E2f1-3 switch from activators in progenitor cells to repressors in differentiating cells

Abstract

In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.

Figure 1. Cell proliferation in the absence of E2f1-3

a Expression of E2F-regulated genes was measured by real-time RT-PCR in proliferating ES and MEFs cells with the indicated genotypes (primer information is provided in Supplementary Fig. 19). b. Growth curves of two sets of DKO and TKO ES cell clones (A and B) and DKO and TKO MEFs. c. DKO and TKO ES cells were injected underneath the skin of athymic nude mice and teratomas were harvested, sectioned and stained with H&E. Representative tissues of DKO and TKO teratomas include muscle (mesoderm), respiratory epithelium (endoderm), skin and neural cells (ectoderm). d. Embryos derived from intercrosses between E2f1^+/−;E2f2^−/−;E2f3^+/− mice were collected at various timepoints during pregnancies. e. Representative E9.5 embryos were photographed immediately upon collection; E2f2^−/− (SKO), E2f2^−/−;E2f3^−/− (DKO), and E2f1^−/−E2f2^−/−E2f3^−/− (TKO) embryos.

Figure 2. Apoptosis of crypt intestinal cells in the absence of E2f1, E2f2, and E2f3

a H&E stained sections from E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP (control) and Ah-cre;E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP (Ah-cre) intestines after 90 days of β-NF administration. b. Analysis of cell differentiation in control and Ah-cre small intestines. Goblet cells were identified by Alcian blue staining (arrows point to positive-stained goblet cells); absorptive cells were identified by anti-Fatty acid binding protein (FABP, green) antibodies; DAPI (blue) was used for staining nuclei. c. BrdU (brown) and phosphorylated histone H3 (P-H3, red) immunohistochemical staining was performed on small intestine sections from β-NF injected control and Ah-cre mice. Quantification of BrdU- and phosphorylated histone H3-positive cells in crypts and villi. n=3, 3 different animals with the indicated genotypes were analyzed (bottom panels); error bars indicate standard deviation. d. Immunohistochemical staining for γ-H2AX, P-ATM¹⁹⁸¹ in control and Ah-cre intestinal crypts and villi. The orange dotted line outlines the luminal side of the villus; the white dotted line outlines the outer side of the villus. DAPI (blue) was used for staining nuclei. e. Examination of γ-H2AX and P-ATM¹⁹⁸¹ in cell extracts from control and Ah-cre intestinal crypts and villi by Western blot assays. f. Sections of small intestines from β-NF injected control and Ah-cre mice were processed for TUNEL (brown) and cleaved caspase-3 (red) assays. DAPI (blue) or hematoxylin was used for staining nuclei. Quantification of TUNEL and cleaved caspase-3 positive cells in crypts and villi (bottom panels). n=3, 3 different animals with the indicated genotypes were analyzed; error bars indicate standard deviation.

Figure 3. Repression of E2F-target genes in E2f1-3 deficient villi

a Scatter plots comparing expression of known E2F-target genes (see Supplementary Fig. 16b) between cell compartments (crypt and villus); E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP (control) and Ah-cre;E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP (Ah-cre). Genes with >1.5-fold increase in expression are depicted as red dots. b. Scatter plots comparing expression of known E2F-target genes between genotypes (control and Ah-cre samples); n=3 for each of the four samples. Red dots indicate genes whose expression increased >1.5-fold and blue dots indicate genes that decreased >1.5-fold. c. Quantitative real-time PCR was performed to compare the relative expression of selected E2F-target genes in control and Ah-cre crypts (left panels) and villi (right panels) using specific primers (Supplementary Fig. 20). d. Immunohistochemical staining of Mcm3 (green) and Pcna (red) in control and Ah-cre villi. DAPI (blue) was used for staining nuclei. Yellow dotted line outlines the luminal side of the villus; white dotted line outlines the outer side of the villus. Note that staining of blood cells in lumens of villi is non-specific. e. Chromatin immunoprecipitation (ChIP) assays using IgG or anti-E2F3 (α-3) antibodies with lysates from wild-type villi (Antibody control; top panels). ChIP assays using anti-E2F3 (α-3) antibodies with lysates from wild type (con) and Ah-cre (TKO) villi (Genotype control: bottom panels). Primers flanking known E2F-binding elements were used to detect the indicated gene promoters (Supplementary Fig. 19). f. Co-immunoprecipitation assays of cell extracts prepared from control villi and crypts. Imunoprecipitations (IP) used anti-E2F3 antibody or IgG. Anti-Rb antibody was used to probe Western blot (WB; left panel). The specificity of the anti-E2F3 antibody used in the left panel was evaluated in intestinal lysates derived from Ah-cre (3^−/−), E2f3a^−/− (3a^−/−), E2f3b^−/− (3b^−/−) and E2f3^+/+ mice. Anti-Rb antibody was used to probe Western blot (WB; right panel).

Figure 4. E2F1-3 contribute to the ectopic cell proliferation caused by Rb-deficiency

a BrdU analysis was performed in E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP (control), Ah-cre;Rb^LoxP/LoxP (RbKO) and Ah-cre;E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP;Rb^LoxP/LoxP (QKO) small intestines. b. Quantification of BrdU incorporation. n=3, 3 different animals with the indicated genotypes were analyzed; error bars indicate standard deviation. c. Scatter plot analysis comparing differentially expressed E2F target genes in control, RbKO, Ah-cre;E2f1^−/−;E2f2^−/−;E2f3^LoxP/LoxP (TKO) and QKO villi; n=3 for each of the eight samples. Red dots indicate genes whose expression increased >1.5-fold and blue dots indicate gene that decreased >1.5-fold. d. Quantitative RT-PCR analysis of selected E2F-target genes in control (con), RbKO, TKO, and QKO villi. The normal basal level of E2F target expression is illustrated as a grey dotted line and the threshold level of E2F target expression required for ectopic proliferation is illustrated as a red dotted line.