E2F4 function in G2: maintaining G2-arrest to prevent mitotic entry with damaged DNA

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.

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.

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)].

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. In bold are targets identified in our report.