e2f target genes: unraveling the...

E2F target genes:unraveling the biologyAdrian P. Bracken1, Marco Ciro1, Andrea Cocito1 and Kristian Helin1,2

1Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy2Biotech Research & Innovation Centre, DK-2100 Copenhagen, Denmark

DB

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DB DIM 385

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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

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 termused for at least seven different transcription factors(E2F1–E2F7), six of which require heterodimerizationwith DP proteins (DP1 and DP2) to be functional(Figure 1). These factors are well studied owing to theirimportance in both cell cycle progression and cancer [1,2].E2F was first described as a cellular activity required byadenovirus E1A proteins for transactivating the adeno-virus E2 promoter [3]. Subsequently, E2F-responsivesites, similar to those found in the E2 promoter, wereidentified in the promoters of two cellular genes, MYC [4]and DHFR [5].

The discovery that the tumor suppressor protein pRBinteracted with E2F and blocked its transcriptionalactivity before the G1/S transition established an earlymodel for cell cycle control [6,7]. In this model (Figure 2a),unphosphorylated pRB binds to E2F in G0/G1, leading tothe repression of E2F target genes. The subsequentinactivation of pRB by cyclin-dependent kinase (CDK)-mediated phosphorylation in late G1 enables E2F totransactivate target promoters, resulting in the synthesisof proteins required for S-phase entry and cell cycleprogression. Analogous to this, the binding and inacti-vation of pRB and its two family members, p107 and p130,by adenovirus E1A or human papilloma virus E7 result inthe transactivation of E2F target genes even in theabsence of growth factor stimulation (Figure 2b). Thismodel of ‘deregulation’ of E2F activity is known tocontribute to tumor development [1].

Several studies have shown that the repressivecapacity of the pRB family members is dependent uponthe recruitment of chromatin modifiers to E2F-regulated

Corresponding author: Kristian Helin ([email protected]).Available online 10 July 2004

www.sciencedirect.com 0968-0004/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved

promoters [8,9]. The recruitment of these chromatin-modifying enzymes (histone deacetylases, histone methyl-transferases and DNA methyltransferases) is thought toresult in the ‘active’ repression of promoters, whichpresumably prevents the binding of other cis-actingproteins required for transactivation. On the basis ofthis knowledge and studies of the five E2F factors(E2F1–E2F5) that are currently known to bind to thepRB family members, a more elaborate model of howE2F target genes are controlled has been suggested(Figure 3a). Incorporated into this model are the findingsthat E2F4 and E2F5 are exported from the nucleus in G1phase, that levels of E2F1–E2F3 increase at the G1/Sboundary, and that E2F4 and E2F5 predominantlyoccupy the E2F-regulated promoters in G0/G1, whereasE2F1–E2F3 are preferentially bound in S phase [10]. Inthis model, E2F target genes are actively repressed inG0/G1, whereas they are transactivated by E2F1–E2F3 inlate G1 and S. Here, we summarize the efforts made to

Review TRENDS in Biochemical Sciences Vol.29 No.8 August 2004

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.

. doi:10.1016/j.tibs.2004.06.006

http://www.sciencedirect.com

Ti BS

G1/S transition

Adenovirus infectionE1A or E7

E2F

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E2F

PCyc CDKGrowth factors

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PP

PP

PP

E1A E1A

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.

E2F1-3

E2F1-3

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Cyc CDK

E2F4-5

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G0 G1

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REPREP

Figure 3. Models of the regulation of E2F target genes. (a) Model of the E2F-mediated regu

in G0 and early G1 by complexes of E2F4 or E2F5 and a pocket protein (PP) in association

in the displacement of this repressor and the subsequent association of E2F1–E2F3, resul

E2F-mediated regulation of genes that accumulate subsequent to G1/S. Genes such as CC

(ACT) because accumulation of their mRNA is delayed with respect to the classical E2F ta

G1. Genes such as MYC and CCND1 are repressed in G0 by complexes of E2F4 or E2F5 and

derepression and the subsequent transactivation of these genes before the accumulatio


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date to identify the target genes of the E2F transcriptionfactors. Recent large-scale approaches have greatlyexpanded the list of E2F target genes revealing severalexciting new aspects of E2F biology, the implications ofwhich are discussed.

Identification of E2F target genes

The discovery of its connection with pRB, andtherefore cancer, greatly increased efforts aimed towardsunderstanding how E2F controls cell proliferation. Whenthe first E2F family members were identified, theywere indeed shown to be capable of transactivatingE2F-responsive promoters and to push immortalizedquiescent cells into the S phase of the cell cycle [11–13].These results were satisfying, because they showed thatthe ectopic expression of key interactors of pRB wassufficient to deregulate cell cycle progression, therebymimicking loss of pRB.

