Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication

Abstract

The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints.

Figure 1. PRC2 regulates MEF proliferation at 3% O₂ with high p16 and p19/Arf expression.

(a) Cumulative population doublings of MEFs grown at 3% or 21% O₂. Insets show a representative picture of the MEF stained for senescence-associated β-galactosidase activity (SA-β-gal) at passage 5. (b) Immunoblots using the indicated antibodies with protein extracts obtained from MEFs grown at 3 and 21% O₂ at the indicated passages. β-tubulin served as loading control. (c) Growth curves, immunoblots and BrdU FACS analysis of Ezh2 fx/fx and Ezh2 −/− MEFs grown at 3% O₂ at the indicated time. Left panels show crystal violet staining of cells at day 5 of the growth curve. The graph represents the quantification of crystal violet absorbance at λ=590 nm at the indicated time points. Error bars indicate s.d., n=3 for all graphs. Middle panel shows immunoblots using the indicated antibodies with protein extracts prepared from Ezh2 fx/fx and Ezh2 −/− MEFs at day 5 of the growth curves. β-tubulin and total histone H3 served as loading controls. Bar plot in the right panel shows the percentage of BrdU incorporation as measured by FACS analysis between Ezh2 fx/fx and Ezh2 −/− MEFs. (d,e) As in c, using MEFs infected with Suz12- (d) or Eed- (e) specific shRNA-expressing lentiviral vectors. Empty or scrambled-expressing (ctrl) vectors were used as negative controls, respectively. Growth curves were performed at 3% O₂.

Figure 2. PRC2 regulates proliferation and embryogenesis independently of Ink4a/Arf.

(a–c) Growth curves measured with crystal violet (λ=590 nm, top panels) and western blot analysis of protein extracts using the indicated antibodies (bottom panels) from Ezh2 fx/fx and Ezh2 −/− Ink4a/Arf −/− MEFs (left panels) or Ink4a/Arf −/− MEFs infected with Suz12- (middle panels) or Eed- (right panels) specific shRNA-expressing lentiviral vectors grown at 3% O₂. Empty and scrambled-expressing (ctrl) vectors were used as negative controls, respectively. Error bars indicate s.d., n=3. (d) Pictures of embryos derived from Suz12 +/−, Ink4a/Arf −/− mating at the indicated developmental stages. PCR genotypes of the single embryos at each developmental stage are presented in the right panels.

Figure 3. PRC2 regulates proliferation in a p53- and pRb-independent manner.

(a–c) Growth curves at 3% O₂, measured with crystal violet (λ=590 nm, top panels) and immunoblots of protein extracts using the indicated antibodies (bottom panels) in p53−/− (a) and pRb−/− (b) MEFs infected with Suz12-specific shRNA-expressing lentiviral vectors, or SV40ER-immortalized Ezh2 fx/fx MEFs grown at 3% O₂ and treated with ethanol (Ezh2 fx/fx) or OHT (Ezh2 −/−). (c) An empty vector was used as a negative control for Suz12 knockdowns. β-tubulin and total histone H3 served as loading controls. Error bars indicate s.d., n=3. (d) Colonigenic assay stained with crystal violet of Ezh2 fx/fx MEFs grown at 3% O₂ and treated with ethanol (Ezh2 fx/fx) or OHT (Ezh2 −/−). Colony quantification was determined using ImageJ. Error bars indicate s.d., n=3. P-value was determined with paired t-test. Scale bar, 2 cm.

Figure 4. PRC2 regulates cellular transformation in a p53- and pRb-independent manner.

(a) Foci formation assay of SV40ER-immortalized Ezh2 fx/fx MEFs that stably expressed H-RASV12, treated with ethanol (Ezh2 fx/fx) or OHT (Ezh2 −/−). PRE indicates that SV40ER-immortalized Ezh2 fx/fx MEFs were exposed to OHT treatment before H-RASV12 expression. The POST condition indicates that H-RASV12 was expressed in SV40ER-immortalized Ezh2 fx/fx MEFs before OHT treatment. Foci were stained with Giemsa and quantified using ImageJ. Error bars indicate s.d., n=3. P-value was determined with paired t-test. All assays were performed at 21% O₂. Scale bar, 2 cm. (b–e) Tumour formation of the cells described in a upon injection in both flanks of athymic nude mice. Pictures of nude mice and isolated tumour masses at 14 days post injection are shown in b and d; the average increase of tumour size at the indicated time (left) and the weights of the tumour masses (right) shown in b and d are given in c and e. Error bars indicate s.d., n=10. P-value was determined using a Mann–Whitney test. Scale bars, 1 cm.

Figure 5. PRC1 is required for proliferation and transformation independently of the Ink4a/Arf-p53-pRb axis.

