Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression

Abstract

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.

Keywords: CDH1; DNA replication; E2F; anaphase‐promoting complex; cell cycle.

Figure 1. High turnover of E2F7 and E2F8 via proteasomal degradation during G1 phase

1. Protein expression E2F7 and E2F8 in RPE cells treated with 100 μg/ml cycloheximide (CHX) and 10 μM MG132. The drugs were added simultaneously.

2. Quantification of (A). E2F/tubulin density ratios were calculated for n = 3 independent experiments, and 0 h was set to 100%. Error bars indicate s.e.m.

3. Protein levels of E2F7 and E2F8 in RPE and U2OS cells after 16 h of treatment with the CDK4/6 inhibitor PD0332991, or the CDK2 inhibitor NU6140.

4. Protein expression of E2F7 and E2F8 after 8 h of PD0332991 treatment, in the presence or absence of the proteasome inhibitor MG132 (10 μM) for 2 h prior to harvesting.

5. Schematic overview of conserved KEN motifs in human/mouse E2F7 and E2F8 proteins.

6. FACS profile showing expression of cell cycle markers in RPE cells with stable expression of the FUCCI system. Encircled areas indicate the gates used to sort cell cycle‐specific populations.

7. Immunoblots of FACS‐sorted RPE‐FUCCI cells. Cells were sorted based on expression of truncated versions of and Azami green‐tagged geminin (amino acids 1–130) and Kusabira orange‐tagged CDT1 (amino acids 30–120), respectively. Blots are representative examples of four independent replicates derived from two different stable RPE‐FUCCI clones.

8. Normalized transcript levels of atypical E2Fs and cyclin B1 in sorted RPE‐FUCCI cells measured by qPCR. Bars represent average ± s.e.m. of fold change, relative to expression in G1 (n = 3).

Figure 2. Atypical E2Fs are targeted for degradation by APC/C^{C dh1}

1. Protein levels of E2F7/8 and two known APC/C^{C dh1} substrates in 293T cells 48 h after transfection with CDH1‐Flag. Immunoblots are representative examples of two independent replicates.

2. Quantification of (A). Band density of indicated proteins was corrected for loading differences by calculating the ratio over tubulin. Bars represent mean ± s.e.m. of n = 2.

3. Expression of EGFP‐tagged E2F7/8 in 293T cells 48 h after transfection of Flag‐tagged CDH1 or empty vector.

4. Effect of CDH1 depletion on protein levels of E2F7/8 in HeLa cells with stable expression of inducible E2F7/8‐EGFP. Overexpression of E2F7 was induced using doxycycline at the onset of release from a thymidine block.

5. Co‐immunoprecipitation of EGFP‐tagged E2F7/8 with CDH1‐Flag after 48 h of co‐expression in 293T cells. Cells were treated with 10 μM MG132 for 5 h prior to harvesting to limit immediate proteasomal degradation of E2F7/8 after binding to CDH1. Asterisks indicate IgG bands; arrow indicates the CDH1‐Flag band.

6. HeLa cells with stable inducible E2F7/8‐EGFP were imaged by fluorescence and differential contrast (DIC) microscopy. Cells were treated with CDH1 siRNA for 10 h, synchronized at the G1‐S border by 16‐h thymidine treatment, followed by thymidine release and induction of E2F7/8‐EGFP by doxycycline. Mean integrated fluorescence of the cells was measured and normalized to the intensity in the frame of nuclear envelope breakdown (NEBD) (set at 100%), as determined by cytoplasmic dispersal of the fluorescent signal. The x‐axis is set to 0 at the onset of anaphase, as observed in the DIC channel. Graphs shown are mean ± s.e.m. Left graph: control n = 15, Cdh1 RNAi n = 14 both from three independent experiments. Right graph: control n = 13, Cdh1 RNAi n = 13 both from two independent experiments.

7. Expression of indicated proteins during mitotic exit of RPE cells treated with siRNA against CDC20 or CDH1. CDK1 inhibitor RO3306 and MPS1 inhibitor reversine were added to force mitotic exit. A scheme of the experimental procedure is shown in Fig EV1B. Blots are representative of two independent experiments.

8. Expression of indicated proteins during mitotic exit of HeLa cells expressing inducible E2F8‐EGFP, treated with siRNA against CDC20 or CDH1. RO3306 and reversine were added to force mitotic exit. Doxycycline was added 12 h prior to mitotic shake‐off. Blots are representative of two independent experiments.

