Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis

Abstract

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4^-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4^-/-p53^+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.

Keywords: DNA polymerase ε; DNA replication; POLE1/2 mutations; genome stability; p53; tumorigenesis.

Graphical abstract

Figure 1

Pole4 Knockout Mice Are Viable in an Outbred Strain and Exhibit Phenotypes Similar to Human Patients with Mutations of POLE1 or POLE2 (A) Schematic representation of the targeting vector used to generate the Pole4-deficient mice. The deletion cassette has been inserted 93 bp after the start of Pole4 exon 1 to replace the entire Pole4 gene. Green arrows represent primers used for genotyping. (B) Left: Pole4 genotyping strategy. Upper band represents the WT allele; lower band, the mutant allele. Right: western blot on 13.5 dpc Pole4^+/+, Pole4^+/−, Pole4^−/− embryos illustrating loss of POLE4. Tubulin was used as loading control. (C) Breeding summary of Pole4^+/− mice intercrosses at birth and during embryogenesis. Numbers and percentages in brackets are expected numbers and ratios. Pole4-deficient mice are represented at expected ratios at 13.5 dpc, whereas only 9.3% Pole4^−/− mice are born. (D) Left: representative images of 13.5 dpc Pole4 embryos. Note the smaller size of Pole4^−/− embryo. Scale bar, 1 mm. Middle: weight analysis of Pole4 embryos at 13.5 dpc. Error bars represent ± SEM of n = 18 Pole4^+/+ and n = 23 Pole4^−/−. Significance: t test, p = 0.0002. Right: weight analysis of Pole4^+/+ and Pole4^−/−mice. Error bars are not shown to render the graph readable; data are from males and females with at least five mice measured at each time point. (E) Top: breeding ability of Pole4 heterozygous × heterozygous versus mutant × heterozygous crosses. Each pair was bred during 5 months, and the number of litters and pups per litter was quantified per 21-day gestation. Significance: t test; litter per 21-day gestation, p = 0.0004; pups per 21-day gestation, p = 0.21. Bottom: testis tubules of 5-day-old neonates were stained with PLZF (germ cells; brown) marker. DNA was counterstained with hematoxylin (blue). Spermatogonia cell quantification per tubule. Error bars represent ± SEM of at least 50 tubules. Significance: t test, p < 0.0001. (F) Representative pictures of 5-month-old Pole4^+/+ and Pole4^−/− littermates illustrating craniofacial abnormalities, belly white patches, and kinks in the tail. (G) Micro-CT scan of tail from Pole4^+/+ and Pole4^−/− mice. Bar, 2.5 mm. (H) Brain-weight analysis of Pole4^+/+ and Pole4^−/− mice. Error bars represent ± SEM of n = 34 Pole4^+/+ and n = 41 Pole4^−/−. Significance: t test, p < 0.0001. (I) Rotarod experiment testing 3-month-old Pole4^+/+ and Pole4^−/− mice coordination. Note the decreased time spent on the rotating rod by Pole4^−/− mice compared to their WT littermates. Error bars represent ± SEM of n = 20 Pole4^+/+ and n = 38 Pole4^−/−. Significance: t test, p = 0.0002. (J) Lymphoma-free survival of Pole4 mice. Significance: Mantel-Cox test, p = 0.5. n = 36 Pole4^+/+ and n = 36 Pole4^−/−. Mice culled due to nonspecific phenotypes (e.g., dermatitis, overgrown teeth, and fits) were excluded from this study. (K) Frequency of Pole4 mice with lymphomas in spleen, thymus, and mesenteric lymph nodes (MLNs). Significance: Fisher’s exact test, p = 0.86 for spleen, p = 0.0652 for thymus, and p = 0.0033 for MLNs.

