network calisthenics

©2011 Landes Bioscience.Do not distribute.

Cell Cycle 10:18, 3086-3094; September 15, 2011; © 2011 Landes Bioscience

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3086 Cell Cycle volume 10 issue 18

Key words: E2F, dynamics, feedback, feedforward, network, DNA replication

Abbreviations: pre-RC, pre-replication complex; ORI, origin of replication; E2F, E2-factor; RB, retinoblastoma; miRNA, micro RNA; I1-FFL, incoherent feedforward loop

Submitted: 07/14/11

Revised: 07/25/11

Accepted: 07/25/11

DOI: 10.4161/cc.10.18.17350

*Correspondence to: Jeffrey V. Wong and Lingchong You; Email: [email protected] and [email protected]

Stimulation of quiescent mamma-lian cells with mitogens induces an

abrupt increase in E2F1–3 expression just prior to the onset of DNA synthe-sis, followed by a rapid decline as repli-cation ceases. This temporal adaptation in E2F facilitates a transient pattern of gene expression that reflects the ordered nature of DNA replication. The chal-lenge to understand how E2F dynamics coordinate molecular events required for high-fidelity DNA replication has great biological implications. Indeed, preco-cious, prolonged, elevated or reduced accumulation of E2F can generate rep-lication stress that culminates in either arrest or death. Accordingly, temporal characteristics of E2F are regulated by several network modules that include feedforward and autoregulatory loops. In this review, we discuss how these net-work modules contribute to “shaping” E2F dynamics in the context of mamma-lian cell cycle entry.

Introduction

In response to mitogenic growth stimu-lation, quiescent mammalian cells can re-enter the cell cycle. Successful division requires faithful and complete duplica-tion of genomic DNA within a narrow time frame of minutes to hours. To deal with the speed and fidelity demanded of this process, eukaryotes have evolved a parallel processing strategy: replication is asynchronously initiated from a subset of several thousand genomic locations called “origins of replication” (ORI). An

Network calisthenicsControl of E2F dynamics in cell cycle entry

Jeffrey V. Wong,1,2,* Peng Dong,3 Joseph R. Nevins,2,4 Bernard Mathey-Prevot5 and Lingchong You1,2,6,*1Department of Biomedical Engineering; 2Institute for Genome Sciences and Policy; 3Computational Biology and Bioinformatics Program; 4Department of Molecular Genetics and Microbiology; 5Deparment of Pharmacology & Cancer Biology; 6Duke Center for Systems Biology;

Duke University; Durham, NC USA

organizing principle of this process is temporal ordering (Fig. 1A): helicase and accessory proteins forming the pre-repli-cation complex (pre-RC) are synthesized, ORI are “licensed” by binding to pre-RC; replication initiation of licensed ORI is triggered by phosphorylation of the pre-RC components, and licenses are removed through a combination of phosphoryla-tion-dependent degradation, inhibition and re-localization of pre-RC machin-ery.1,2 Temporal coordination ensures that DNA is faithfully duplicated “once and only once” during each cell cycle. Deregulation of this process commonly results in replication stress, i.e., aberrant re-initiation and DNA breakage resulting from uncoordinated progression of repli-cation forks,3 which has been speculated to generate the genomic instability that underlies malignant transformation.4

E2F transcription factors play an inte-gral role in coordination of DNA replica-tion events. The first member, E2-factor 1 (E2F1), was identified through its physical association with the retinoblastoma (RB) tumor suppressor,5,6 which acts to seques-ter E2F1.7 Growth factor stimulation induces RB phosphorylation, permitting release and activation of E2F1 activity.8 E2F1, in association with DP1, behaves as a sequence-specific transcriptional activa-tor of cellular genes, including those asso-ciated with growth and proliferation (e.g., c-Myc,9,10 Dihydrofolate reductase, c-Myb and Epidermal growth factor receptor8). This is consistent with observations that ectopic E2F1 stimulated DNA synthesis in quiescent cells.11-13 This early evidence


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classification implies opposing roles by the two groups, it is increasingly clear that the activities of E2Fs are context-dependent.16,17 Regardless, the functions of various E2Fs in both normal18 and pathological circumstances19 have been

also genes at G2/M that encode mitotic

activities.In the last two decades, eight E2F

family members have been identified14,15 and divided into “activators” (E2F1–3) and “repressors” (E2F4–8). While this

supports the view of RB-E2F as a link between growth signals and cell cycle gene expression. Recent genome-scale measures of gene expression further revealed a role for E2F in activating not only genes at G

