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

Chong JL, Wenzel PL, Sáenz-Robles MT, Nair V, Ferrey A, Hagan JP, Gomez YM, Sharma N, Chen HZ, Ouseph M, Wang SH, Trikha P, Culp B, Mezache L, Winton DJ, Sansom OJ, Chen D, Bremner R, Cantalupo PG, Robinson ML, Pipas JM, Leone G - Nature (2009)

Bottom Line: In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.

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.

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Apoptosis of crypt intestinal cells in the absence of E2f1, E2f2, and E2f3a H&E stained sections from E2f1−/−;E2f2−/−;E2f3LoxP/LoxP (control) and Ah-cre;E2f1−/−;E2f2−/−;E2f3LoxP/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.
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Figure 2: Apoptosis of crypt intestinal cells in the absence of E2f1, E2f2, and E2f3a H&E stained sections from E2f1−/−;E2f2−/−;E2f3LoxP/LoxP (control) and Ah-cre;E2f1−/−;E2f2−/−;E2f3LoxP/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.

Mentions: To explore whether E2F1-3 might have cell cycle-related functions in tissues that arise later in embryonic and postnatal development, we exploited the highly organized cellular architecture of the small intestine. Maintenance of structural and functional integrity of the small intestine requires continuous epithelial regeneration13. Intestinal stem cells are housed at the base of crypts of Lieberkühn and give rise to transit-amplifying cells. As these cells migrate up from the base and into the finger-like extensions called villi, they exit the cell cycle and differentiate13. Western blot assays showed that E2f1, E2f2 and both isoforms of E2f3 (E2F3a and E2F3b) are expressed in the crypt and villus (Supplementary Fig. 6). We used Ah-cre mice14 to ablate E2f1-3 in the small intestine in utero or in adult mice (Ah-cre;E2f1−/−;E2f2−/−;E2f3LoxP/LoxP, TKO). Induction of Ah-cre expression by intraperitoneal injection of β-napthoflavone (β-NF) led to the efficient deletion of E2f3LoxP in crypt stem cells and transit-amplifying cells by one day post-injection, and in the entire intestinal epithelium within 3–4 days (crypt and villus; Supplementary Fig. 7a–c). Loss of E2f1-3 did not result in a compensatory increase of other E2F family members, except for a modest increase in E2f8 (Supplementary Fig. 7d). Whether E2f3LoxP was deleted in utero at E15.5 or in the adult at 2 months of age, the architecture of TKO small intestines remained relatively intact and animals were asymptomatic for 90 days following β-NF administration (Fig. 2a, Supplementary Fig. 8a, 8b). Cell-type specific marker analysis demonstrated that all differentiated epithelial cell-types were appropriately represented in TKO small intestines (Fig. 2b, Supplementary Fig. 9). Remarkably, cell proliferation was identical in TKO and control intestines (Fig. 2c), however, we noted a marked increase in γ-H2AX and P-ATM1981 staining in TKO crypts and villi (Fig. 2d, 2e, Supplementary Fig. 10a). A parallel analysis of retinal (Chen et al, accompanying manuscript) and lens (P.W. unpublished observations) progenitors also revealed increased γ-H2AX staining in TKO samples (Supplementary Fig. 10b, 10c). Together, these observations suggest that counter to current dogma, E2F1-3 are dispensable for the proliferation of embryonic stem cells and their mesodermal, endodermal, and ectodermal derivatives, and cells in at least some adult tissues.


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

Chong JL, Wenzel PL, Sáenz-Robles MT, Nair V, Ferrey A, Hagan JP, Gomez YM, Sharma N, Chen HZ, Ouseph M, Wang SH, Trikha P, Culp B, Mezache L, Winton DJ, Sansom OJ, Chen D, Bremner R, Cantalupo PG, Robinson ML, Pipas JM, Leone G - Nature (2009)

Apoptosis of crypt intestinal cells in the absence of E2f1, E2f2, and E2f3a H&E stained sections from E2f1−/−;E2f2−/−;E2f3LoxP/LoxP (control) and Ah-cre;E2f1−/−;E2f2−/−;E2f3LoxP/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.
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Figure 2: Apoptosis of crypt intestinal cells in the absence of E2f1, E2f2, and E2f3a H&E stained sections from E2f1−/−;E2f2−/−;E2f3LoxP/LoxP (control) and Ah-cre;E2f1−/−;E2f2−/−;E2f3LoxP/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.
Mentions: To explore whether E2F1-3 might have cell cycle-related functions in tissues that arise later in embryonic and postnatal development, we exploited the highly organized cellular architecture of the small intestine. Maintenance of structural and functional integrity of the small intestine requires continuous epithelial regeneration13. Intestinal stem cells are housed at the base of crypts of Lieberkühn and give rise to transit-amplifying cells. As these cells migrate up from the base and into the finger-like extensions called villi, they exit the cell cycle and differentiate13. Western blot assays showed that E2f1, E2f2 and both isoforms of E2f3 (E2F3a and E2F3b) are expressed in the crypt and villus (Supplementary Fig. 6). We used Ah-cre mice14 to ablate E2f1-3 in the small intestine in utero or in adult mice (Ah-cre;E2f1−/−;E2f2−/−;E2f3LoxP/LoxP, TKO). Induction of Ah-cre expression by intraperitoneal injection of β-napthoflavone (β-NF) led to the efficient deletion of E2f3LoxP in crypt stem cells and transit-amplifying cells by one day post-injection, and in the entire intestinal epithelium within 3–4 days (crypt and villus; Supplementary Fig. 7a–c). Loss of E2f1-3 did not result in a compensatory increase of other E2F family members, except for a modest increase in E2f8 (Supplementary Fig. 7d). Whether E2f3LoxP was deleted in utero at E15.5 or in the adult at 2 months of age, the architecture of TKO small intestines remained relatively intact and animals were asymptomatic for 90 days following β-NF administration (Fig. 2a, Supplementary Fig. 8a, 8b). Cell-type specific marker analysis demonstrated that all differentiated epithelial cell-types were appropriately represented in TKO small intestines (Fig. 2b, Supplementary Fig. 9). Remarkably, cell proliferation was identical in TKO and control intestines (Fig. 2c), however, we noted a marked increase in γ-H2AX and P-ATM1981 staining in TKO crypts and villi (Fig. 2d, 2e, Supplementary Fig. 10a). A parallel analysis of retinal (Chen et al, accompanying manuscript) and lens (P.W. unpublished observations) progenitors also revealed increased γ-H2AX staining in TKO samples (Supplementary Fig. 10b, 10c). Together, these observations suggest that counter to current dogma, E2F1-3 are dispensable for the proliferation of embryonic stem cells and their mesodermal, endodermal, and ectodermal derivatives, and cells in at least some adult tissues.

Bottom Line: In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.

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.

Show MeSH
Related in: MedlinePlus