diverge further from E2F1–E2F5 and do not contain a DIM domain; instead, they contain two DB domains, both of which are required for binding to the E2F DNA-binding consensus site. DP1 and DP2 are distantly related to the E2F family, sharing homology in the DB and DIM domains.
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date to identify the target genes of the E2F transcription factors. Recent large-scale approaches have greatly expanded the list of E2F target genes revealing several exciting new aspects of E2F biology, the implications of which are discussed.
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Identification of E2F target genes The discovery of its connection with pRB, and therefore cancer, greatly increased efforts aimed towards understanding how E2F controls cell proliferation. When the first E2F family members were identified, they were indeed shown to be capable of transactivating E2F-responsive promoters and to push immortalized quiescent cells into the S phase of the cell cycle [11–13]. These results were satisfying, because they showed that the ectopic expression of key interactors of pRB was sufficient to deregulate cell cycle progression, thereby mimicking loss of pRB. These observations have led many laboratories to search for the transcriptional targets of E2F, the hypothesis being that these genes themselves would be key regulators of cell cycle progression. Initially, E2F target genes were identified by searching for E2F-binding sites in the promoters of genes already known to be induced at the G1/S transition. In this way, E2F was found to regulate
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Figure 2. Classical models that link E2F with cell cycle progression and cancer. (a) The G1/S transition and entry into S phase. The retinoblastoma (pRB) family of proteins, also called the pocket proteins (PP), bind and negatively regulate the transcriptional activity of E2F in G0 and early G1. Upon stimulation by growth factors, complexes of cyclin (Cyc) and cyclin-dependent kinase (CDK) accumulate and phosphorylate (P) pocket proteins, resulting in the release of E2F and the transactivation of target genes. This enables cells to progress into S phase. (b) Virally transformed cells enter S phase even in the absence of growth factors, in part, because the viral proteins bind and block the capacity of the pocket proteins to inhibit E2F transcriptional activity. The pocket proteins are bound by adenovirus E1A proteins, the E7 proteins from human papilloma viruses, and the large T protein from SV40. E1A is shown here as an example.
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Figure 3. Models of the regulation of E2F target genes. (a) Model of the E2F-mediated regulation of genes at the G1/S transition. Genes such as CCNE1 and CDC6 are repressed in G0 and early G1 by complexes of E2F4 or E2F5 and a pocket protein (PP) in association with co-repressors (REP). Phosphorylation (P) of the pocket protein in late G1 results in the displacement of this repressor and the subsequent association of E2F1–E2F3, resulting in the transactivation of E2F target genes at the G1/S transition. (b) Model of the E2F-mediated regulation of genes that accumulate subsequent to G1/S. Genes such as CCNA2 and CDC2 are presumably regulated by additional repressors and/or activators (ACT) because accumulation of their mRNA is delayed with respect to the classical E2F target genes shown in (a). (c) Model of the E2F-mediated regulation of genes in early G1. Genes such as MYC and CCND1 are repressed in G0 by complexes of E2F4 or E2F5 and a pocket protein in association with co-repressors. The mechanisms involved in the derepression and the subsequent transactivation of these genes before the accumulation of cyclin (Cyc)/ cyclin-dependent kinase (CDK) activity are not well understood. www.sciencedirect.com
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several additional genes with functions in cell cycle control (e.g. CCNE1 and CDC25A) and DNA replication (e.g. MCM2-7 and CDC6) [1,2]. In the past three years, large-scale systematic approaches have been used to identify many hundreds of E2F target genes. Such approaches include detecting gene expression changes in E2F-overexpressing cells by using DNA oligonucleotide microarrays [14–18], and chromatin immunoprecipitation (ChIP) assays using E2F-specific antibodies [19–21]. Both of these approaches are advantageous in that they are unbiased and can be done on a large scale. Predictably, they have shown that the expression of many additional DNA replication and cell cycle control genes is