1/S

that encode DNA replication proteins, but

Figure 1. temporal correspondence between DNa replication and e2F. (a) (top) Overview of successive temporal events in DNa replication. Gene products regulated by e2Fs are shown in blue. the licensed pre-replication complex (pre-rC) contains CYCLiN e, OrC, CDC6, CDt1 and MCM situated at origins of replication (Ori). initiation involves MYC and CYCLiN a:CDK-mediated activation of pre-rC helicase activity. Delicensing occurs through in-hibition of CDt1 by protein sequestration by GeMiNiN along with SKP2 and PCNa-mediated ubiquitination. a temporal delay between CYCLiN e- and CYCLiN a-associated CDK activity is mediated by aPC/CCDH1. aPC/C, anaphase-promoting complex/cyclosome with CDH1; GeM, GeMiNiN; OrC, ori-gin recognition complex; CYC, CYCLiN complexed with cyclin-dependent kinase (CDK); MCM, minichromsome maintenance proteins 2–7; Pol, DNa polymerase. (Bottom) typical temporal pattern for e2F activators (e2F1–3) as cells re-enter the cell cycle from quiescence (G0) following growth factor stimulation. the temporal dynamics summarized in this review are indicated: (1) Delayed e2F increase relative to immediate early genes; (2) switching OFF to ON; (3) amplitude modulation and (4) switching ON to OFF. (B) (Left) Genes induced by e2F1–3 and their associated network modules. (right) Overview of network logic involving modules of the rB-e2F network. iN, upstream signals originating from growth factor signaling and MYC.



in stability mediated by ERK and PI3K, respectively.42-44 Though MYC is required for transcription of E2f1–3, the rise in E2f is delayed, occurring in concert with the second peak of MYC.25 This lag likely reflects the time required to remove complexes that otherwise silence E2F expression.

A critical aspect in E2F biology is nega-tive regulation: E2F activators and many downstream target genes are repressed in quiescence but de-repressed during cell cycle entry (Fig. 2A). E2F1–3 are seques-tered by several “pocket” proteins: retino-blastoma (RB), p107 and p130.45,46 RB is constitutively expressed, functioning as a bona fide tumor suppressor that is often disrupted during the genesis of many types of human cancers.47 In contrast, p107 and p130 are dominant in cycling and quiescent cells, respectively, and nei-ther is altered in cancers despite their ability to compensate for aspects of RB function.48 Extensive work has shown that phosphorylation mediated by CDKs is a primary means to alleviate pocket protein inhibition of E2F activators.49-51

E2F transcription is also negatively regulated by pocket proteins. In qui-escent fibroblasts, a protein complex of p130:E2F4/5 maintains low tran-scription of E2f1 as well as other E2F-regulated cell cycle genes (e.g., Cdc

6,

Myb and Cyclin A)52 through direct bind-ing to upstream regulatory sequences. Expression silencing is achieved in part through E2F4/5:p130-mediated recruit-ment of histone deacetylases (HDAC) that maintain a non-permissive chromatin state.53 Germline deletion of both p107 and p130 expressed higher basal levels of E2F and E2F-regulated targets and were constitutively acetylated,54 confirming the notion that continual HDAC activity is required to maintain low expression from these loci.

In the presence of growth factors, p130 levels decrease sharply between 6–10 h, coincident with the increase in E2F acti-vator mRNA levels.55 Decreased p130 is dependent upon CDK

4,6 phosphorylation,

which signals SCFSKP2-mediated ubiqui-tination, reducing the p130 half-life to ~1 h.56,57 In the absence of p130, which nor-mally tethers E2F4/5 in the nucleus, E2F4 is found predominantly in the cytoplasm,

temporal pattern of expression induces double-strand breaks resulting from re-replication, followed by a p53-mediated checkpoint activation.29 Second, E2F is part of an intricate regulatory cascade that activates Cyclin A/Skp2, but in a delayed manner relative to Cyclin E. This differ-ential temporal control presumably pro-vides a “window of opportunity” between ORI licensing and initiation/delicensing, respectively.30-32 Third, persistent levels of E2F1 are unable to drive DNA synthe-sis to completion in quiescent fibroblasts but, rather, trigger a p53-mediated DNA damage checkpoint.33 Fourth, deletion of E2f1–3 in mice did not prevent cell cycling consistent with the existence of pathways parallel to RB-E2F;34,35 however, it did result in DNA damage attributed to replicative stress.17 Indeed, while E2F tar-gets retain their overall expression pattern in the absence of E2f1–3, their dynamics are altered and unable to reach the same peak levels.36 Fifth, decoupling of E2F from control mechanisms that leads to either precocious37 or prolonged38 activity triggers DNA stress and a p53-mediated checkpoint. This is reminiscent of the impact of tumor-related disruptions of the RB-E2F pathway:39,40 deregulation of RB can lead to abnormal replication fork dynamics, DNA strand breakage and genomic instability.41