Figure EV1. Detection of E2F7/8 during mitosis and mitotic exit

1. Montages of representative cells from HeLa cells with inducible expression of E2F7/8‐EGFP. Time (min) indicates time from onset of anaphase.

2. Schematic overview of mitotic release experiments shown in Fig 2G and H.

Figure EV2. Generation of KEN mutant E2F7 and E2F8

1. Sanger sequencing result after site‐directed mutagenesis of the nucleotides encoding the KEN domains in E2F7 and E2F8. The middle panel shows the N‐terminal KEN sequence starting at amino acid 5, the right panel shows the KEN domain starting at amino acid 374 in E2F8.

2. Quantification of EGFP levels determined with FACS analysis in HeLa cell lines with stable inducible expression of indicated constructs, with or without CDH1 RNAi transfection. Boxes indicate 25^th and 75^th percentiles, whiskers indicate 5^th and 95^th percentiles. For every condition, 10,000 cells were measured. Statistics were performed with Kruskal‐Wallis tests followed by Dunn's individual group comparison method. The inlay shows how we first gated the G1 cells (2C DNA content) prior to EGFP measurement.

3. Montages of representative HeLa cells expressing KEN mutant versions of E2F7/8. Time (min) indicates time from onset of anaphase.

Figure 3. Mutation of KEN domains results in marked stabilization of E2F7 and E2F8

1. Protein expression of wild‐type and KEN mutant E2F7 and E2F8 in 293T cells in the presence or absence of CDH1‐Flag 48 h after transfection. For E2F8, KEN motifs starting at amino acid 5 (KEN5) or 374 (KEN374) were mutated separately into three consecutive alanines or in combination (K/K). Blots are representative of two independent experiments.

2. Immunoblots showing repression of the E2F target genes CDC6 and cyclin A in HeLa cells with stable doxycycline‐inducible expression of wild‐type and KEN double‐mutant E2F7/8 after 16 h of doxycycline treatment.

3. FACS plots showing DNA content on the x‐axis (propidium iodide) of HeLa cells with stable expression of wild‐type or KEN mutant E2F7, after 24 h of doxycycline. Thresholds for EGFP positivity were set by applying the same gate to vehicle‐ and doxycycline‐treated cells.

4. HeLa cells expressing inducible E2F7‐EGFP (left destruction graph) or E2F8‐EGFP (right destruction graph) were blocked with thymidine for 16 h and then released in fresh medium with doxycycline. Imaging was performed as in Fig 2E. The x‐axis is set to 0 at the frame of anaphase onset. Graphs show mean ± s.e.m., wild‐type (wt) shown from Fig 2E in gray. E2F7^KEN‐EGFP: n = 13 from two independent experiments, E2F8^K/K‐EGFP: n = 26 from three independent experiments.

Figure EV3. E2F8 binds CDC20

1. Co‐immunoprecipitation of EGFP‐tagged E2F7 and E2F8 with endogenous CDH1. GFP‐binding agarose beads were used to pull down the fusion proteins. Cells were treated with 10 μM MG132 for 5 h prior to harvesting to prevent potential immediate proteasomal degradation of E2F7/8 after binding to CDH1.

2. Schematic overview of putative D‐boxes (RXXL) in mouse E2F8, and alignment of human and mouse E2F8 sequences.

3. Co‐immunoprecipitation of EGFP‐tagged E2F8^WT or E2F8^K/Kmut with CDC20‐Flag after 48 h of co‐expression in 293T cells. Cells were treated with 10 μM MG132 for 5 h prior to harvesting to prevent potential immediate proteasomal degradation of E2F7/8 after binding to CDC20.

4. Co‐immunoprecipitation as in (B), performed with GFP‐binding agarose beads. Note that CDC20 only interacted with E2F8, in a KEN‐independent manner.

Figure 4. Mutating the KEN domain of E2F7 inhibits S phase entry and progression

1. BrdU incorporation in HeLa cells with inducible wild‐type and KEN mutant E2F7/8 expression, measured by flow cytometry. Bars represent mean ± s.e.m. (n = 3). *P < 0.05 versus vehicle, determined by one‐way ANOVA followed by Holm‐Sidak pairwise comparisons.

2. Proliferation curves of HeLa cells with stable inducible wild‐type E2F7‐EGFP and E2F8‐EGFP expression after doxycycline treatment. Dots represent average ± s.e.m. of two replicates.