regulated by E2F (Table 1). Several unexpected findings have, however, also emerged. For example, many newly identified E2F target genes are not induced in late G1, but accumulate in the early G1 or S/G2 phases of the cell cycle [10,22]. Perhaps most surprisingly, E2F also regulates many genes with functions in processes that are unrelated to cell cycle progression such as DNA damage repair, apoptosis, differentiation and development. Furthermore, many negative regulators of cell growth are induced by E2F and numerous genes are repressed upon its overexpression. How does E2F regulate genes outside G1/S? CCND1 and MYC mRNAs accumulate in early G1 upon the stimulation of quiescent cells to enter the cell cycle, whereas CDC2 and CCNA2 mRNAs accumulate later in S/G2 (Table 1), suggesting that different mechanisms of regulation must exist for these genes. The best-studied example of an E2F target gene that accumulates in S phase is CCNA2 [23]. In non-growing cells, the pocket proteins p107 and p130 form complexes with E2F4 or E2F5 on the CCNA2 promoter, which coincides with gene repression [24]. Upon entry into the cell cycle, accumulation of CCNA2 mRNA is delayed with respect to CDC6 or other ‘classical’ E2F target genes [24]. This late accumulation of CCNA2 mRNA could be due to the delayed release of E2F4 or another co-repressor from the CCNA2 promoter, and the subsequent delayed association of, and transactivation by, E2F1–E2F3 [25]. However, E2F1–E2F3 seem to associate with the CCNA2 promoter at the G1/S transition [24,26]. Assuming that this E2F association is required for CCNA2 transactivation, then its occurrence at the G1/S phase suggests that transcription factors or chromatin remodeling events in addition to E2F1–E2F3 are also required for transactivation (Figure 3b). This suggestion is consistent with the ‘combinatorial control’ model recently proposed by Nevins and colleagues [27] for E2F target gene regulation. In this model, individual E2F-dependent promoters are transactivated by the combination of E2F and additional specific transcription factors. Such a model could explain the delayed upregulation of S/G2 genes such as CCNA2 by E2F (Figure 3b). Less is known about the nature of E2F’s regulation of genes that accumulate in early G1. Recent data have shown that an E2F4/E2F5–p107–SMAD3 protein complex represses the MYC promoter in cells treated with transforming growth factor-b (TGF-b) [28,29]. It would www.sciencedirect.com
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be interesting to know whether this complex is associated with the repressed MYC promoter in quiescent cells and, if so, whether it is subsequently dissociated from the promoter upon serum stimulation. A mechanism can be envisaged in which the cytoplasmic relocalization of SMAD3 is a key event in the derepression of the MYC promoter [30,31]. Transcription factors other than E2F might be also required to transactivate the gene subsequent to derepression (Figure 3c). In summary, E2F family members are required to control the timely activation and repression of numerous genes that are essential for ordered progression through the cell cycle. This control is executed in several phases of the cell cycle and not just at the G1/S transition. Mechanistically, it is likely to be achieved at many levels and involve the action of several E2F members, some of which repress (when associated with pocket proteins and other co-repressors) and others of which transactivate (possibly in combination with additional non-related transcription factors). It is clear, however, that a substantial effort is still required to understand the mechanisms by which E2F exerts its regulation of target genes in different phases of the cell cycle. Can E2F1–E2F3 execute pocket-protein-independent gene repression? Genome-wide expression approaches have pinpointed an unexpectedly large number of genes whose expression is repressed upon overexpression of E2F [16–18,32]. For example, Mu¨ller et al. [16] identified several hundred genes that were repressed upon overexpression of E2F1–E2F3. The regulation of six of these genes (PAI-1, CTGF, EPLIN, TGFB2, BCL3 and INHBA) was subsequently shown to be reduced even in the absence of protein synthesis. This suggests that E2F1–E2F3 can function as direct repressors of transcription independently of pocket proteins. In fact, overexpression of pRB results in an increase in mRNA levels of both PAI-1 and CTGF [16]. It is therefore likely that E2F1–E2F3 negatively regulate the transcription of numerous target genes by mechanisms that remain to be defined. One possibility is that