E2F4 Function in G₂

Abstract

Mammalian cells undergo cell cycle arrest in response to DNA damage through multiple checkpoint mechanisms. One such checkpoint pathway maintains genomic integrity by delaying mitotic progression in response to genotoxic stress. Transition though the G₂ phase and entry into mitosis is considered to be regulated primarily by cyclin B1 and its associated catalytically active partner Cdk1. While not necessary for its initiation, the p130 and Rb-dependent target genes have emerged as being important for stable maintenance of a G₂ arrest. It was recently demonstrated that by interacting with p130, E2F4 is present in the nuclei and plays a key role in the maintenance of this stable G₂ arrest. Increased E2F4 levels and its translocation to the nucleus following genotoxic stress result in downregulation of many mitotic genes and as a result promote a G₀-like state. Irradiation of E2F4-depleted cells leads to enhanced cellular DNA double-strand breaks that may be measured by comet assays. It also results in cell death that is characterized by caspase activation, sub-G₁ and sub-G₂ DNA content, and decreased clonogenic cell survival. Here we review these recent findings and discuss the mechanisms of G₂ phase checkpoint activation and maintenance with a particular focus on E2F4.

Initiating A G₂ Checkpoint: The Main Players

In response to DNA damage, such as that caused by ionizing radiation, the most prominent arrest is in the G₂ phase,¹ which occurs particularly in tumor cells that almost invariably lack an effective G₁ arrest. In normal, nontransformed cells that have a functional p53 pathway, both a G₁ and a G₂ arrest can take place in response to DNA damage.^2,3 It has been established that the G₂ checkpoint initiation is primarily regulated through the control of Cdk1 (Cdc2) activity, which is regulated at multiple levels (Fig. 1), including periodic association with the B-type cyclins, reversible phosphorylation, and intracellular compartimentalization.⁴ Cyclin B1, the prototypical cyclin B (B2 and B3 are less abundant) reaches maximum levels in G₂, when it enters into the nucleus to form a complex with Cdk1 in a phosphorylation-dependent manner.⁵ The complex is activated by phosphorylation of Cdk1 on threonine 161 (T161) by Cdk-activating kinase (CAK), but is kept inactive by its phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15) by the Myt1 and Wee1 kinases, respectively.⁶

Figure 1.

Schematic representation of the G₂ checkpoint. Cdk1 is the key control point for G₂ checkpoint initiation, which is dependent on both positive and negative regulators. To be active, Cdk1 requires binding of its catalytic partner, cyclin B1 (also B2 and B3 in some circumstances), which is regulated transcriptionally and by its nuclear localization. Cdk1 is regulated by its phosphorylation, as determined by a balance of kinase and phosphatase activities, as well as at the level of transcription. G₂ progression to mitosis is triggered by the Cdc25C (or B)-mediated dephosphorylation of the Cyclin B/Cdk1 complex. Cyclin B/Cdk1 is activated by phosphorylation of T161 by CAK (Cyc H/Cdk7 complex) and the dephosphorylation of T14 and Y15 by Cdc25C. The complex is kept in an inactive state due to the phosphorylation of T14 and Y15 by the Myt1 and Wee1 kinases that can in turn be regulated by Plk1. The stable maintenance of the G₂ arrest is determined by the activity of E2F, Rb, or p53 family of transcription factors through their transcriptional targets, which include many of the components described here. p53 regulates the stability of the checkpoint through levels of its mitosis target genes cdk1, cyclin B1, but also p21 (CDKN1A) and 14-3-3σ. The activity of Cdc25C is also regulated by Chk1 or Chk2-mediated phosphorylation, leading to its inactivation through binding to and sequestration by 14-3-3σ. ATM/ATR kinases transduce the DNA damage signal to the effector kinases Chk1 and Chk2. Chk1 and Plk1, by regulating Wee1, also play a role in the recovery from the arrest.