Figure 2

Loss of POLE4 Leads to Failed Lymphoid Progenitor Maturation following p53 Activation (A) White blood cell (left), red blood cell (middle), and platelet (right) counts in Pole4^+/+ and Pole4^−/− mice. n = 26 Pole4^+/+ and n = 41 Pole4^−/−. Significance: t test, p < 0.0001 for white blood cells (WBCs) and red blood cells (RBCs), p < 0.05 for platelets. (B) White blood cell distribution. Note the lower number of lymphocytes in Pole4^−/− mice. (C) Representative flow cytometry plots of Pole4^+/+ and Pole4^−/− mice spleen gated on T lymphocytes and demonstrating a significant reduction of CD4⁺ and CD8⁺ single-positive cells and an increase in CD4⁻CD8⁻ double-negative population. At least three animals have been analyzed per condition, and analyses were done in triplicates. CD5 was used as T cell marker (D) Frequency of Pole4^+/+ and Pole4^−/− mice presenting abnormal thymus. Note the size of Pole4^−/− thymus is greatly reduced compared to WT littermates. Significance: Fisher’s exact test, p = 0.026 for thymus absence, p < 0.0001 for thymus with medulla only. n = 36 Pole4^+/+ and n = 36 Pole4^−/−. (E) Representative flow cytometry plots of Pole4^+/+ and Pole4^−/− mice spleen gated on B lymphocytes and demonstrating a significant reduction of CD19⁺ cells and an increase in IgD⁻ population. At least 3 animals have been analyzed per condition, and analyses were done in triplicates. (F) Top: representative picture of spleen from Pole4 mice. Note the reduction in size following deletion of Pole4. Bottom: frequency of Pole4^+/+ and Pole4^−/− mice presenting spleen with underdeveloped follicles. n = 36 Pole4^+/+ and n = 36 Pole4^−/−. Significance: Fisher’s exact test, p = 0.001. (G) Left: representative flow cytometry plots of Pole4^+/+ and Pole4^−/− mice bone marrow gated on Lin-Sca1⁺cKit⁺ hematopoietic stem cells (HSCs) and demonstrating an increase of HSCs in Pole4^−/− mice. Right: Pole4^+/+ and Pole4^−/− mice bone marrow gated on CD34 and Flt3 illustrating a decrease of long-term and short-term HSCs and an increase of multipotent progenitors in Pole4^−/− mice. At least three animals have been analyzed per conditions, and analyses were done in triplicates. (H) Western blot analysis of proliferation and apoptosis in Pole4^+/+ and Pole4^−/− embryos at 13.5 dpc. Note the increased expression of p53 and caspase 3 cleavage in Pole4^−/− extracts. SMC1 was used as loading control. (I) Immunohistochemistry quantification of γH2AX, cleaved caspase 3, and p53 in forebrain, and liver section of 13.5 dpc embryos. Significance: t test.

Figure 3

Pole4^−/− Mouse Cells Exhibit Polε Complex Instability and Heightened Replication Stress (A) Western blot analysis of replication proteins from total, soluble, and chromatin fractions of Pole4^+/+ and Pole4^−/− MEFs. Tubulin and histone H3 were used as loading controls. (B) Western blot analysis of replication proteins from Pole4^+/+ and Pole4^−/− mice testis extracts. Tubulin was used for normalization. (C) Results of the iPOND-SILAC-MS experiment reported as logarithmic fold change of heavy/light ratio. Polδ and Polε major subunits are indicated in the plot as blue and red dots, respectively. (D) Pole4^+/+ and Pole4^−/− cells were labeled with EdU for 10 min and subjected or not to 30-min chase in media containing thymidine before being processed for iPOND. Captured proteins were analyzed by SDS-PAGE and western blot using the indicated antibodies. (E) Analysis of fork symmetry in Pole4^+/+ and Pole4^−/− MEFs reported as left/right moving fork ratio. Data were obtained from three different Pole4^+/+ and Pole4^−/− MEF clones; error bars ± SEM are included. Scale bar, 5 μM. (F) Bar graphs showing inter-origin distance distribution in Pole4^+/+ and Pole4^−/− cells. Data were obtained from three different Pole4^+/+ and Pole4^−/− MEF clones; error bars ± SEM are included. (G) Bar graphs showing replication fork speed distribution in Pole4^+/+ and Pole4^−/− cells. A total of ∼1,000 fiber tracts/condition were analyzed from three different Pole4^+/+ and Pole4^−/− MEF clones; error bars ± SEM are included.

Figure 4

Human POLE1 Patient Cells Exhibit Polε Instability and Replication Stress (A) Western blot analysis of Polε subunits and replisome components from total extracts of control- (CTR1 and CTR2) and patient-derived (P1 and P2) cell lines. Tubulin was used for normalization. (B) Western blot analysis of total, soluble, and chromatin fractions from P2 mutant and CTR1 cells. Tubulin and histone H3 were used as loading controls. (C) Analysis of fork symmetry in POLE1 mutant (P1 and P2) and CTR1 cells reported as left/right moving fork ratio. In the top panel, a scheme of the labeling strategy is presented, together with representative pictures of symmetric and asymmetric DNA fibers from CTR1 and P2 cells, respectively. Scale bar, 5 μM. (D) Bar graphs showing replication fork speed distribution in POLE1 mutant and control cells. A total of ∼1,000 fiber tracts/condition were analyzed from triplicate experiments; error bars ± SEM are included. (E) Bar graphs showing inter-origin distance distribution in POLE1 mutant and control cells. Data plotted were obtained from triplicate experiments; error bars ± SEM are included. (F) Representative scheme of the nucleotide labeling strategy together with representative pictures of the replication structures (ongoing forks and initiation events) analyzed. Scale bar, 5 μM. (G) Bar graph showing the percentage of initiation events in POLE1 mutant and control cells. Data were obtained from triplicate experiments (^∗p < 0.05; ^∗∗p < 0.01); error bars ± SEM are included.