E2F1–E2F3 recruit co-repressors to block transcription (Figure 4a) in a manner similar to the capacity of MYC to repress the expression of some target genes directly [33]. An even more intriguing possibility has emerged from recent observations that suggest that antisense regulation of the human genome is a widespread event. Using a set of computational tools, one study has identified about 1600 antisense RNA transcripts and mapped them to the gene locus of their target mRNA [34]. In addition, Cawley et al. [35] have shown that many transcription factors frequently bind at the start site of antisense mRNA sequences located within or at the end of coding gene loci. It is therefore possible that E2F1–E2F3 bind to the promoters of antisense transcripts and activate their transcription. These antisense transcripts could subsequently bind to the corresponding target mRNA and downregulate its expression in the absence of protein synthesis (Figure 4b). Although these hypotheses await experimental validation, it is already clear that E2F transcription factors very probably
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Table 1. E2F target genesa
exert part of their biological effect via the downregulation of target genes. Does E2F control negative growth regulators to finetune cell cycle progression? Of the newly identified E2F target genes, several encode negative regulators of cell proliferation (Table 1). This www.sciencedirect.com
might seem surprising, because E2F is best known for its role in regulating the transcription of genes that positively affect cell cycle progression. CDKN2C (encoding p18), CDKN2D (p19), CDKN1C (p57), E2F7, RB1 (pRB) and RBL1 (p107) are upregulated by E2F during S phase, yet these genes are known to regulate cell growth negatively when overexpressed [36–39]. Their induction could,
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Table 1. E2F target genesa (continued)
however, function to ensure orderly progression through the cell cycle. For example, the induction of CDK inhibitors (p18, p19 and p57) would be predicted to decrease CDK activity in S phase, which in turn would result in both the firing of fewer replication origins and a decrease in E2F activity during the latter stages of S phase. Recently, the E2F repressor E2F7 has been identified as an E2F target gene that is induced in S phase [38,40]. Notably, E2F7 represses only a subset of E2F target genes, such as CCNE1 and CDC6, and does not repress latertranscribed genes such as CCNA2 and CDC2 [38]. This potentially provides another way in which the E2F transcription factors can control orderly progression through the cell cycle, because the downregulation of CCNE1 and CDC6 expression in late S phase is important for preventing the refiring of origins and genomic instability [41]. www.sciencedirect.com
A role for E2F in the DNA damage response? The discovery that DNA damage results in ATM-mediated phosphorylation of E2F1 and its subsequent stabilization has suggested that E2F might have a role in mediating the DNA damage response [42]. Such a role is supported by the presence of many checkpoint (CHK1, TP53, ATM, BRCA1 and BRCA2) and DNA damage repair (RPA1–RPA3, RAD51, RAD54, MSH2–MSH6 and MLH1) genes among E2F targets (Table 1). But checkpoint and DNA repair proteins have been also proposed to function in non-stressed cells to suppress genomic rearrangements resulting from DNA replication or chromosome segregation errors during the normal cell cycle [43]; therefore, their upregulation could be simply another facet of the role of E2F in regulating normal progression through the cell cycle. This point is well illustrated by the E2F-mediated upregulation of CHK1, which encodes an essential
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Figure 4. Models of pocket-protein-independent repression by E2F1–E2F3. (a) Direct transcriptional repression of E2F target genes is achieved independently of retinoblastoma protein (pRB) through the recruitment of co-repressors (REP) by E2F1–E2F3, in a manner analogous to how the transactivator MYC represses some target genes by the recruitment of co-repressors to promoters (reviewed in Ref. [33]). (b) E2F1–E2F3 bind within a gene locus and activate the expression of a noncoding antisense transcript (red). Subsequent binding of the antisense transcript to the sense transcript (black) negatively affects gene expression by any of several possible mechanisms, including activation of the RNA interference (RNAi) machinery, nuclear retention of the mRNA, and heterochromatization of the corresponding gene loci (reviewed in Ref. [73]).
