Abstract

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis1,2. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world’s biomass3,4. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase5,6. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modelling, we show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4CDT2 ubiquitin ligase, and this in turn mediates the destruction of E2F1 (ref. 7). We propose that it is the transient loss of E2F1 during S phases that creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, we find that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants1,2,8, indicating that elements of this mechanism act in most growth-dependent cell cycles.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Endoreplication cell cycles: more for less. Cell 105, 297–306 (2001)

  2. 2.

    & New insights into cell cycle control from the Drosophila endocycle. Oncogene 24, 2765–2775 (2005)

  3. 3.

    & “Big it up”: endoreduplication and cell-size control in plants. Curr. Opin. Plant Biol. 6, 544–553 (2003)

  4. 4.

    , & Prokaryotes: the unseen majority. Proc. Natl Acad. Sci. USA 95, 6578–6583 (1998)

  5. 5.

    , & Fluctuations in cyclin E levels are required for multiple rounds of endocycle S phase in Drosophila. Curr. Biol. 8, 235–238 (1998)

  6. 6.

    , , & Continuous cyclin E expression inhibits progression through endoreduplication cycles in Drosophila. Curr. Biol. 8, 239–242 (1998)

  7. 7.

    et al. Intrinsic negative cell cycle regulation provided by PIP box- and Cul4Cdt2-mediated destruction of E2f1 during S phase. Dev. Cell 15, 890–900 (2008)

  8. 8.

    et al. Cullin 4-ring finger-ligase plays a key role in the control of endoreplication cycles in Arabidopsis trichomes. Proc. Natl Acad. Sci. USA 107, 15275–15280 (2010)

  9. 9.

    Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786 (2004)

  10. 10.

    , , , & The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles. Genes Dev. 22, 1690–1703 (2008)

  11. 11.

    et al. Dampened activity of E2F1-DP and Myb-MuvB transcription factors in Drosophila endocycling cells. J. Cell Sci. 123, 4095–4106 (2010)

  12. 12.

    et al. Cyclin E controls S-phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 77, 107–120 (1994)

  13. 13.

    & The Drosophila endocycle is controlled by cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev. 10, 2514–2526 (1996)

  14. 14.

    et al. APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle. Development 135, 1451–1461 (2008)

  15. 15.

    , , & Coordination of growth and cell division in the Drosophila wing. Cell 93, 1183–1193 (1998)

  16. 16.

    & Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90, 671–681 (1997)

  17. 17.

    , , & The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions. Development 131, 3169–3181 (2004)

  18. 18.

    & Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster. Chromosoma 91, 279–286 (1985)

  19. 19.

    et al. The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles. EMBO J. 26, 2071–2082 (2007)

  20. 20.

    & Developmental control of the G1 to S transition in Drosophila: cyclin E is a limiting downstream target of E2F. Genes Dev. 9, 1456–1468 (1995)

  21. 21.

    , & Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program. Genes Dev. 11, 1999–2011 (1997)

  22. 22.

    , & Mutations of the Drosophila dDP, dE2F, and cyclin E genes reveal distinct roles for the E2F-DP transcription factor and cyclin E during the S-phase transition. Mol. Cell. Biol. 18, 141–151 (1998)

  23. 23.

    , & Ectopic E2F expression induces S-phase and apoptosis in Drosophila imaginal discs. Genes Dev. 10, 1422–1432 (1996)

  24. 24.

    & Negative regulation of dE2F1 by cyclin-dependent kinases controls cell cycle timing. Cell 117, 253–264 (2004)

  25. 25.

    , , & Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila. BMC Genet. 4, 9 (2003)

  26. 26.

    , , & Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development. EMBO J. 22, 3865–3875 (2003)

  27. 27.

    et al. Functional antagonism between E2F family members. Genes Dev. 15, 2146–2160 (2001)

  28. 28.

    & Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125, 2149–2158 (1998)

  29. 29.

    , , , & Drosophila’s insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2, 239–249 (2002)

  30. 30.

    , , , & The Drosophila F box protein archipelago regulates dMyc protein levels in vivo. Curr. Biol. 14, 965–974 (2004)

  31. 31.

    et al. Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 87, 1225–1235 (1996)

  32. 32.

    et al. A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2. Genetics 155, 233–244 (2000)

  33. 33.

    , & dDP is needed for normal cell proliferation. Mol. Cell. Biol. 25, 3027–3039 (2005)

  34. 34.

    , , , & The transcription factor E2F is required for S phase during Drosophila embryogenesis. Genes Dev. 9, 1445–1455 (1995)

  35. 35.

    , , & The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis. Genes Dev. 15, 2741–2754 (2001)

  36. 36.

    , , & The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation. Development 120, 535–544 (1994)

  37. 37.