Following DNA damage, Chk1 and Chk2 are phosphorylated and thus activated by ATM and ATR,^7,8 related members of the phosphatidylinositol 3-kinase (PI3K) family. In response to DNA damage, active Chk1 and Chk2 then phosphorylate the Cdc25C protein phosphatase at serine 216.^9,10 Once phosphorylated, Cdc25C can then be bound by 14-3-3σ and sequestered in the cytoplasm.⁹ Cdc25C, only when it is in the nucleus, dephosphorylates the T14 and Y15 residues and thus activates the Cdk1/cyclin B1 complex.^11-13 Additional phosphatases, Wip1 (PPM1D)¹⁴ and PP2A,¹⁵ may also regulate the checkpoint by dephosphorylating other factors that contribute to the activation of Cdk1. Cdk1/cyclin B can phosphorylate Cdc25C, further activating it and initiating a positive feedback loop.¹⁶ A nuclear export signal (NES) in Cyclin B1 facilitates rapid export of the Cdk1/cyclin B1 complex from the nucleus.¹⁷ Phosphorylation of the NES blocks the nuclear export by interfering with the binding of the nuclear export receptor CRM1.¹⁸

Sustaining A G₂ Checkpoint: An Unexpected Role for P130

The strength of a G₂ arrest elicited by a particular DNA damage signal depends on how long the arrest takes and how fast the recovery is. A role for the maintenance, but not the initiation of the G₂ arrest has been earlier attributed to p53. By inactivating the p53 pathway, which occurs in the majority of tumor cells, the G₁ arrest is abolished while the G₂ arrest is only attenuated.^19-22 Clearly, the G₂-arrest occurs through checkpoint pathways implicating the DNA damage sensors ATM/ATR and effectors Chk1/Chk2 to converge on the regulation of the Cdc25C phosphatase and its downstream target, Cdk1.⁷ Surprisingly, a G₂ arrest still occurs in ATM, ATR, Chk1, Chk2, and p53-deficient prostate cancer cells, suggesting that there are other pathways, at least in the context of prostate epithelial tumor cells, that contribute to an effective and stable G₂ arrest (Ray, Shyam, Fraizer, Almasan, unpublished data).

Recent studies have specifically implicated p130, a retinoblastoma (Rb)-family pocket protein in the mechanism of G₂ arrest following treatment with DNA-damaging chemotherapeutics.^23,24 Rb family of proteins, comprised of Rb, p130, and p107, associate with a wide variety of transcription factors and chromatin remodeling enzymes forming transcriptional repressor complexes that control gene expression.²⁵ Rb, a tumor suppressor critical for the control of cell cycle progression, regulates E2F activity. It has been known for some time that Rb-deficient cells have increased levels of mRNA and protein levels of genes that are under the transcriptional control of activating E2Fs.^26,27 Recently, it has become clear that the inactivation of Rb-family members complexing with these activating E2Fs reduces the cellular ability to also undergo G₂ arrest in response to DNA damage.²³ A small molecule based on the Rb2/p130 spacer domain proved to be effective in inhibiting Cdk2 activity, cell cycle arrest, and tumor growth reduction in vivo.²⁸

p53 can contribute to a sustained G₂ arrest by inducing the transcription of p21 and 14-3-3σ.² 14-3-3σ sequesters Cdk1/cyclin B1 and Cdc25C in the cytoplasm,^29,30 while p21 primarily inhibits the phosphorylation of p130 and p107 by Cdks, allowing them to repress the transcription of a large number of genes required for transit through G₂.^3,20 Similar to p53, p130 can repress transcription of cyclin B1 and cdk1 and therefore, enforce the G₂ arrest. p130, just like p53 in certain cell type contexts, has additional targets that do not affect Cdk1 but also contribute to G₂ arrest. For example, Plk1 is phosphorylated by Wee1 and may degrade it in a proteasome-mediated manner. Chk1 and Plk1 have been also suggested to be important for the checkpoint adaptation, a phenomenon that allows cells to escape from a G₂ checkpoint into mitosis with damaged DNA.³¹ The cellular fate leading to survival or elimination by mitotic catastrophe during the next few cell divisions remains to be established. A surprising recent finding is that Artemis, previously known to be implicated in NHEJ DNA repair following its phosphorylation by DNA-PK can have an impact on the G₂ recovery through direct interaction with cyclin B1. When it is phosphorylated by ATM, at lower doses of radiation, Artemis affects cyclin B1 complex formation with Cdk1 by sequestering it to the centrosome and by preventing Wee1-dependent phosphorylation.³²