component of the DNA damage signaling pathway. In addition to its role in the DNA damage response, CHK1 accumulates in S phase in non-stressed cycling cells and ensures that DNA replication has been successfully completed before entry into M phase [44]. Mechanistically, this function is partly achieved by regulating the levels of CDC25A phosphatase, which is required for activating the CDC2/cyclin B complex and entry into mitosis. Thus, regulation of CHK1 expression might be another way in which E2F facilitates orderly progression through the cell cycle. In Table 1 we have separated the genes encoding DNA damage repair and checkpoint proteins from those encoding replication proteins. There are, however, several examples of functional overlap between these two groups, and thus this separation could be inaccurate or misleading. For example, the RPA1–RPA3 family of replication proteins is also involved in the DNA damage response [45,46], whereas the RFC1–RFC4 and POLE2 DNA damage repair proteins might be essential for replication [47]. This overlap significantly blurs the definition of E2F as a regulator of DNA damage checkpoint and DNA damage repair. What is clear is that E2F regulates the expression of a host of factors that function during S phase in a DNA synthesis or a DNA repair context, or both. It is not clear, however, whether the E2F transcription factors regulate DNA repair and DNA damage genes because these genes have a role in ‘normal’ DNA synthesis or because they are important for the cellular response to DNA damage and are involved in DNA repair.
physiological role for E2F in apoptosis is suggested by the observation that mice deficient in E2f1 have defects in thymocyte apoptosis [50]. Many key apoptotic genes, including APAF1, CASP3, CASP7 and TP73, are induced by E2F1 [16,51–53] (Table 1). Furthermore, several reports have highlighted the importance of TP73 in E2F1-mediated apoptosis [52–54], and ChIP analysis has recently shown that E2F1 is recruited to the TP73 promoter in apoptotic cells [55]. A possible link between the E2F-dependent regulation of apoptosis genes and cell cycle progression has been recently demonstrated [56]. Consistent with their genes being ‘classical’ E2F targets, both caspase-3 and caspase-7 proteins are upregulated at the G1/S transition [56]. Furthermore, E2F1 binds to the CASP7 promoter in nonapoptotic cells [56] and, despite the fact that caspase-3 and caspase-7 accumulate in S phase, they do so in an inactive state because additional posttranslational modifications are required for their activation [57]. It is therefore possible that endogenous E2F1 does not induce apoptosis under normal physiological conditions, but instead might function to ‘prime’ growing cells for apoptotic signals. In a model analogous to that of the E2F-mediated regulation of DNA repair and checkpoint proteins, the transcriptional regulation of apoptotic genes could constitute part of the cell’s capability to maintain an intact genome. In this model, growing cells that have undergone serious DNA damage could activate the already highly expressed, but inactive caspases, independently of E2F1 and subsequently undergo apoptosis.
Is E2F involved in regulating apoptosis, development and differentiation? Many newly identified E2F target genes have been found in diverse functional groups such as apoptosis, development and differentiation (Table 1). Below, we discuss the potential role of E2F in some of these biological processes.
Development Several genes that are essential for normal development are induced by E2F1–E2F3 [16,18], consistent with the observation that E2f1 and E2f3 knockout mice show developmental defects [58]. In mammalian embryogenesis, the correct spatial expression of homeobox (Hox) genes is crucial for proper development. The polycomb group (PcG) and trithorax group proteins provide a transcriptional memory mechanism that functions to ensure maintenance of the correct transcription patterns of the Hox genes, thereby guaranteeing the proper execution of developmental programs [59–61]. Notably, several of these Hox and PcG genes are among the developmental genes upregulated in E2F1–E2F3
Apoptosis As in the case of the DNA damage repair genes, the E2F-mediated upregulation of pro-apoptotic genes might be an aspect of the role of E2F in normal cell cycle progression. Contrary to this hypothesis is the wellestablished fact that overexpression of E2F1 results in the induction of apoptosis [48,49]. In addition, a www.sciencedirect.com
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expression arrays [16,18], suggesting that E2F might be also involved in the transcriptional control of embryonic development. In support of this notion is the finding in Xenopus that xE2F is required both for the correct expression of some Hox genes and for proper development [62]. It is not clear, however, whether the developmental defects observed in E2F-depleted organisms are actually related to specific faults in proliferation. For example, many Hox genes have been shown to have important roles in cellular proliferation [63–65] and the PcG proteins EZH2 and EED are required for normal cell proliferation [66]. Thus, although E2F regulates several genes involved in development, these genes are also required for normal cell proliferation. Development, differentiation and cell proliferation are very closely linked, and E2F family members might be the regulators that connect these three