These observations have led many laboratories tosearch for the transcriptional targets of E2F, the hypoth-esis being that these genes themselves would be keyregulators of cell cycle progression. Initially, E2F targetgenes were identified by searching for E2F-binding sites inthe promoters of genes already known to be induced at theG1/S transition. In this way, E2F was found to regulate

Ti BS

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E2F1-3

E2F4-5

E2F4-5

2F4-5

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P

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ACT

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lation of genes at the G1/S transition. Genes such as CCNE1 and CDC6 are repressed

with co-repressors (REP). Phosphorylation (P) of the pocket protein in late G1 results

ting in the transactivation of E2F target genes at the G1/S transition. (b) Model of the

NA2 and CDC2 are presumably regulated by additional repressors and/or activators

rget genes shown in (a). (c) Model of the E2F-mediated regulation of genes in early

a pocket protein in association with co-repressors. The mechanisms involved in the

n of cyclin (Cyc)/ cyclin-dependent kinase (CDK) activity are not well understood.


Review TRENDS in Biochemical Sciences Vol.29 No.8 August 2004 411

several additional genes with functions in cell cyclecontrol (e.g. CCNE1 and CDC25A) and DNA replication(e.g. MCM2-7 and CDC6) [1,2].

In the past three years, large-scale systematicapproaches have been used to identify many hundreds ofE2F target genes. Such approaches include detecting geneexpression changes in E2F-overexpressing cells by usingDNA oligonucleotide microarrays [14–18], and chromatinimmunoprecipitation (ChIP) assays using E2F-specificantibodies [19–21]. Both of these approaches are advan-tageous in that they are unbiased and can be done on alarge scale. Predictably, they have shown that theexpression of many additional DNA replication and cellcycle control genes is regulated by E2F (Table 1).

Several unexpected findings have, however, alsoemerged. For example, many newly identified E2Ftarget genes are not induced in late G1, but accumulatein the early G1 or S/G2 phases of the cell cycle [10,22].Perhaps most surprisingly, E2F also regulates manygenes with functions in processes that are unrelatedto cell cycle progression such as DNA damage repair,apoptosis, differentiation and development. Furthermore,many negative regulators of cell growth are inducedby E2F and numerous genes are repressed upon itsoverexpression.

How does E2F regulate genes outside G1/S?

CCND1 and MYC mRNAs accumulate in early G1 uponthe stimulation of quiescent cells to enter the cell cycle,whereas CDC2 and CCNA2 mRNAs accumulate later inS/G2 (Table 1), suggesting that different mechanisms ofregulation must exist for these genes. The best-studiedexample of an E2F target gene that accumulates in Sphase is CCNA2 [23]. In non-growing cells, the pocketproteins p107 and p130 form complexes with E2F4 orE2F5 on the CCNA2 promoter, which coincides with generepression [24]. Upon entry into the cell cycle, accumu-lation of CCNA2 mRNA is delayed with respect to CDC6 orother ‘classical’ E2F target genes [24]. This late accumu-lation of CCNA2 mRNA could be due to the delayed releaseof E2F4 or another co-repressor from the CCNA2 promoter,and the subsequent delayed association of, and transactiva-tion by, E2F1–E2F3 [25]. However, E2F1–E2F3 seem toassociate with the CCNA2 promoter at the G1/S transition[24,26]. Assuming that this E2F association is required forCCNA2 transactivation, then its occurrence at the G1/Sphase suggests that transcription factors or chromatinremodeling events in addition to E2F1–E2F3 are alsorequired for transactivation (Figure 3b). This suggestion isconsistent with the ‘combinatorial control’ model recentlyproposed by Nevins and colleagues [27] for E2F target generegulation. In this model, individual E2F-dependent pro-moters are transactivated by the combination of E2F andadditional specific transcription factors. Such a model couldexplain the delayed upregulation of S/G2 genes such asCCNA2 by E2F (Figure 3b).

Less is known about the nature of E2F’s regulation ofgenes that accumulate in early G1. Recent data haveshown that an E2F4/E2F5–p107–SMAD3 protein complexrepresses the MYC promoter in cells treated withtransforming growth factor-b (TGF-b) [28,29]. It would


be interesting to know whether this complex is associatedwith the repressed MYC promoter in quiescent cells and, ifso, whether it is subsequently dissociated from thepromoter upon serum stimulation. A mechanism can beenvisaged in which the cytoplasmic relocalization ofSMAD3 is a key event in the derepression of the MYCpromoter [30,31]. Transcription factors other than E2Fmight be also required to transactivate the gene subse-quent to derepression (Figure 3c).

