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

Mentions: To explore the functions of the E2F activator subclass, we derived E2f1−/−;E2f2−/−;E2f3LoxP/− ES cells (Supplementary Fig. 1a, 1b) and compared the consequences of inactivating the conditional E2f3LoxP allele in these cells versus in E2f1−/−;E2f2−/−;E2f3LoxP/LoxP MEFs. The expression of E2f1, E2f2 and E2f3 in wild type ES cells was generally higher than in MEFs and the loading of E2F3 protein on classic E2F target promoters was comparable between the two proliferating cell types (Supplementary Fig. 2a-c). Consistent with previous observations, the ablation of E2f1-3 in MEFs with standard cre-expressing vectors led to the induction of p53 activity, the loading of E2F4-p130 repressor complexes on E2F target promoters and a marked decrease in E2F target expression (Fig. 1a, Supplementary Fig. 3a–c)5–7. Consequently, triply deficient MEFs underwent a complete cell cycle arrest (Fig. 1b)5–7. In contrast, E2f1−/−;E2f2−/−;E2f3Δ/−(TKO) ES cells failed to activate p53 or form E2F4/p130 repressive complexes, and as a result, E2F target expression was unaffected and cells proliferated equally well as E2f1−/−;E2f2−/−;E2f3LoxP/− (DKO) control cells (Supplementary Fig. 3a-c).


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)

Cell proliferation in the absence of E2f1-3a 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2806193&req=5

Figure 1: Cell proliferation in the absence of E2f1-3a 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.
Mentions: To explore the functions of the E2F activator subclass, we derived E2f1−/−;E2f2−/−;E2f3LoxP/− ES cells (Supplementary Fig. 1a, 1b) and compared the consequences of inactivating the conditional E2f3LoxP allele in these cells versus in E2f1−/−;E2f2−/−;E2f3LoxP/LoxP MEFs. The expression of E2f1, E2f2 and E2f3 in wild type ES cells was generally higher than in MEFs and the loading of E2F3 protein on classic E2F target promoters was comparable between the two proliferating cell types (Supplementary Fig. 2a-c). Consistent with previous observations, the ablation of E2f1-3 in MEFs with standard cre-expressing vectors led to the induction of p53 activity, the loading of E2F4-p130 repressor complexes on E2F target promoters and a marked decrease in E2F target expression (Fig. 1a, Supplementary Fig. 3a–c)5–7. Consequently, triply deficient MEFs underwent a complete cell cycle arrest (Fig. 1b)5–7. In contrast, E2f1−/−;E2f2−/−;E2f3Δ/−(TKO) ES cells failed to activate p53 or form E2F4/p130 repressive complexes, and as a result, E2F target expression was unaffected and cells proliferated equally well as E2f1−/−;E2f2−/−;E2f3LoxP/− (DKO) control cells (Supplementary Fig. 3a-c).

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