Underscoring the critical impor-tance of the E2F temporal program, the RB-E2F network is governed by multiple layers of feedback and feedforward regu-lation (Fig. 1B). In this review, we sum-marize the regulatory mechanisms that may contribute to precise control of E2F expression and activity during cell cycle entry. We emphasize evidence from mam-malian cells and the dynamics of E2F1–3 activators, since they positively correlate with replication in this context. In the future, coupling mathematical model-ing and experiments will be essential for quantitative understanding of E2F tempo-ral dynamics.

Delayed E2F: Derepression

Growth stimulation initiates a cascade of signaling events20 that generates an early peak in MYC (<60 min) along with a late peak (~8 h) due to changes

extensively analyzed; this information has defined the “wiring diagram” of the wider RB-E2F regulatory network.20,21

Our group has shown that the RB-E2F pathway plays a central role in discrimi-nating between different types of growth stimulation. Arthur Pardee coined the term restriction point (R-point) to describe the time at which cells commit to the cell cycle by discontinuing their dependence on mitogenic stimulation.22 The R-point can enforce one of two cell states (quiescence and proliferation) in accord with environmental conditions. Consistent with this notion, we have shown that the RB-E2F network acts as a bistable switch to convert graded growth inputs into an “all-or-none” response.23,24 Further, we have shown that the RB-E2F network can discriminate between nor-mal and aberrant growth signaling from proto-oncogenes such as c-Myc (Myc). MYC is a critical mediator of physiologi-cal growth signals that facilitates E2F expression during cell cycle entry.25 The Myc locus is often amplified in human cancers, presumably to “short circuit” the need for external growth stimulation. In normal cells, however, overexpression of MYC fails to induce DNA replication or division,26,27 suggesting that cells can somehow respond specifically to MYC expressed in a physiological context. These differing responses are reconciled by the observation that E2F1 is only upregulated when MYC levels are within a narrow window comparable to levels achieved fol-lowing growth factor stimulation.28

The bistable and biphasic responses in the dose domain represent the culmina-tion of successive temporal events initiated by growth signals. However, the temporal dynamics of E2F are equally important. In response to strong growth stimulation of quiescent cells (G

0), E2F1–3 expre-

sion will rise and peak just prior to the onset of DNA synthesis (S phase) fol-lowed by inactivation just prior to the onset of mitosis (M) (Fig. 1A). This tem-poral program may be pivotal in coor-dinating the ordered molecular events required for high-fidelity DNA replica-tion. First, E2F controls the expression of genes that constitute the pre-RC and licensing machinery which are absent in quiescent cells; perturbing their normal



promoting assembly and licensing of pre-RC.71,72 Moreover, constitutive expression of Cyclin E results in cell cycle arrest and chromosomal instability,73 underscoring the interwoven nature of positive auto-regulation and DNA replication.

Another source of positive feedback involves p53. Growth stimulation and E2F activation are required to eliminate a p53-p21WAF1-mediated block in cell cycle entry in late G

1.74 p21WAF1 is a transcrip-

tional target of p53 75 and promotes RB activity by inhibiting its phosphorylation by CDKs. One proposed link between E2F and p53 is Sirt1, which is induced by E2F1 and encodes a deacetylase that inactivates p53 activity.76,77 Another pos-sible link is the E2F-mediated induction of the Arf tumor suppressor that inacti-vates MDM2, a ubiquitin ligase of p53. However, it remains unclear if modula-tion of Arf expression and activity is cell cycle-dependent.36,78,79 Regardless of spe-cific mechanisms, suppression of the p53-p21WAF1 axis in an E2F-dependent fashion represents an additional means to alleviate E2F sequestration by pocket proteins.