(a) Growth curves, immunoblots and BrdU FACS analysis of Ring1a −/−, Ring1b fx/fx and Ring1a −/− and Ring1b −/− MEFs grown at 3% O₂ for the indicated times. Left panels show the crystal violet staining of cells at day 5 of the growth curve. Quantification of crystal violet absorbance at λ=590 nm at the indicated time points is depicted in the graph. Middle panel shows immunoblots using the indicated antibodies of protein extracts derived from Ring1a −/− Ring1b fx/fx and Ring1a −/−, Ring1b −/− MEFs at day 5. β-tubulin and total histone H2A served as loading controls. Right panel shows percentage of BrdU incorporation measured by FACS analysis between Ring1a −/−, Ring1b fx/fx and Ring1a −/− and Ring1b −/− MEFs. In all growth curves, error bars indicate s.d., n=3. (b) Growth curves and immunoblots as in a, using Ink4a/Arf −/−, Ring1a −/−, Ring1b fx/fx or Ring1b −/− MEFs at 3% O₂. β-tubulin and total histone H2A served as loading controls. (c) Growth curves as in a with SV40ER-immortalized Ring1a −/− or Ring1b fx/fx MEFs treated with ethanol (Ring1b fx/fx) or OHT (Ring1b −/−) at 3% O₂. (d) Immunoblots were performed with the indicated antibodies using protein extracts prepared from SV40ER-immortalized Ring1a −/− or Ring1b fx/fx MEFs in which OHT treatment and H-RASV12 expression was combined with the order indicated in the figure. β-tubulin and total histone H2A served as loading controls. (e,f) Tumour formation of the cells described in d by injection in both flanks of athymic nude mice. Pictures of mice and of the isolated tumour masses at 14 days post injection are shown in e. The average increase of tumour size at the indicated time (left) and the weights of the tumour masses shown in e are presented in f. Error bars indicate s.d., n=10. P-value was determined using a Mann–Whitney test. Scale bar, 1 cm.

Figure 6. PcGs regulate S-phase entry.

(a) Venn diagrams representing the overlap of genes upregulated in either Ezh2 fx/fx or Ink4a/Arf −/− Ezh2 fx/fx MEF with OHT treatment (Ezh2 −/−) as compared with ethanol treatment (Ezh2 fx/fx), with a minimal fold difference of 1.5. The diagrams on the right represent the same overlap as previously characterized H3K27me3-enriched promoters in WT MEFs. (b) FACS analyses of SV40ER-immortalized Ezh2 fx/fx MEFs that stably expressed H-RASV12 7 days after ethanol (Ezh2 fx/fx) and OHT (Ezh2 −/−) treatment and fixed 30 min after release from a double-thymidine G1/S block in the presence of BrdU. The bar plots represent the relative percentage of cells with 2C DNA content present in the highlighted gates (BrdU negative, intermediate and BrdU positive). (c) Confocal immunofluorescence images of Ink4a/Arf −/−; Ezh2 fx/fx and Ink4a/Arf −/− Ezh2 −/− MEFs 2 h after release from double-thymidine G1/S block following BrdU staining with the indicated antibodies. Bar plots indicate the average Mender’s colocalization coefficient among all images of the Z-stack for each cell. Error bars indicate s.d. n is indicated in the figure. Scale bars, 2 μm. (d) Proximity ligation assay co-incubating Suz12 and Pcna-specific antibodies in Ezh2 fx/fx, Ink4a/Arf −/− (n=64) or by incubating Ring1b and Pcna-specific antibodies in Ezh2 fx/fx, Ink4a/Arf −/− (n=50) or in Ezh2 −/−, Ink4a/Arf −/− (n=46). (e) Accelerated native isolation of proteins on nascent DNA assay (aniPOND) performed in SV40, H-RASV12 transformed MEFs pulsed for 10 min with 10 μM of EdU. No Click-IT reaction served as negative control. Scale bars, 2 μm.

Figure 7. PcGs supervise DNA replication.

(a) Distribution of the replication speed for all forks between SV40ER-immortalized Ezh2 fx/fx MEFs that stably expressed H-RASV12 7 days after ethanol (Ezh2 fx/fx) or OHT (Ezh2 −/−) treatment. n is indicated in the figure. P-value was determined by λ² test. (b) Representative spinning disk confocal microscopy images of DNA combing performed in the cells presented in a and stained with the indicated antibodies. DNA fibres were visualized using ssDNA-specific antibodies. Scale bar, 40 kb. (c) Scatter plot of the fork distribution based on left/right fork speed ratio in the cells presented in a. Dotted lines indicate the 30% ratio tolerance applied to define symmetric fork progression. Box plots show the percentage distribution of fork symmetry between Ezh2 fx/fx and Ezh2 −/− cells described in a. (d) Distribution of replication speed for all symmetric forks forming a replicon between Ezh2 fx/fx and Ezh2 −/− cells described in a. n is indicated in the figure. P-value was determined by λ² test. (e) Distribution of inter-origin distances measured for all DNA fibres between Ezh2 fx/fx and Ezh2 −/− cells described in a. Bar plots show the average inter-origin distances of Ezh2 fx/fx and Ezh2 −/− cells. Error bars indicate s.d. n is indicated in the figure. P-value was determined by λ² test.

Figure 8. Model of PcG regulation of cellular proliferation.

(a) Normalized growth curves quantified by crystal violet staining of SV40 ER immortalized and SV40, H-RASV12-transformed Ezh2 fx/fx MEFs treated with ethanol (Ezh2 fx/fx) or 4-OHT (Ezh2 −/−) for 7 days. Error bars indicate s.d., n=3. (b) Bar plots indicating the number of population doublings after 5 days of growth curve calculated from a. Fold differences are indicated in the panel. P-values were determined by student's t-test. (c) The model highlights the role of PcGs in regulating DNA replication and cell cycle checkpoints in normal and neoplastic cells. Also, the frequent genetic deletions and oncogenic activities that occur in tumours cells are highlighted, to emphasize the requirement of PcG activities for the proliferation of cancer cells.