3. Quantitative PCRs showing expression of KEN mutant E2F7/8 and two classic E2F target genes in inducible HeLa cell lines after a mitotic shake‐off. Doxycycline was added 4 h prior to the nocodazole release. Bars represent average ± s.d. of two independent replicates, which were both measured in duplo.

4. Schematic overview of live microscopy using mCherry‐tagged PCNA to monitor cell cycle progression. Onset of S phase is marked by nuclear dots formation, which disappear when S phase is completed. PCNA leakage into the cytosol was always followed by apoptosis.

5. Cumulative progression into S phase of HeLa cells expressing KEN mutant E2F7/8‐EGFP, monitored by nuclear PCNA dot formation. P‐value indicates significant change of EGFP‐positive versus negative cells from the same cell line, determined by log‐rank (Mantel‐Cox) tests.

6. Cumulative cell death of HeLa cells expressing KEN mutant E2F7/8‐EGFP, monitored by cell blebbing and cytosolic PCNA‐mCherry. Statistical analysis was performed as described in (E).

7. Quantification of cell fates in PCNA live imaging experiments from (C–E), specified per cell cycle phase. Explanation of the legend: cell death in S phase, cell dies after appearance of PCNA dots; cell death in G, cell death after mitosis, but prior to PCNA dot formation; cell death in G1 or G2, cell death without observing mitosis or PCNA dot formation.

8. Schematic overview of synchronization experiment. HeLa cells were arrested at the onset of S phase with 2 mM hydroxyurea (HU), and induction of E2F8^WT or E2F8^K/Kmut was started 12 h prior to release from HU. FACS plat shows a representative example of fitting the different phases with a Watson exact model.

9. Quantification of DNA content of synchronized cells described under (H) measured by flow cytometry. Data points represent average ± s.e.m. of two independent experiments. In case of doxycycline‐treated cells, only EGFP‐positive cells were counted. The gating strategy and quantification of EGFP in both cell lines is shown in Fig EV5B and C.

Figure EV4. Stabilization of KEN mutant E2F7/8 inhibits DNA replication and triggers cell death

1. FACS data showing scatterplots of DNA synthesis (anti‐BrdU‐FITC) versus DNA content (propidium iodide) in cells with stable expression of indicated inducible constructs. Gates were used to calculate the percentage of BrdU‐positive cells shown in Fig 4A.

2. Montages of PCNA dot formation and cell death in cells with inducible expression of indicated constructs, and effect of CDH1 RNAi. Arrowheads indicate a cell that enters S phase, but undergoes cell death. Time in hh:mm from the onset of imaging and doxycycline induction.

Figure EV5. Stabilization of KEN mutant E2F7/8 inhibits DNA replication and triggers cell death; continued

1. Apoptosis, measured by FACS analysis of annexin V‐stained HeLa cells expressing the indicated inducible constructs. Apoptosis staining was done using the annexin V‐APC detection kit (Thermo Scientific), according to the manufacturer's instructions. Doxycycline was added 24 h prior to harvesting, and only EGFP‐positive cells were measured to avoid bias from differences in percentages of E2F‐expressing cells. Bars represent average ± s.e.m. of four independent replicates.

2. FACS plots of DNA content versus E2F8‐EGFP intensity in HU‐arrested cells, 12 h after doxycycline induction.

3. Quantification of EGFP levels in cells with 2C DNA content, determined with FACS analysis in HeLa cell lines with stable inducible expression of indicated constructs. Boxes indicate 25^th and 75^th percentiles, whiskers indicate 5^th and 95^th percentiles. For every condition, 10,000 cells were measured.

4. Representative FACS DNA content plots of HeLa cells with stable inducible expression of indicated E2F8 constructs. Gray overlays indicate the fraction of E2F8‐EGFP‐positive cells within each doxycycline‐treated cell line.

Figure 5. Stabilization of ectopic E2F7/8 in G1 by CDH1 depletion causes massive cell death

1. Effect of CDH1 RNAi on percentages of BrdU‐positive HeLa cells after induction of E2F7/8 expression, measured by flow cytometry. Bars represent mean ± s.e.m. (n = 4).