processes. To determine whether E2F transcriptional control is involved directly in the regulation of development, we need to design systems in which development can be discriminated from cell proliferation. Differentiation E2F is also thought to have a role in differentiation; indeed, mouse mutant strains lacking functional E2f4, E2f5, p107, p130 and Rb all show defects in the differentiation of several cell lineages [8]. An obvious explanation for these differentiation defects is that many E2F target genes such as CCNE1 and CCNA2 remain highly expressed in the absence of the E2F repressors and thus prevent exit from the cell cycle. The recent demonstration that the activating E2F members E2F1–E2F3 upregulate the expression of several differentiation factors suggests, however, that they might have a more central role in differentiation (Table 1). For example, the TEAD4 transcription factor is a regulator of muscle-specific genes [67] and its gene is a transcription target of E2F1–E2F3 [16]. Notably, its expression is known to increase during mitogenic stimulation of quiescent fibroblasts and also during myogenic differentiation, suggesting that TEAD4 functions in both cell cycle and differentiation [68]. A possible explanation for this dual upregulation is that cells that have been stimulated to differentiate undergo several additional cell cycles before ultimately stopping and terminally differentiating [69]. This process, which is sometimes referred to as ‘clonal expansion’, has been recently shown in differentiating adipocyte cells to involve the upregulation of E2F target genes such as CCNA2 and the concomitant upregulation of tissue-specific factors [70]. One of these factors, peroxisome proliferator-activated receptor-g (PPARg), is crucial for the control of terminal adipocyte differentiation and is regulated in an E2F-dependent manner. Significantly, the PPARg gene is not expressed with other E2F-responsive genes in cycling cells of other cell types. It is therefore possible that unknown mechanisms function to block the ability of E2F to transactivate the PPARg gene in non-adipocyte cells. Alternatively, E2F might require the combined action of other coactivators to activate the PPARg gene in cycling adipocyte cells. This latter hypothesis expands on the www.sciencedirect.com
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combinatorial model (Figure 3c) discussed above and predicts that E2F could regulate many different target genes in specific tissues types during the process of clonal expansion before terminal differentiation. As such, it will be interesting to determine whether E2F also regulates the expression of other tissue-specific factors essential for the differentiation of various tissues. In conclusion, the identification of differentiation genes as E2F targets indicates that E2F might have a specific role in promoting differentiation.
Perspectives and future directions The E2F transcription factors are crucial for regulating cell cycle progression and tumorigenesis. Therefore, to understand these biological processes, we need to unravel the mechanisms by which these factors exert control. The nature and function of the several hundred E2F target genes identified by genome-wide approaches have already disclosed, and will continue to open up, many directions in the field of E2F biology. Several potential pitfalls in the recently developed genome-wide approaches remain, however, to be overcome. It is not clear whether all of the reported E2F target genes are directly regulated by E2F and, if so, whether this regulation is relevant in vivo for the basic cell processes in which E2F is involved. It is also not clear whether the genomic fragments that have been shown to bind to E2F by ChIP correspond to real E2F targets, and whether E2F binding to these sites is ratelimiting for target gene expression. To resolve these issues, it will be useful to combine ChIP experiments with expression arrays in the same cellular systems. Nevertheless, the genome-wide screens published so far have identified many diverse types of E2F target gene, which provide significant insights into how the E2Fs control cellular homeostasis. Moreover, on the basis of the presence of several E2F target genes involved in tumorigenesis, we anticipate the addition of several novel oncogenes and tumor suppressors to the list of E2F target genes. Several years of research will be required to address some of the issues in this field. For example, the involvement of E2F in processes not linked to the cell cycle needs further investigation. Little is known about the biological functions of E2F6 and E2F7, and additional family members are likely to be identified. The emerging concepts of both combinatorial control and E2F1–E2F3 as repressors still require experimental validation. The E2F story looks set to continue to provide crucial links for understanding cellular homeostasis and the development of cancer.
Acknowledgements We thank Claire Attwooll, Emmanuelle Trinh, Eros Lazzerini Denchi and Luisa Di Stefano for critically reading the manuscript and helpful discussions. This work was supported by grants from the Association for International Cancer Research, Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Italiana per la Ricerca sul Cancro (FIRC), The European Union Fifth Framework Program, The Italian Health Ministry, and the Danish Ministry of Research.
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