Figure 5

Defective Origin Activation Leads to Chromosomal Instability in Pole4^−/− and POLE1 Patient Cells (A) Pole4^−/− and Pole4^+/+ cells were subjected to IR or HU (hydroxyurea) treatment, with the described doses, and cell viability was assessed after 5 days (ns, not significant; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001); error bars ± SD are included. (B) POLE1 mutant (P1 and P2) and control (CTR1 and CTR2) cells were subjected to IR or HU (hydroxyurea) treatment, with the described doses, and cell viability was assessed after 5 days (ns, not significant; ^∗∗p < 0.01; ^∗∗∗p < 0.001); error bars ± SD are included. (C) Upper: representative scheme of the hydroxyurea treatment and CldU-IdU labeling scheme used for fiber stretching assay. Lower: representative images of inter-origin distances (IODs) from Pole4^+/+ and Pole4^−/− cells. Scale bar, 5 μM. (D) Mean inter-origin distance values from HU-treated Pole4^+/+ and Pole4^−/− MEFs (^∗∗∗p < 0.001). (E) Mean inter-origin distance values from HU-treated CTR and POLE1 mutant cells. (F) Representative chromosome spreads from Pole4^+/+ or Pole4^−/− MEFs treated or not with APH (0.3 μm for 24 hr). Yellow arrows indicate scored chromosomal aberrations such as breaks or radials. Scale bar, 10 μM. Right: bar graph showing the main number of chromosomal abnormalities identified in Pole4^+/+ or Pole4^−/− MEFs treated or not with APH (^∗∗p < 0.01); error bars ± SD are included. (G) Representative chromosome spreads from CTR or POLE1 mutant cells treated or not with APH (0.3 μm for 24 hr). Yellow arrows indicate scored chromosomal aberrations such as breaks or radials. Scale bar, 10 μM. Right: bar graph showing the main number of chromosomal abnormalities identified in the described conditions (^∗p < 0.05; ^∗∗p < 0.01); error bars ± SD are included.

Figure 6

p53 Deficiency Rescues Embryonic Lethality and Drive Tumorigenesis in Pole4^−/− Mice (A) Representative picture of Pole4/p53 mice. Note the similar size between Pole4^+/+p53^−/− and Pole4^−/−p53^−/− animals. (B) Rotarod experiment testing Pole4^+/+ and Pole4^−/− mice coordination in a p53^+/− or p53^−/− background. Note the decreased time spent on the rotating rod by Pole4^−/−p53^+/− mice compared to their control littermates. Error bars represent ± SEM of n = 27 Pole4^+/+p53^+/−, n = 18 Pole4^−/−p53^+/−, n = 6 Pole4^+/+p53^−/−, n = 6 Pole4^−/−p53^−/−. Significance: t test; Pole4^+/+p53^+/− versus Pole4^−/−p53^+/−, p < 0.0001; Pole4^−/−p53^+/− versus Pole4^+/+p53^−/−, p < 0.0001; Pole4^−/−p53^+/− versus Pole4^−/−p53^−/−, p = 0.0046; Pole4^+/+p53^+/− versus Pole4^−/−p53^−/−, p = 0.0031; Pole4^+/+p53^+/− versus Pole4^+/+p53^+/−, p < 0.0001. (C) Hematology analysis of Pole4/p53 mice. White blood cell count (left) and distribution (right). n = 24 Pole4^+/+p53^+/−, n = 21 Pole4^−/−p53^+/−, n = 8 Pole4^+/+p53^−/−, n = 11 Pole4^−/−p53^−/−. Significance: t test; Pole4^+/+p53^+/− and Pole4^−/−p53^−/− versus Pole4^−/−p53^+/−, p < 0.0001; Pole4^+/+p53^+/− versus Pole4^−/−p53^−/−, p = 0.633 and Pole4^+/+p53^+/− versus Pole4^−/−p53^+/−, p = 0.4245. Note the normal distribution of myeloid population in double mutant compared to Pole4^−/−p53^+/− animals. (D) Representative flow cytometry plots of Pole4/p53 mice spleen gated on T lymphocytes and demonstrating a similar number CD4⁺ and CD8⁺ single-positive cells and CD4⁻CD8⁻ double-negative population. At least three animals have been analyzed per condition, and analyses were done in triplicates. CD5 was used as T cell marker. (E) Representative flow cytometry plots of Pole4/p53 mice spleen gated on B lymphocytes and demonstrating a similar number CD19⁺ and IgD⁻ cells. At least three animals have been analyzed per condition, and analyses were done in triplicates. (F) Lymphoma-free survival of Pole4/p53 mice. n = 16 Pole4^+/+p53^+/−, n = 16 Pole4^−/−p53^+/−, n = 13 Pole4^+/+p53^−/−, n = 11 Pole4^−/−p53^−/−. Significance: Mantel-Cox test; Pole4^+/+p53^+/−versus Pole4^−/−p53^+/−, p < 0.0001; Pole4^+/+p53^−/− versus Pole4^−/−p53^−/−, p = 0.3066; Pole4^−/−p53^+/− versus Pole4^−/−p53^−/−, p = 0.0746. Mice culled due to nonspecific phenotypes (e.g., dermatitis, overgrown teeth, and fits) were excluded from this study. (G) Lymphoma frequency of Pole4/p53 mice. Significance: Fisher’s exact test; Pole4^+/+p53^+/− versus Pole4^−/−p53^+/−, p = 0.0051; Pole4^+/+p53^−/− versus Pole4^−/−p53^−/−, p = 0.1261.