2. Effect of CDH1 RNAi on cumulative S phase entry of cells with inducible expression of E2F7/8^WT, measured by co‐expressed PCNA‐mCherry. P‐values indicate significant change of CDH1 RNAi versus control. Font colors of P‐values match with the line colors of the significantly changed conditions.

3. Effect of CDH1 RNAi on cumulative death of cells with inducible expression of E2F7/8^WT. P‐values indicate significant change of CDH1 RNAi versus control.

4. Quantification of cell fates in PCNA live imaging experiments from (B) and (C), specified per cell cycle phase. Explanation of the legend: cell death in S phase, cell dies after appearance of PCNA dots; cell death in G1, cell death after mitosis, but prior to PCNA dot formation; cell death in G1 or G2, cell death without observing mitosis or PCNA dot formation.

Figure 6. Atypical E2Fs can activate APC/C^{C dh1} via Emi1‐ and CDK2‐associated cyclins

1. Immunoblots showing Emi1 protein levels in HeLa cells with stable inducible expression of E2F7 or E2F8 after 16 h of doxycycline treatment.

2. Quantitative PCR of FBXO5 transcripts in FACS‐sorted cells with doxycycline‐induced E2F7/8 expression, after 8 h of doxycycline treatment. To avoid bias from cell cycle defects, cells were released from HU arrest at the onset of doxycycline treatment, resulting in a strong enrichment of cells in late S or G2 of both vehicle‐ and doxycycline‐treated cells (see Fig EV3C). Bars in indicate mean ± s.e.m. (n = 3); asterisks indicate P < 0.05 versus vehicle and scrambled siRNA, respectively.

3. Immunoblots of protein lysates from HeLa cells treated with siRNA against E2F7 and E2F8. Cells were harvested 6 h after release from HU block to enrich for cells in mid‐ to late S phase, where E2F7/8 expression was previously found to be high.

4. Quantitative PCR of FBXO5 transcripts in HeLa cells treated with siRNA against E2F7 and E2F8. Cells were harvested 6 h after release from HU block to enrich for cells in mid‐ to late S phase. Bars indicate mean ± s.e.m. (n = 3); asterisks indicate P < 0.05 versus vehicle and scrambled siRNA, respectively.

5. Cell cycle profiles of RPE cells determined by FACS analysis, during PD0332991 treatment, or 6 h after release. Percentages indicate the numbers of BrdU‐positive cells for each condition in, or 6 h after removing the drug (n = 3). BrdU was added to the cell culture medium 1 h prior to harvesting. A schematic overview of the experiment is shown in Fig EV6C.

6. Immunoblots showing increased levels of E2F7 and other APC/C^{C dh1} substrates after CDH1 RNAi in RPE cells, during late G1 and early S phase.

7. Immunoblots showing the effect of Emi1 RNAi on E2F7 and E2F8 expression in RPE cells. Hydroxyurea (2 nM) was added to arrest cells in S phase, the cyclin‐dependent kinase 2 inhibitor NU6140 (100 nM) was added to block APC/C inhibition by cyclin‐dependent kinase 2. Emi1 RNAi transfections were performed 48 h prior to harvesting, and drugs were added 24 h prior to harvesting.

8. Overlap between E2F target genes and APC/C substrates. The APC/C substrates are taken from a curated list 36, 37 with the addition of E2F7 and E2F8 based on the current paper; E2F7/8 targets from 8, 10. Asterisk: cyclin B1 is indicated in red, because we only found it to be directly regulated by E2F7/8 in a hepatoblastoma cell line, but not HeLa cells.

9. Simplified working model of interaction between atypical E2Fs and APC/C to coordinate DNA replication, based on the work described in this paper. Asterisks indicate indirect effect via transcriptional regulation of Emi1 and cyclin A/E.

Figure EV6. Schematic overviews of experiments to show feedback between atypical E2Fs and APC/C^Cdh1

1. Experimental scheme of FACS sorting in synchronized cells with stable expression of E2F7/8 in the presence or absence of doxycycline induction.

2. Example of gating strategy to sort late S/G2 cells with detectable E2F7/8‐EGFP.

3. Schematic overview of synchronization using the CDK4/6 inhibitor PD0332991. RPE cells were transfected with CDH1 RNAi, then treated overnight with 0.5 μM of the CDK4/6 inhibitor PD0332991, and released by washing away the drug.

4. Schematic overview of Emi1 RNAi experiments in RPE cells. Hydroxyurea and the CDK2 inhibitor NU6140 were added simultaneously.