In summary, E2F family members are required tocontrol the timely activation and repression of numerousgenes that are essential for ordered progression throughthe cell cycle. This control is executed in several phases ofthe cell cycle and not just at the G1/S transition.Mechanistically, it is likely to be achieved at many levelsand involve the action of several E2F members, some ofwhich repress (when associated with pocket proteins andother co-repressors) and others of which transactivate(possibly in combination with additional non-relatedtranscription factors). It is clear, however, that a sub-stantial effort is still required to understand the mechan-isms by which E2F exerts its regulation of target genes indifferent phases of the cell cycle.

Can E2F1–E2F3 execute pocket-protein-independent

gene repression?

Genome-wide expression approaches have pinpointed anunexpectedly large number of genes whose expression isrepressed upon overexpression of E2F [16–18,32]. Forexample, Muller et al. [16] identified several hundredgenes that were repressed upon overexpression ofE2F1–E2F3. The regulation of six of these genes (PAI-1,CTGF, EPLIN, TGFB2, BCL3 and INHBA) was subse-quently shown to be reduced even in the absence of proteinsynthesis. This suggests that E2F1–E2F3 can function asdirect repressors of transcription independently of pocketproteins. In fact, overexpression of pRB results in anincrease in mRNA levels of both PAI-1 and CTGF [16]. It istherefore likely that E2F1–E2F3 negatively regulate thetranscription of numerous target genes by mechanismsthat remain to be defined.

One possibility is that E2F1–E2F3 recruit co-repres-sors to block transcription (Figure 4a) in a manner similarto the capacity of MYC to repress the expression of sometarget genes directly [33]. An even more intriguingpossibility has emerged from recent observations thatsuggest that antisense regulation of the human genome isa widespread event. Using a set of computational tools,one study has identified about 1600 antisense RNAtranscripts and mapped them to the gene locus of theirtarget mRNA [34]. In addition, Cawley et al. [35] haveshown that many transcription factors frequently bind atthe start site of antisense mRNA sequences located withinor at the end of coding gene loci. It is therefore possiblethat E2F1–E2F3 bind to the promoters of antisensetranscripts and activate their transcription. These anti-sense transcripts could subsequently bind to the corre-sponding target mRNA and downregulate its expressionin the absence of protein synthesis (Figure 4b). Althoughthese hypotheses await experimental validation, it isalready clear that E2F transcription factors very probably


Table 1. E2F target genesa


exert part of their biological effect via the downregulationof target genes.

Does E2F control negative growth regulators to fine-

tune cell cycle progression?

Of the newly identified E2F target genes, several encodenegative regulators of cell proliferation (Table 1). This


might seem surprising, because E2F is best known for itsrole in regulating the transcription of genes that positivelyaffect cell cycle progression. CDKN2C (encoding p18),CDKN2D (p19), CDKN1C (p57), E2F7, RB1 (pRB) andRBL1 (p107) are upregulated by E2F during S phase, yetthese genes are known to regulate cell growth negativelywhen overexpressed [36–39]. Their induction could,


Table 1. E2F target genesa (continued)


however, function to ensure orderly progression throughthe cell cycle. For example, the induction of CDKinhibitors (p18, p19 and p57) would be predicted todecrease CDK activity in S phase, which in turn wouldresult in both the firing of fewer replication origins anda decrease in E2F activity during the latter stages ofS phase.

Recently, the E2F repressor E2F7 has been identified asan 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 later-transcribed genes such as CCNA2 and CDC2 [38]. Thispotentially provides another way in which the E2Ftranscription factors can control orderly progressionthrough the cell cycle, because the downregulation ofCCNE1 and CDC6 expression in late S phase is importantfor preventing the refiring of origins and genomicinstability [41].


A role for E2F in the DNA damage response?

The discovery that DNA damage results in ATM-mediatedphosphorylation of E2F1 and its subsequent stabiliza-tion has suggested that E2F might have a role inmediating the DNA damage response [42]. Such a role issupported 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 andDNA repair proteins have been also proposed to functionin non-stressed cells to suppress genomic rearrangementsresulting from DNA replication or chromosome segre-gation errors during the normal cell cycle [43]; therefore,their upregulation could be simply another facet of therole of E2F in regulating normal progression through thecell cycle.