E2F Family Proteins: from DNA Damage to A Stable G₂ Arrest

As the Rb family members interact with multiple binding partners and can control a wide range of signaling pathways,³³ the role of individual E2Fs has been of great interest. The E2F family of transcription factors controls the expression of ten genes encoded by eight independent loci that are divided, based on sequence homology, into three groups.³⁴ E2F1-3, which are highly regulated during cell growth and cell cycle generally, act as transcriptional activators. E2F4-5, which are constitutively expressed, are transcriptional repressors when bound to p130 or Rb. E2F6-8 act also as transcriptional repressors but do so independently of Rb.³⁵ Various E2Fs have opposite biological effects but they can also complement each other.³⁶ E2F4 and 5 are unique as they are regulated through their predominantly cytoplasmic cellular localization, which is facilitated by a NES.³⁷ However, they do not have a nuclear localization signal (NLS); therefore it is believed that nuclear translocation is dependent on binding to p130³⁸ or the other pocket proteins, Rb or p107.

Deregulation of the Rb/E2F pathway in human fibroblasts results following a genotoxic insult in E2F1-mediated apoptosis. This deregulation was first reported in Rb-deficient cells.²⁶ E2F1-dependent apoptosis has now been shown to be dependent not only on p53, but also on ATM, NBS1, and Chk2. E2F1 expression results in MRN foci formation that are similar to irradiation-induced foci, as they colocalize with 53BP1 and γH2AX foci.³⁹ These results suggest that E2F1-induced foci generate a cell cycle checkpoint that, with sustained E2F1 activity, eventually leads to apoptosis. E2F4 and E2F5, in complex with p130, are known to be present primarily in quiescent cells,⁴⁰ with decreasing levels of E2F4 found associated with Rb and p107-Cdk2 during S-phase progression.^41,42 Interestingly, we found that a typical genotoxic agent, ionizing radiation, induces complex formation of E2F4, but not E2F5, with the unphosphorylated form of p130.⁴³ Examining the dynamics of its subcellular localization indicated that E2F4 and p130 were present in the nucleus a short time following irradiation; their colocalization reached a peak at 24 h. In contrast, the protein levels of E2F1, which may counterbalance the activity of E2F4,³⁶ were dramatically downregulated. This pattern of decreased E2F1 levels was sustained following irradiation or treatment with the topoisomerase II inhibitor, VP16.

To examine the role of E2F4, we have chosen a transient, siRNA-mediated depletion of E2F4. This approach has been recently shown to be particularly fortuitous, as long-term, chronic loss of E2F activity, as produced in knockouts or stable knockdowns, does lead to compensation by other family members.³⁴ In contrast, the analyses of acute loss of function reveal specific and distinct roles of these proteins. To assess the DNA damage and its repair, we used alkaline comet assays.⁴⁴ This assay indicated that irradiation of C4-2 prostate carcinoma cells depleted of E2F4 by siRNA (siE2F4) markedly elevated DNA double strand break (DSB) levels as quantification of DNA damage showed more pronounced tail lengths as well as tail moments (Fig. 2A). Compared to radiation or siE2F4 treatments alone (Fig. 2B), these cells underwent apoptotic cell death that was characterized by both a sub-G₁ and a sub-G₂ DNA content. The appearance of sub-G₂ DNA content is an interesting finding, as it can be easily confused with late S-phase cells by propidium iodide staining-based flow cytometry. When cells treated with siE2F4 were irradiated, the DNA content distributions demonstrated a large accumulation of cells with sub-G₁ content, indicative of apoptotic cells and an accumulation of cells with an apparent late S phase DNA content. This finding is consistent with either a decrease in S phase transit in late S or apoptosis originating from cell with a 4C DNA content. BrdU incorporation in irradiated cells, used to distinguish S phase cells from those with the same DNA content that were not synthesizing DNA, decreased, consistent with cell cycle arrest in G₁ and G₂. A small sub-4C (sub-G₂) population of BrdU-negative cells was markedly enhanced in E2F4 knockdown irradiated cells, suggesting that cell death from G₂ arrest was elevated. Clonogenic cell survival studies provided an assessment of the long-term impact of the E2F4 knockdown. These cells became more sensitive to radiation, as indicated by a marked decrease in their LD50 values and clonogenic survival.³ A 4C DNA-content and low levels of cyclin A2 and B1 have been suggested also for cells encountering a G₁ arrest, as these features are characteristic of endoreduplication.²⁵ Alternatively, those cells that proceed through mitosis inappropriately, as a result, undergo mitotic catastrophe. However, the combined analyses of DNA content profiles, BrdU-incorporation, mitotic morphology, specific (cyclin A2 and B1), and general (MPM-2) mitotic markers excluded this possibility. Taken together, these data suggest that E2F4 activity helps to sustain G₂ arrest and prevent cell death.

