Canonical and atypical E2Fs regulate the mammalian endocycle

Abstract

The endocycle is a variant cell cycle consisting of successive DNA synthesis and gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals.

Figure 1. Loss of E2f activators promotes TGC and hepatocyte endocycles

a, NanoString analysis of TGC-specific E2f1-3 expression in laser capture microdissected wild type cells, n=2 placentas analyzed per time point. b, Quantitative RT-PCR analysis of E2f activators in pre- (E13.5) and post-natal (P0-12mo) wild type livers, with n per time point as indicated. 3wk*, 1 day post weaning (3wk). c, Representative E9.5 H&E placenta sections showing control and E2f1^−/−;E2f2^−/−;E2f3^−/− (123tko) TGCs. Arrows point to selected TGCs. Scale bar, 12.5µm. d, Feulgen quantification of ploidy in E9.5 control and 123tko TGCs, n=3 per genetic group. One-way ANOVA, * p≤0.05, ** p≤0.01, *** p≤0.001. e, Representative H&E sections showing control and Mx-cre;E2f1^−/−;E2f2^−/−;E2f3^f/f (Mx-123tko) livers of 8-week-old mice 10 days after pIpC injection. Scale bar, 10µm. f, Measurements showing enlarged Mx-123tko livers (top graph) and hepatocytes (bottom graph) relative to control tissues and hepatocytes, with n per genetic group as indicated. Two-tailed Student T-test, ** p≤0.01. g, Flow cytometry of control and Mx-123tko liver nuclei from 8-week-old mice, with n per genetic group as indicated. One-way ANOVA, ** p≤0.01, *** p≤0.001. c control, wild type TGCs; e control, pIpC injected Mx-cre;E2f1^−/−;E2f2^−/−;E2f3^+/+ livers; f-g control, wild type livers. Data in a, b, d, f and g reported as average values, ± SD are included when n>2 samples were analyzed.

Figure 2. E2f7 and E2f8 promote TGC endocycles

a, NanoString analysis of TGC-specific E2f4-6 (left panel) and E2f7/E2f8 (right panel) expression in laser capture microdissected wild type cells, n=2 placentas analyzed per time point. b, Immunohistochemistry demonstrating E2F7 (top left) and E2F8 (bottom left) expression in wild type E10.5 TGCs but not mutant controls. Arrows point to selected TGCs. Scale bar, 10µm. c, Representative H&E sections of E10.5 control and E2f7^−/−;E2f8^−/− (78dko) placentas. Inset, a 78dko TGC in metaphase. d, Feulgen quantification of genome ploidy in E10.5 TGCs, n=3 placentas analyzed per genetic group. e–g, Immunostaining and quantification of S and M phase proteins in E10.5 control and 78dko TGCs. Arrows point to TGCs. n=3 placentas analyzed per genetic group. Scale bar in e–f, 12.5µm. h, Co-immunofluorescence showing E10.5 78dko TGCs in anaphase (left, P-H3) and metaphase (right, PL-1). DAPI stained total DNA. Scale bar, 5µm. i, Representative confocal images of nuclei in E10.5 control and 78dko TGCs (top) and 3D reconstruction of a binucleated 78dko TGC (bottom). Draq5 stained total DNA pseudocolored in green. Scale bar, 10µm. j, Left, transmission electron micrograph of a 78dko E10.5 TGC (left top, arrows indicate two nuclei; left bottom, enlarged view of boxed area showing separation between nuclear envelopes). Right, quantification of binucleated E10.5 TGCs, n=3 placentas analyzed per genetic group. k, X-gal staining of E10.5 control and Plf^cre/+ placentas carrying the reporter allele Rosa26^LoxP. l, PCR genotyping of genomic DNA isolated from laser capture microdissected E10.5 control and Plf^cre/+ TGCs. e, f and i control, E2f7^+/+;E2f8^+/+; e, f and h–j 78dko, E2f7^−/−;E2f8^−/−; k control, Plf^+/+;Rosa26^LoxP/+; l control, Plf^+/+;E2f7^f/f;E2f8^f/f; d, g, and j Plf-78dko, Plf^cre/+;E2f7^f/f;E2f8^f/f. PL-1, placental lactogen 1; N.E., nuclear envelope. Data in a, d, g and j reported as average values, ± SD are included when n>2 samples were analyzed. One-way ANOVA, * p≤0.05; ** p≤0.01; ***p≤0.001.

