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The E2F1–3 transcription factors are essential for cellular proliferation

Nature volume 414, pages 457–462 (2001)Cite this article

Abstract

The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities1,2. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition3. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

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Figure 1: Targeted disruption of the E2F3 locus.
Figure 2: E2Fs are required for cellular proliferation.
Figure 3: E2F activators regulate G1/S-specific target gene activation.
Figure 4: E2Fs are required for S phase entry and cell cycle progression.
Figure 5: Loss of E2Fs leads to an elevation of p21Cip1.

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Acknowledgements

We are grateful to R. Premont for providing the loxP vectors. We thank C. Bock for assistance in generating the E2F3 chimaeric mice; C. Brown and L. Jakoi for various technical assistance; and M. Weinstein for critical comments on the manuscript. This work was supported by grants from the National Institutes of Health (NIH) (G.L.). L.W. was supported by an NIH award, C.T. was supported by the Up on the Roof Human Cancer Genetics Posdoctoral Fellowship, J.R.N. is an investigator of the Howard Hughes Medical Institute, and G.L. is a V-Foundation and Pew Charitable Trust Scholar.

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Authors and Affiliations

  1. Division of Human Cancer Genetics, Department of Molecular Virology, and Department of Molecular Genetics, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, 43210, USA

    Lizhao Wu, Cynthia Timmers, Baidehi Maiti, Harold I. Saavedra, Ling Sang, Gabriel T. Chong, Fred A. Wright & Gustavo Leone

  2. Division of Molecular and Human Genetics, Children's Research Institute, The Ohio State University, Columbus, 43210, Ohio, USA

    Michael L. Robinson

  3. Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Faison Nuckolls, Paloma Giangrande & Joseph R. Nevins

  4. Department of Neuroscience, Harvard Medical School, Boston, Massachusetts, 02115

    Seth J. Field & Michael E. Greenberg

  5. Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Stuart Orkin

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Correspondence to Gustavo Leone.

Supplementary information

Supplementary Table 1

(GIF 3.18 KB)

Supplementary Figure 1

(GIF 11.6 KB)

Loss of E2F3 leads to defects in cellular proliferation and S-phase entry. Growth curves (a) and BrdU incorporation assays (b) of E2F-deficient primary MEFs (closed symbols) in comparison with their wild-type counterparts (open symbols).

Supplementary Figure 2

(GIF 8.03 KB)

Ectopic expression of E2F1, E2F2, or E2F3 can rescue the proliferation defects of TKO cells. Colony formation assays of E2F1-/-E2F2-/-E2F3f/f MEFs infected with the indicated retrovirus as well as pBpuro-Cre. (con): pBhygro; (E2F1, E2F2, E2F3): pBhygro-HA-tagged E2F1, E2F2, and E2F3a, respectively. Similar results were obtained in four independent experiments; a representative experiment is shown. (inset) Western blot using an antibody against HA-epitope.

Supplementary Figure 3

(JPG 23 KB)

Northern blot analysis of various E2F-responsive genes in E2F3f/f (a) or E2F2-/-E2F3f/ (b) MEFs that were infected with a control or a Cre-retrovirus. The experiment was performed as described in Figure 3a of the main text. (con): pBhygro; (E2F3a): pBhygro-myc-tagged E2F3a. c, Deletion of E2F1, E2F2, and E2F3 does not affect early mitogenic events. Western blot of retrovirus-infected E2F1-/-E2F2-/-E2F3f/f MEFs using antibodies against phosphorylated forms of Erk1/2 or Akt; the same blots were re-probed with antibodies against total Erk1/2 and Akt to verify equal loading (data not shown).

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Wu, L., Timmers, C., Maiti, B. et al. The E2F1–3 transcription factors are essential for cellular proliferation. Nature 414, 457–462 (2001). https://doi.org/10.1038/35106593

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