Figure 2.

E2F4 depletion leads to DNA strand breaks. (A) Comet assays were performed under alkaline conditions to determine the amount of double-strand DNA breaks. Cells treated with siRNA against E2F4 (100 nM, 24 h), or vehicle alone and ionizing radiation (IR) were trypsinized and washed in PBS before being added to preheated (37°C) low-melting point agarose. The solution was pipetted onto slides precoated with 1% agarose. The chilled slides were allowed to lyse for 40 min at 4°C in 2.5 M NaCl, 100 mM NaEDTA (pH 10), 10 mM Tris Base, 1% SDS, 1% Triton X-100 prior to immersion in alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH 13). After 30 min, slides were placed into a horizontal electrophoresis chamber samples for ∼30 min (1 V/cm at 4°C). The slides were washed with deionized H₂O to remove the alkaline buffer, dehydrated in 70% ice-cold EtOH and air-dried overnight. Slides were stained with PI (50 μg/ml) and examined by microscopy. B) Tail moment (TM) and tail length (TL) were used to quantify the DNA damage. Image analysis and quantification has been performed with NIH ImageJ. TM = % of DNA in the tail × TL; where % of DNA in the tail = tail area (TA) × tail average intensity (TAI) × 100/(TA × TAI) + [head area (HA) × head area intensity (HAI)].

In our study, cyclin B1, Bub1B, CENP-E, Chk1, HEC, Mad2L1, and PTTG1, all known to be regulated by E2F4 and to have a critical function in mitosis, were found to be associated with E2F4 function. Cdc6, known to be essential for DNA replication could also play an important role in mitosis.⁴⁵ Moreover, chromatin immunoprecipitation (ChIP) analyses demonstrated specific E2F4 recruitment to the promoter binding sites of PTTG1 and BUB3. In contrast, E2F4 was bound constitutively to Cdc6, an observation similar to the constitutive chromatin binding by p53 to its target promoters,⁴⁶ indicating the involvement of other factors or post-transcriptional regulation for their activation. PTTG1, BUB1, BUB3, Mad2, and CENP-E are mitotic checkpoint proteins (Fig. 3). Stathmin,⁴⁷ HEC, and Ect-2, are involved in spindle formation.⁴⁸ The abundance of these targets and their individual role proposed in the DNA damage response is continuously expanding. For example, p130 regulates telomere length by interacting with RINT-1, previously identified as a Rad50-interacting protein. By forming a complex with Rad50 through RINT-1 p130 is likely to block the recombination dependent and telomerase-independent telomere lengthening in normal cells.⁴⁹ Another example is DDB2, a gene mutated in XPE patients and involved in global genomic repair, especially the repair of cyclobutane pyrimidine dimers (CPDs). In Rb-null cells, where we have shown that E2F activity is elevated,⁵⁰ global DNA repair is increased and removal of CPDs is more efficient than in wild-type cells. Endogenous E2F1 and E2F3 bind to the DDB2 promoter and treatment with E2F1-antisense or E2F1-siRNA decreases DDB2 transcription, demonstrating that E2F1 is a transcriptional regulator of DDB2.⁵¹

Figure 3.

Transcriptional programs implicating E2F and Rb family proteins in G₂ control. E2F4 repressor activity is mainly regulated by p130, with Rb playing a role only when p130 and p107 are absent. The E2F4/p130 complex is translocated to the nuclei shortly after DNA damage where it binds to the promoters of and downregulates many transcriptional targets important for mitosis (cylin B1), mitotic checkpoints (Bub1, PTTG-1), or DNA damage checkpoints (Chk1). Repression of these targets assures maintenance of a stable G₂ arrest for a tight control of mitotic entry. Activating E2Fs, such as E2F1, when released from Rb, following their activation through ATM and Chk2, lead to increased expression of genes important for DNA damage, DNA repair, but also apoptosis. Ultimately, a balance between the repressor and activating E2Fs may determine the physiological outcome.³⁶ In bold are targets identified in our report.³