Figure 3. E2f8 is sufficient to promote hepatocyte endocycles

a, Representative H&E sections of aged (12mo) livers. Scale bar, 12.5µm. b, Quantitative RT-PCR analysis of E2f7 and E2f8 in pre- (E13.5) and post-natal (P0-12mo) wild type livers, with n per time point analyzed as indicated. 3wk*, 1 day post weaning (3wk). c, Left graph, assessment of nuclei volume by confocal imaging and 3-D reconstruction in 6-month-old control and Alb-78dko hepatocytes. n, number of nuclei evaluated per genotype. Right panels, representative confocal images of DAPI-stained liver sections. Scale bar, 10µm. d, Assessment of hepatocyte size in young (3wk), adult (2-6mo) and aged (12mo) livers by quantifying number of hepatocytes per image field, with n per genetic group analyzed as indicated. e, Left graph, quantification of binucleated hepatocytes in young, adult and aged livers, with n per genetic group analyzed as indicated. Right panels, immunofluoresence with anti-E-cadherin marking the boundary of mono- or binucleated hepatocytes. Scale bar, 10µm. f, Left column, flow cytometry of liver nuclei in young, adult and aged livers. n, number of livers analyzed per genotype and age group. Right column, representative FACS profiles of liver nuclei from aged (12mo) mice. g, Immunohistochemistry and quantification of hepatocyte proliferation in 2-month-old livers, with n per genetic group analyzed as indicated. Scale bar, 10µm. control, E2f7^f/f;E2f8^f/f; Alb-7ko, Alb-cre;E2f7^f/f; Alb-8ko, Alb-cre;E2f8^f/f; Alb-78dko, Alb-cre;E2f7^f/f;E2f8^f/f. FACS, fluorescence activated cell sorting. Data in b–g reported as average values, ± SD are included when n>2 samples were analyzed. One-way ANOVA, * p≤0.05; ** p≤0.01; ***p≤0.001.

Figure 4. Canonical activator and atypical repressor E2Fs regulate key transcriptional networks coordinating endocycles

a, Heatmap of approximately 4,500 differentially expressed genes in weaning age (3wk) livers. Class I: genes regulated by E2F7 and E2F8. Class II: genes regulated by either E2F7 or E2F8. Class III: genes synergistically regulated by E2F7/E2F8. n=3–4 livers analyzed per genetic group. control, E2f7^f/f;E2f8^f/f; Alb-78dko, Alb-cre;E2f7^f/f;E2f8^f/f; Alb-8ko, Alb-cre;E2f8^f/f; Alb-7ko, Alb-cre;E2f7^f/f. b, Top, heatmap of TGC gene expression in a custom NanoString mRNA codeset. RNA was isolated from laser capture microdissected E10.5 TGCs in frozen placental tissues. Bottom, G₂/M-related genes in NanoString codeset with deregulated expression in TGCs. n=2 placentas analyzed per genetic group. control, E2f7^+/+;E2f8^+/+; 78dko, E2f7^−/−;E2f8^−/−; 8ko, E2f7^+/+;E2f8^−/−; 7ko, E2f7^−/−;E2f8^+/+. c, Heatmap of downregulated genes in Mx-123tko livers with significantly deregulated expression in Alb-78dko livers. n=3–4 livers analyzed per genetic group. Alb-78dko, Alb-cre;E2f7^f/f;E2f8^f/f; Mx-123tko, Mx-cre;E2f1^−/−;E2f2^−/−;E2f3^f/f. d, Top five Molecular & Cellular Functions revealed through Ingenuity Pathway Analysis (IPA) of genes antagonistically regulated by E2F1-3 and E2F7/E2F8.

Figure 5. Atypical repressors E2F7/E2F8 directly bind gene targets involved in endocycle control

a, Immunoblot of transfected HepG2 cells showing exogenous expression of flag-tagged E2F7 (F7) and E2F8 (F8) proteins with Tubulin as control. Arrows indicate tagged protein. b, Representative chromatin immunoprecipitation (ChIP) assay in transfected HepG2 cells with anti-Flag antibodies demonstrating enhanced occupancy of F7 and F8 proteins on E2F binding sites in the promoter of G₁/S (E2f1 and Ccne2) and G₂/M (Ccna2 and Chek1) genes. The Tubulin (Tub) gene, a non-E2F target, demonstrates specific recruitment of F7/F8 to target promoters containing consensus E2F binding sequences. n=3 independent transfection-ChIP experiments performed. c, Immunoblot of transfected Rcho-1 TS cells showing exogenous expression of F7 and F8 proteins with Tubulin as control. Arrows indicate tagged protein. d, Representative ChIP assays in transfected Rcho-1 TS cells of G₁/S and G₂/M genes as in HepG2 cells. n=3 independent transfection-ChIP experiments performed. e, Immunoblot of liver lysates from 6-week-old mice demonstrating expression of tagged E2F8 protein. A total of 10µg of plasmid DNA was delivered through tail vein injection. Lysates were prepared from livers 24 hours after injection. Tubulin served as loading control. n=3 and n=5 wild type mice were injected with control (empty) plasmid and plasmid expressing Flag-E2F8, respectively. One out of 5 had detectable expression of F8 as shown by Western blot (Fig. 5e). f, ChIP assay in liver lysates with anti-Flag antibodies demonstrating enhanced occupancy of F8 on E2F binding sites in the promoter of G₁/S (E2f1 and Ccne2) and G₂/M (Cdk1) genes. a–f con., empty plasmid; F7, plasmid expressing flag-tagged E2F7; F8, plasmid expressing flag-tagged E2F8. Data in b, d and f from the representative experiment are reported as average values from triplicate quantitative RT-PCR reactions ± SD.