This point is well illustrated by the E2F-mediatedupregulation of CHK1, which encodes an essential


Ti BS

Start site Start site

Nuclear retention

RNAi machinery+

gene silencingE2F1-3 REP

(a) (b)

E2F1-3

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. Inaddition to its role in the DNA damage response, CHK1accumulates in S phase in non-stressed cycling cells andensures that DNA replication has been successfullycompleted before entry into M phase [44]. Mechanistically,this function is partly achieved by regulating the levels ofCDC25A phosphatase, which is required for activating theCDC2/cyclin B complex and entry into mitosis. Thus,regulation of CHK1 expression might be another way inwhich E2F facilitates orderly progression through the cellcycle.

In Table 1 we have separated the genes encoding DNAdamage repair and checkpoint proteins from those encod-ing replication proteins. There are, however, severalexamples of functional overlap between these two groups,and thus this separation could be inaccurate or mislead-ing. For example, the RPA1–RPA3 family of replicationproteins is also involved in the DNA damage response[45,46], whereas the RFC1–RFC4 and POLE2 DNAdamage repair proteins might be essential for replication[47]. This overlap significantly blurs the definition of E2Fas a regulator of DNA damage checkpoint and DNAdamage repair. What is clear is that E2F regulates theexpression of a host of factors that function during S phasein a DNA synthesis or a DNA repair context, or both. It isnot clear, however, whether the E2F transcription factorsregulate DNA repair and DNA damage genes becausethese genes have a role in ‘normal’ DNA synthesis orbecause they are important for the cellular response toDNA damage and are involved in DNA repair.

Is E2F involved in regulating apoptosis, development

and differentiation?

Many newly identified E2F target genes have been foundin diverse functional groups such as apoptosis, develop-ment and differentiation (Table 1). Below, we discuss thepotential role of E2F in some of these biological processes.

Apoptosis

As in the case of the DNA damage repair genes, theE2F-mediated upregulation of pro-apoptotic genes mightbe an aspect of the role of E2F in normal cell cycleprogression. Contrary to this hypothesis is the well-established fact that overexpression of E2F1 results inthe induction of apoptosis [48,49]. In addition, a


physiological role for E2F in apoptosis is suggested bythe observation that mice deficient in E2f1 have defects inthymocyte apoptosis [50]. Many key apoptotic genes,including APAF1, CASP3, CASP7 and TP73, are inducedby E2F1 [16,51–53] (Table 1). Furthermore, severalreports have highlighted the importance of TP73 inE2F1-mediated apoptosis [52–54], and ChIP analysis hasrecently shown that E2F1 is recruited to the TP73promoter in apoptotic cells [55].

A possible link between the E2F-dependent regulationof apoptosis genes and cell cycle progression has beenrecently demonstrated [56]. Consistent with their genesbeing ‘classical’ E2F targets, both caspase-3 and caspase-7proteins are upregulated at the G1/S transition [56].Furthermore, E2F1 binds to the CASP7 promoter in non-apoptotic cells [56] and, despite the fact that caspase-3 andcaspase-7 accumulate in S phase, they do so in an inactivestate because additional posttranslational modificationsare required for their activation [57]. It is thereforepossible that endogenous E2F1 does not induce apoptosisunder normal physiological conditions, but instead mightfunction to ‘prime’ growing cells for apoptotic signals.

In a model analogous to that of the E2F-mediatedregulation of DNA repair and checkpoint proteins, thetranscriptional regulation of apoptotic genes could con-stitute part of the cell’s capability to maintain an intactgenome. In this model, growing cells that have undergoneserious DNA damage could activate the already highlyexpressed, but inactive caspases, independently of E2F1and subsequently undergo apoptosis.

Development

Several genes that are essential for normal developmentare induced by E2F1–E2F3 [16,18], consistent with theobservation that E2f1 and E2f3 knockout mice showdevelopmental defects [58]. In mammalian embryo-genesis, the correct spatial expression of homeobox (Hox)genes is crucial for proper development. The polycombgroup (PcG) and trithorax group proteins provide atranscriptional memory mechanism that functions toensure maintenance of the correct transcription patternsof the Hox genes, thereby guaranteeing the properexecution of developmental programs [59–61]. Notably,several of these Hox and PcG genes are among thedevelopmental genes upregulated in E2F1–E2F3



expression arrays [16,18], suggesting that E2F might bealso involved in the transcriptional control of embryonicdevelopment. In support of this notion is the findingin Xenopus that xE2F is required both for thecorrect expression of some Hox genes and for properdevelopment [62].