Regulation of E2F by multiple posi-tive feedback loops is critical for the con-trol of cell cycle entry. Positive feedback is a hallmark of bistable responses80 and

E2f2 and E2F3a employ similar regu-latory mechanisms to modulate their expression.61-63 In vivo evidence suggests cross-association between E2F activators at promoter binding sites during the exit from quiescence.52,53 It remains unclear how individual E2F activators contribute to overall E2F expression. At least in terms of development, a single E2F activator can suffice.64

Another major player in positive autoregulation is Cyclin E, a regulatory subunit for CDK

2, which has similar

dynamics as E2F1.65,66 The activities of RB, CYCLIN E and E2F are deeply inter-connected (Fig. 2A). First, as for E2f1–3, pocket proteins negatively regulate Cyclin E transcription, and their disruption leads to increased Cyclin E expression even in quiescent cells.54,67-69 Second, pocket pro-teins are phosphorylated by CYCLIN E:CDK

2 complexes at the conclusion of

G1, leading to disruption of their E2F

binding.5 Third, Cyclin E is a direct tran-scriptional target of E2F1; mutation of two canonical, promoter-proximal E2F binding sites results in qualitatively similar temporal dynamics in response to serum but with a premature peak and overall ele-vated levels.70 Cyclin E is essential for exit from quiescence, likely owing to its role in

thus restricting its association with gene regulatory sequences.53,58,59 It is likely that the dynamics of p130 degradation (~5 h after CDK increase) are rate limiting for subsequent stages of E2F regulation.

From OFF to ON: Positive Feedback

Johnson et al.60 showed that a growth regulated region of the human E2f1 gene is activated by E2F1–3, which physically associate with two consensus E2F bind-ing sites (TTTSSCGC, where S is either a G or a C) situated in the proximal pro-moter.52-54 Mutation of E2F binding sites simultaneously abrogated E2F protein binding and resulted in constitutively high promoter activity, consistent with a role in mediating repression by p130:E2F4/5 complexes. Further dissection of each binding site revealed subtle differences: The upstream site mediates repression, whereas the proximal site activation.59 The functional distinction between sites is reflected in their association with differ-ent protein complexes.53 The specific role that E2F transactivation (vs. de-repres-sion) plays in the transcriptional dynam-ics of E2f1–3 or other downstream targets remains to be seen.

Figure 2. Switching OFF to ON: Derepression and positive feedback on e2F. (a) events following growth factor receptor engagement that increases e2F expression. in the exit from quiescence, a repressive p130:e2F4/5 complex (designated by rBp:e2FrNuc) situated on e2F target genes is removed via phosphorylation and degradation of p130; e2F4/5 is exported from the nucleus (Nuc) to the cytoplasm (Cyt) in the absence of p130. rB, on the other hand, cycles through states of hypo- and hyperphosphorylation during the cell cycle. repression of p53 is achieved through e2Fa-mediated upregulation of the Sirt1 deacetylase. Green arrows indicate positive feedback. GF, growth factors; e2Fa, activators e2F1–3; e2Fr, repressors e2F4–5; rBp, hypophosphorylated pocket proteins; rBpp, hyper-phosphorylated pocket proteins; p21waF1, cyclin-dependent kinase inhibitor; (B) effect of posi-tive feedback on gene expression. Cell-cell variability in isogenic cells (noise) can manifest in differences in expression dynamics. Depicted are time courses from distinct cells with a gene subject to weak (gray) or strong (black) positive feedback. Positive feedback can decrease the time needed to exceed basal expression (time to cross horizontal dotted line; τDelay) and the coherence across a population (difference in τDelay between cells).



induction (3–6 h) along with an increase in maximal E2F1 expression.

Like miR-17, the Arf tumor suppressor is regulated by E2F and MYC, and ARF protein can enhance proteasomal-medi-ated degradation of E2F.105-111 However, Arf expression during the cell cycle may be context-dependent. Arf expression was described as constitutive throughout the cell cycle in rat and human cell lines stimulated by serum.78 In contrast, serum-stimulated MEFs showed a decrease in Arf transcription during the period coincident with S phase.79 Our work has shown that Arf is, in fact, induced in rat fibroblasts with kinetics similar to E2f1.28 Thus, the qualitative and quantitative contribution of Arf toward E2F dynamics is unclear. It is possible that the influence of ARF may be context-dependent112 or involve post-translational, cell cycle-dependent modifications rather than changes in its expression.