Several expression profiling experiments have documented that similar to p130, E2F4 regulates many genes that are critical for mitotic entry. Our results are consistent with previous studies based on combined cross-linking DNA immunoprecipitation and micro-array experiments,⁵² which have identified some of the same targets we have uncovered as E2F4 target genes with a putative role in various phases of the cell cycle including G₂ and M.⁵³ Similar targets were independently identified in studies examining specific p130 or Rb-regulated gene expression.²⁴ At the same time, Rb-deficient U2OS cells showed an analogous dependence on E2F4-regulated genes,⁴⁸ suggesting a context-dependence for p130 or Rb requirement for regulating mitotic entry. From our results and other studies we conclude that in response to DNA damage, these tumor cells undergo cell cycle exit initiated in G₂.^3,24,54 Independent profiling of the radiation response in human diploid fibroblasts has yielded similar results, with a prominent induction of additional markers of G₀.⁵⁵ Hypoxia also induces p130 dephosphorylation and nuclear accumulation, leading to the formation of E2F4/p130 complexes and increased occupancy of E2F4 and p130 at the RAD51 and BRCA1 promoters that suggest a coordinated transcriptional program responsible for an integral response to hypoxic stress to regulate DNA repair.^56,57

Interestingly, a number of E2F4-bound genes originally identified through array analyses were also bound by E2F1,⁵³ an observation extended to the promoters of genes regulated during G₂ and M containing distinct E2F4 and E2F1 binding sites.^52,58 Some of these promoters may display collaborative binding of several E2F family members to the same elements, such as in case of ect-2,⁴⁸ while others distinct positive and negative E2F-dependent regulatory elements.⁵⁸ In addition, E2F-dependent transcriptional activity may require binding of other transcription factors, such NF-Y or Myb to regulate the transcription of Cdk1.⁵⁸ The balance between the E2Fs that occupy these promoters determines the physiological outcome, with more E2F4 bound resulting in cell cycle arrest.^3,36

Conclusion

A working model, summarizing recent findings on the G₂ initiation and maintenance discussed here, is presented in Figure 1. The salient feature is that while its initiation is dependent on cyclinB1/Cdk1 its stable maintenance is governed primarily by the activity of Rb and p53 family proteins. Together, these proteins may integrate the type and severity of the genotoxic stress with the proper G₂ response to presumably assure that there is sufficient time for effective repair of the DNA damage. Earlier reports have indicated that Rb deficiency sensitizes cells to genotoxins by allowing S-phase entry under inappropriate conditions.^26,50 This can be mimicked, for example by expressing typical Rb/E2F targets such as cyclin E1,⁵⁹ which drives cells through S-phase. Our recent report³ suggests that, in a similar way, E2F4 deficiency allows inappropriate G₂ progression that in response to DNA damage results in apoptosis without reaching mitosis. Interestingly, siRNA-mediated depletion of the classical radiation-induced checkpoint regulators ATM, ATR, Chk1, or Chk2 had no effect on the stability of the G₂ checkpoint in these cells (Ray, Shyam, Fraizer and Almasan, unpublished data). ATR and Chk1 depletion impacts the G₂ arrest only when p53 is rendered nonfunctional by expression of a dominant-negative mutant. In these cells, ATR seems to be hyperactivated, with a more limited role attributable to ATM.

Cellular radioresistance in tumor cells can be attributed to a strong G₂ checkpoint activation that can be abolished by agents that inhibit the G₂ checkpoint. Cell cycle G₂ checkpoint abrogation is also an attractive strategy for sensitizing cancer cells to DNA-damaging anti-cancer agents without increasing adverse effects on normal cells.⁶⁰ Interestingly, a recent report has suggested that G₂ checkpoint inhibitors may be most effective at an earlier stage in the cell cycle,⁶¹ a stage that may correspond to what has been reported to be regulated by E2F4.³ Genome-wide analysis of E2F-dependent transcriptional regulation has identified many potential gene targets. Whether such individual targets play a major role in a specific cell type or biological context or that their cooperativity is needed to achieve tight control of the G₂ to M-phase transition still needs to be established.