Figure 6. Loss of E2f1 restores endocycles in E2f7/E2f8 deficient TGCs and hepatocytes

a, Representative H&E sections of E10.5 TGCs. Scale bar, 12.5µm. b, Feulgen quantification of genome ploidy in E10.5 TGCs, n=3 placentas per genetic group analyzed. Note significant increase in proportions of 64C and 128C Plf-178tko TGCs relative to Plf-78dko TGCs. c, Quantification of E10.5 TGCs undergoing karyokinesis and expressing S/M phase markers, n=3 placentas per genetic group analyzed. Note significant reduction in proportion of Plf-178tko TGCs undergoing karyokinesis relative to Plf-78dko TGCs. d, Representative H&E liver sections from 6-month-old mice. Scale bar, 10µm. e, Measurements showing no significant change in percent hepatic mass in 6-month-old mice and a significant decrease in number of hepatocytes per image field in Alb-178tko relative to Alb-78dko livers, with n per genetic group analyzed as indicated in Fig. 6f. f, Flow cytometry of liver nuclei in 6-month-old mice, with n per genetic group analyzed as indicated in legend. Note significant increase and decrease in proportion of 4C and 2C nuclei, respectively, in Alb-178tko livers relative to Alb-78dko livers. g, Quantification of binucleated hepatocytes in 2-month-old livers, with n per genetic group analyzed as indicated in Fig. 6f. h, Quantification of hepatocyte proliferation in 2-month-old livers, with n per genetic group analyzed as indicated in Fig. 6f. a–c control, E2f7^+/+;E2f8^+/+; d-h control, E2f7^f/f;E2f8^f/f; 1ko, E2f1^−/−; Plf-178tko, Plf^cre/+;E2f1^−/−;E2f7^f/f;E2f8^f/f; Alb-178tko, Alb-cre;E2f1^−/−;E2f7^f/f;E2f8^f/f. Data in b, c and e-h reported as average ± SD. b and f One-way ANOVA, * p≤0.05; ** p≤0.01; ***p≤0.001. c, e, g and h, Two-tailed Student T-test, * p≤0.05; ** p≤0.01; ***p≤0.001.

Figure 7. Cyclin A ablation reinstates genome ploidy of E2f7/E2f8 deficient TGCs and hepatocytes

a, H&E images of E10.5 TGCs. The combined loss of Ccna1 and Ccna2 (Plf-A¹²dko) led to TGCs similar in appearance to control TGCs. The additional loss of Ccna1 and Ccna2 in E2f7/E2f8 deficient TGCs (Plf-A¹²78qko) led to TGCs with normal (wildtype-like) appearance, in contrast to Plf-78dko TGCs that appeared small or were binucleated (yellow arrows). Scale bar, 12.5µm. b, Feulgen quantification of genome ploidy in E10.5 TGCs. The intensities (i.e. estimated genome content) of 120–140 TGCs were quantified per placenta sample, with n placenta samples analyzed per genetic group as indicated. A number of TGCs quadruply deficient for Ccna1, Ccna2, E2f7 and E2f8 reach ploidy levels of 128C and 256C. control, E2f7^f/f;E2f8^f/f; Plf-A¹², Plf^cre/+;Ccna1^−/−;Ccna2^f/f; Plf-78dko, Plf^cre/+;E2f7^f/f;E2f8^f/f; Plf-A¹²78qko, Plf^cre/+;Ccna1^−/−;Ccna2^f/f;E2f7^f/f;E2f8^f/f. c, H&E images of 3-month-old liver sections, which were stained with the Feulgen technique to facilitate quantification of DNA content, showing an increase in both cellular and nuclear size of hepatocytes lacking Ccna1 and Ccna2 (Alb-A¹²dko) when compared with control hepatocytes. Hepatocytes quadruply deficient for Ccna1, Ccna2, E2f7 and E2f8 (Alb-A¹²78qko) had nuclei resembling control hepatocytes. Scale bar, 12.5µm. d, Feulgen quantification of genome ploidy in livers of 3-month-old mice. The intensities (i.e. estimated genome content) of 100 hepatocyte nuclei were quantified per liver sample, with n liver samples analyzed per genetic group as indicated. Ccna1/Ccna2 deficient livers had increased proportion of hepatocytes with higher ploidy levels (16C, 32C and 64C) relative to control livers. Quadruple deficient livers had significantly elevated number of hepatocytes with 16C genomes relative to Alb-78dko livers. control, E2f7^f/f;E2f8^f/f; Alb-A¹², Alb-cre;Ccna1^−/−;Ccna2^f/f; Alb-78dko, Alb-cre;E2f7^f/f;E2f8^f/f; Alb-A¹²78qko, Alb-cre;Ccna1^−/−;Ccna2^f/f;E2f7^f/f;E2f8^f/f. Data in b and d reported as average values, ± SD are included when n>2 samples were analyzed. One-way ANOVA, * p≤0.05; ***p≤0.001.