ON to OFF: Delayed Negative Feedback

Concurrent with the entry into S phase, E2F activity is downregulated as persis-tent expression of E2F1 induces apopto-sis.11-13,33,113 Intuitively, abrupt suppression

fold-change detection95 and biphasic dose-response.96-98 Negative feedback can gen-erate adaptation, oscillations99 and expand the dynamic range of a dose-response.100 Both I1-FFL and negative feedback can increase response speed:101-103 repression of steady state levels by either module can be offset through an increased production rate, which reduces the time required to achieve half-maximal levels (Fig. 3B).

Several lines of evidence suggest that repression due to miR-17 may act as a mech-anism to facilitate accelerated E2F induc-tion. First, theoretical work demonstrates that miRNA downregulates steady state E2F output.104 Second, miR-17 expres-sion is rapid relative to E2F1–3, peaking within 1 h following growth stimulation.90 Third, once induced, levels of miR-17 are relatively stable throughout the cell cycle. These properties suggest that the miRNA functions to attenuate overall E2F levels rather than playing a role in turning E2F OFF following DNA replication. Indeed, inhibition of miR-17-5p and miR-20a in human fibroblasts by antisense RNA led to a reduction in de novo DNA synthesis arising from engagement of a p53-depen-dent DNA damage checkpoint.37 While the overall pattern of E2F1 expression was maintained, there was precocious E2F1

may underlie the self-sustaining behavior of the RB-E2F network. Furthermore, coupled slow and fast positive feedback loops can generate rapid transit from the OFF to ON state yet remain noise-resistant when ON.81 Using a synthetic mammalian gene circuit, Longo et al. demonstrated that positive feedback can reduce both the time needed to surpass basal expression levels and cell-cell vari-ability in gene expression (Fig. 2B). These findings are similar to those gathered by our group using single-cell measurements of E2f1 expression coupled with stochas-tic simulations of an RB-E2F network model:83 feedback mediated by CDK

2

reduced both the minimum time for E2F to surpass a basal threshold (“time delay”) and the variability of ON-switching across a population (“transition rate”). An intriguing result of this work is the cor-respondence between variability in E2F activation and cell-cell variability in the time between G

0 and division previously

described using phenomenological mod-els.84,85 This suggests that E2F dynam-ics may have direct consequence on both DNA replication and the rate of cell pro-liferation. Whether positive autoregula-tion arising through cross-regulation or p53 has an effect on time delay, coherence and noise in E2F or the relative contri-bution of each feedback loop is unclear. Importantly, how positive feedback may constrain the temporal pattern of pre-RC synthesis, assembly, loading and licensing remains to be seen.

Amplitude Modulation: Rapid Negative Feedback/Feedforward

There is a growing appreciation for the role of micro RNA (miRNA) in modu-lating RB-E2F activity, target genes and replication stress.86-89 The miR-17 cluster is regulated by both MYC and E2F. Two mature products of this locus, miR-17-5p and miR-20a, target E2F mRNA and downregulate translation.90 The relation-ship between MYC, E2F and miRNA represents an incoherent feedforward loop (I1-FFL), while that between E2F and miRNA represents negative feedback91,92 (Fig. 3A).

I1-FFL can generate distinct net-work behaviors,93 including adaptation,94

Figure 3. Switching ON to OFF—negative feedback and feedforward. (a) Negative feedback and incoherent feedforward (i1-FFL) modules. Direct regulation of miR-17–92 cluster of mirNas along with the tumor suppressor Arf by both MYC and e2F represent incoherent feedforward/negative feedback loops. CYCLiN a and SKP2 are involved in suppression of DNa binding and stability of e2F1–3, respectively. E2f7/8 are transcriptional targets of e2F that bind to and suppress E2f1–3 transcription in the late phases of the cell cycle. (B) effect of rapid incoherent feedforward (i1-FFL) and negative feedback (NF). repression provided by i1-FFL/NF can reduce steady state but when offset by increased production rate, it can allow a shorter rise time (τrise) compared with a circuit without iFF/NF.



regard. First is the development of an experimental platform sensitive enough to detect endogenous levels of multiple genes in individual cells with high temporal resolution. For the most part, observations of E2F dynamics have been made using population-average methods that mask cell-cell differences likely to have pro-found phenotypic consequences. A second challenge is the development of appropri-ate modeling tools, such as stochastic dif-ferential equations (SDEs), that can both describe overall network behavior and capture the cell-cell variability in gene expression. A development cycle involving modeling and quantitative experiments provides a synergistic platform for both refining model parameters (based upon experimental measurements) and making testable predictions (using model simula-tions) about how genetic or environmen-tal perturbations may deregulate network function. Quantitative frameworks will be invaluable for the systematic investigation of E2F function in normal and pathologi-cal circumstances. Ultimately, it may pro-vide opportunities for the rational design of targeted cancer therapeutics aimed at quantitative modulation of network behavior.