It is not clear, however, whether the developmentaldefects observed in E2F-depleted organisms are actuallyrelated to specific faults in proliferation. For example,many Hox genes have been shown to have important rolesin cellular proliferation [63–65] and the PcG proteinsEZH2 and EED are required for normal cell proliferation[66]. Thus, although E2F regulates several genes involvedin development, these genes are also required for normalcell proliferation. Development, differentiation and cellproliferation are very closely linked, and E2F familymembers might be the regulators that connect these threeprocesses. To determine whether E2F transcriptionalcontrol is involved directly in the regulation of develop-ment, we need to design systems in which developmentcan 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 thedifferentiation of several cell lineages [8]. An obviousexplanation for these differentiation defects is that manyE2F target genes such as CCNE1 and CCNA2 remainhighly expressed in the absence of the E2F repressors andthus prevent exit from the cell cycle. The recent demon-stration that the activating E2F members E2F1–E2F3upregulate the expression of several differentiationfactors suggests, however, that they might have a morecentral role in differentiation (Table 1).

For example, the TEAD4 transcription factor is aregulator of muscle-specific genes [67] and its gene is atranscription target of E2F1–E2F3 [16]. Notably, itsexpression is known to increase during mitogenic stimu-lation of quiescent fibroblasts and also during myogenicdifferentiation, suggesting that TEAD4 functions in bothcell cycle and differentiation [68]. A possible explanationfor this dual upregulation is that cells that have beenstimulated to differentiate undergo several additional cellcycles before ultimately stopping and terminally differ-entiating [69]. This process, which is sometimes referredto as ‘clonal expansion’, has been recently shown indifferentiating adipocyte cells to involve the upregulationof E2F target genes such as CCNA2 and the concomitantupregulation of tissue-specific factors [70]. One of thesefactors, peroxisome proliferator-activated receptor-g(PPARg), is crucial for the control of terminal adipocytedifferentiation and is regulated in an E2F-dependentmanner. Significantly, the PPARg gene is not expressedwith other E2F-responsive genes in cycling cells of othercell types. It is therefore possible that unknown mechan-isms function to block the ability of E2F to transactivatethe PPARg gene in non-adipocyte cells.

Alternatively, E2F might require the combined actionof other coactivators to activate the PPARg gene in cyclingadipocyte cells. This latter hypothesis expands on the


combinatorial model (Figure 3c) discussed above andpredicts that E2F could regulate many different targetgenes in specific tissues types during the process of clonalexpansion before terminal differentiation. As such, it willbe interesting to determine whether E2F also regulatesthe expression of other tissue-specific factors essential forthe differentiation of various tissues. In conclusion, theidentification of differentiation genes as E2F targetsindicates that E2F might have a specific role in promotingdifferentiation.

Perspectives and future directions

The E2F transcription factors are crucial for regulatingcell cycle progression and tumorigenesis. Therefore, tounderstand these biological processes, we need to unravelthe mechanisms by which these factors exert control. Thenature and function of the several hundred E2F targetgenes identified by genome-wide approaches have alreadydisclosed, and will continue to open up, many directions inthe field of E2F biology. Several potential pitfalls in therecently developed genome-wide approaches remain,however, to be overcome. It is not clear whether all ofthe reported E2F target genes are directly regulated byE2F and, if so, whether this regulation is relevant in vivofor the basic cell processes in which E2F is involved. It isalso not clear whether the genomic fragments that havebeen shown to bind to E2F by ChIP correspond to real E2Ftargets, and whether E2F binding to these sites is rate-limiting for target gene expression. To resolve theseissues, it will be useful to combine ChIP experimentswith expression arrays in the same cellular systems.Nevertheless, the genome-wide screens published so farhave identified many diverse types of E2F target gene,which provide significant insights into how the E2Fscontrol cellular homeostasis. Moreover, on the basis of thepresence of several E2F target genes involved in tumori-genesis, we anticipate the addition of several noveloncogenes and tumor suppressors to the list of E2F targetgenes.

Several years of research will be required to addresssome of the issues in this field. For example, theinvolvement of E2F in processes not linked to the cellcycle needs further investigation. Little is known aboutthe biological functions of E2F6 and E2F7, and additionalfamily members are likely to be identified. The emergingconcepts of both combinatorial control and E2F1–E2F3 asrepressors still require experimental validation. The E2Fstory looks set to continue to provide crucial links forunderstanding cellular homeostasis and the developmentof cancer.

Acknowledgements

We thank Claire Attwooll, Emmanuelle Trinh, Eros Lazzerini Denchi andLuisa Di Stefano for critically reading the manuscript and helpfuldiscussions. This work was supported by grants from the Association forInternational Cancer Research, Associazione Italiana per la Ricerca sulCancro (AIRC), Fondazione Italiana per la Ricerca sul Cancro (FIRC), TheEuropean Union Fifth Framework Program, The Italian Health Ministry,and the Danish Ministry of Research.



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