    , , & The role of RBF in developmentally regulated cell proliferation in the eye disc and in cyclin D/Cdk4 induced cellular growth. Development 129, 1345–1356 (2002)

  38. 38.

    et al. Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nature Cell Biol. 5, 566–571 (2003)

  39. 39.

    & Rca1 inhibits APC-Cdh1(Fzr) and is required to prevent cyclin degradation in G2. Dev. Cell 2, 29–40 (2002)

  40. 40.

    , , & Drosophila G1-specific cyclin E homolog exhibits different modes of expression during embryogenesis. Development 119, 673–690 (1993)

  41. 41.

    & Developmental control of a G1-S transcriptional program in Drosophila. Development 120, 1503–1515 (1994)

  42. 42.

    , , , & DNA-binding and trans-activation properties of Drosophila E2F and DP proteins. Proc. Natl Acad. Sci. USA 91, 6359–6363 (1994)

  43. 43.

    et al. Multiplex detection of RNA expression in Drosophila embryos. Science 305, 846 (2004)

  44. 44.

    & A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85 (1989)

  45. 45.

    , & A Caenorhabditis elegans nutrient response system partially dependent on nuclear receptor NHR-49. Proc. Natl Acad. Sci. USA 102, 13496–13501 (2005)

Download references

Acknowledgements

Supported by NIH GM51186 to B.A.E., a DAAD fellowship to N.Z., NIGMS 5 P50 GM66050 and NSF MCB0090835 to G.v.D. and K.J.K, DFG LE987/5-1 to C.F.L., CIHR MOP-86622 to S.G., and NIH GM57859 to R.J.D. We thank Y. Liu for help with statistics.

Author information

Author notes

    • Norman Zielke
    • , Kerry J. Kim
    •  & Vuong Tran

    These authors contributed equally to this work.

Affiliations

  1. German Cancer Research Center (DKFZ)-Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH) Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany

    • Norman Zielke
    • , Monique van Straaten
    •  & Bruce A. Edgar
  2. Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109, USA

    • Norman Zielke
    • , Vuong Tran
    • , Maria-Jose Bravo
    • , Brigitte Woods
    •  & Bruce A. Edgar
  3. Center for Cell Dynamics, Friday Harbor Labs, University of Washington, 620 University Road, Friday Harbor, Washington 98250, USA

    • Kerry J. Kim
    •  & George von Dassow
  4. Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA

    • Shusaku T. Shibutani
    •  & Robert J. Duronio
  5. Clark H. Smith Brain Tumor Center, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive, Calgary, Alberta T2N 4N1, Canada

    • Sabarish Nagarajan
    •  & Savraj S. Grewal
  6. Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive, Calgary, Alberta T2N 4N1, Canada

    • Sabarish Nagarajan
    •  & Savraj S. Grewal
  7. Institute of Molecular and Life Sciences (IMLS), Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland

    • Carmen Rottig
    •  & Christian F. Lehner

Authors

  1. Search for Norman Zielke in:

  2. Search for Kerry J. Kim in:

  3. Search for Vuong Tran in:

  4. Search for Shusaku T. Shibutani in:

  5. Search for Maria-Jose Bravo in:

  6. Search for Sabarish Nagarajan in:

  7. Search for Monique van Straaten in:

  8. Search for Brigitte Woods in:

  9. Search for George von Dassow in:

  10. Search for Carmen Rottig in:

  11. Search for Christian F. Lehner in:

  12. Search for Savraj S. Grewal in:

  13. Search for Robert J. Duronio in:

  14. Search for Bruce A. Edgar in:

Contributions

The E2F1-based oscillator was conceived by B.A.E.; N.Z. developed the framework for licensing control and E2F2-mediated repression of mitotic genes. K.J.K. did most of the computational modeling, which was initiated by G.v.D. Initial experiments were done by V.T., who, with help from B.W. and K.J.K., contributed Figs 1d–f, 2a, b, 4a, b and Supplementary Fig. 13. N.Z. carried out much of the later experimental work with help from M.v.S., and contributed Figs 2b, c, 3c–j, 4c and Supplementary Figs 1, 3, 11, 12 and 14–20. S.T.S and R.J.D. contributed the GFP–E2F1PIP3A transgenics and controls. M.-J.B. contributed Figs 1a–c, 3a, b and Supplementary Fig. 15g, h. S.N. and S.S.G. contributed Fig 4d. C.R. and C.F.L. contributed Supplementary Fig. 2 and the cdk2−/− data in Fig. 2b. B.A.E. directed the project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Bruce A. Edgar.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    The file contains, Supplementary Text including Supplementary Methods and a Supplementary Discussion (see Contents for details), Supplementary Tables 1-2, Supplementary Figures 1-20 with legends and additional references.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature10579

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.