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CYCLIN A:CDK2 and is required for its

ability to promote DNA replication.127,128 This physical coupling may represent a way to integrate the initiation and deli-censing machinery, potentially minimiz-ing the window of time in which aberrant re-initiation may occur. Thus, in addition to its role in replication, delayed negative feedback from CYCLIN A/SKP2 down-regulates E2F, underscoring the inextri-cable coupling of negative feedback and DNA replication events.

Another source of negative feedback involves E2F7 and E2F8, the most distantly related members of the E2F family.129,130 Although they can form homo- and het-erodimers on E2F DNA binding sites, E2F7/8 do not interact with DP proteins, and their expression is delayed, rising at the conclusion of S phase. Work by Li et al.131 found that these genes are direct targets of E2F1, and germline disruption of E2f7/8 led to both higher and pro-longed levels of E2f1 mRNA beginning at S phase. Deletion of E2f7/8 was accompa-nied by massive apoptosis that was depen-dent upon the presence of intact E2f1 and p53. These observations were initially sur-prising, because downregulation of E2f1 was fully dependent upon E2f7/8 despite the presence of Cyclin A and Skp2.132 However, this could be expected in light of the fact that E2F7/8 target transcrip-tion, while CYCLIN A/SKP2 act at the post-translational level, although the pres-ence of positive feedback complicates this interpretation. These sorts of discrepan-cies emphasize the need to understand how different regulatory modules impact E2F at both the transcriptional and post-transcriptional level.

A Quantitative Framework of E2F Dynamics

We have presented evidence indicating that E2F dynamics encode information from growth signals, enabling the coor-dinated activity of cell cycle modules involved in DNA replication. A framework to describe the quantitative relationship between E2F dynamics and the replica-tion machinery would aid in determining how coordination is specifically achieved and ways it can become deregulated. Two important challenges lie ahead in this

of E2F and licensing proteins may prevent aberrant re-initiation of replication, which otherwise triggers a cell cycle checkpoint. As discussed, rapid activation of moderate negative feedback/incoherent feedforward (e.g., miRNA) can modulate steady state levels of gene expression. On the other hand, strong delayed negative feedback permits levels of an upstream node to overshoot before it is repressed.114 Multiple sources of delayed negative feedback may play a role in quenching E2F activity fol-lowing S-phase entry.

CYCLIN A:CDK2 activity is essential

for DNA replication.115 Similar to Cyclin E, the transcription of Cyclin A is growth regulated and under negative control through E2F binding sites.116 Importantly, Cyclin A expression is delayed relative to E2F and Cyclin E,36,117 and temporal staggering is enforced at both the tran-scriptional30 and post-translational lev-els.31 These successive interactions have a functional role in allowing pre-RC assembly (Cyclin E) to precede replication initiation and delicensing (Cyclin A).32 CYCLIN A also downregulates E2F DNA binding by phosphorylating and inhibit-ing the obligate DNA binding partner, DP.118,119 Prolonged DNA binding activity of an E2F1 mutant resistant to CYCLIN A:CDK

2 triggers a DNA damage check-

point in conjunction with apoptosis.38 A subtle observation is that both E2F1 and E2F3 are required for cell cycle entry,120 but in subsequent cell cycles, only E2F3 binding activity is required.121 The mech-anism and significance of this selectiv-ity is unclear. It will be important to understand how precisely prolonged or unscheduled E2F DNA binding activity (i.e., E2F1 during subsequent cell cycles) impacts the operation of the DNA replica-tion machinery.

E2F protein stability is modulated through ubiquitin-mediated proteasomal degradation.122-124 E2F1 directly acti-vates transcription of Skp2 gene,125 which encodes a subunit of the SCFSKP2 ubiqui-tin ligase that targets E2Fs for destruc-tion.126 Changes in SKP2 levels are cell cycle-dependent and, importantly, are delayed with respect to E2F1 through a mechanism similar to the one leading to delayed increase in Cyclin A.127 Moreover, SKP2 exists in a protein complex with



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