Chromatin regulates origin activity in Drosophila follicle cells


It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds1. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes2. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Histone hyperacetylation and ORC2 co-localize at chorion origins.
Figure 2: ChIP analysis indicates that nucleosomes are hyperacetylated at the 3rd chromosome chorion origin.
Figure 3: Rpd3 loss-of-function clones show hyperacetylation, increased replication and altered ORC2 distribution.
Figure 4: Sodium butyrate induces extra replication, which is not blocked by α-amanitin.
Figure 5: Tethering chromatin modifiers to chorion origins alters their activity.


  1. 1

    Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002)

    CAS  Article  Google Scholar 

  2. 2

    Calvi, B. R., Lilly, M. A. & Spradling, A. C. Cell cycle control of chorion gene amplification. Genes Dev. 12, 734–744 (1998)

    CAS  Article  Google Scholar 

  3. 3

    Claycomb, J. M., Benasutti, M., Bosco, G., Fenger, D. D. & Orr-Weaver, T. L. Gene amplification as a developmental strategy: isolation of two developmental amplicons in Drosophila. Dev. Cell 6, 145–155 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Spradling, A. & Mahowald, A. Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc. Natl Acad. Sci. USA 77, 1096–1100 (1980)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Calvi, B. R. & Spradling, A. C. Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control. Methods 18, 407–417 (1999)

    CAS  Article  Google Scholar 

  6. 6

    Calvi, B. R. & Spradling, A. C. The nuclear location and chromatin organization of active chorion amplification origins. Chromosoma 110, 159–172 (2001)

    CAS  Article  Google Scholar 

  7. 7

    Royzman, I., Austin, R. J., Bosco, G., Bell, S. P. & Orr-Weaver, T. L. ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP. Genes Dev. 13, 827–840 (1999)

    CAS  Article  Google Scholar 

  8. 8

    Whittaker, A. J., Royzman, I. & Orr-Weaver, T. L. Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts. Genes Dev. 14, 1765–1776 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Bandura, J. L. & Calvi, B. R. Duplication of the genome in normal and cancer cell cycles. Cancer Biol. and Ther. 1, 8–13 (2002)

    CAS  Article  Google Scholar 

  10. 10

    Austin, R. J., Orr-Weaver, T. L. & Bell, S. P. Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element. Genes Dev. 13, 2639–2649 (1999)

    CAS  Article  Google Scholar 

  11. 11

    de Cicco, D. & Spradling, A. Localization of a cis-acting element responsible for the developmentally regulated amplification of Drosophila chorion genes. Cell 38, 45–54 (1984)

    CAS  Article  Google Scholar 

  12. 12

    Lu, L., Zhang, H. & Tower, J. Functionally distinct, sequence-specific replicator and origin elements are required for Drosophila chorion gene amplification. Genes Dev. 15, 134–146 (2001)

    CAS  Article  Google Scholar 

  13. 13

    Claycomb, J. M., MacAlpine, D. M., Evans, J. G., Bell, S. P. & Orr-Weaver, T. L. Visualization of replication initiation and elongation in Drosophila. J. Cell Biol. 159, 225–236 (2002)

    CAS  Article  Google Scholar 

  14. 14

    Vogelauer, M., Rubbi, L., Lucas, I., Brewer, B. J. & Grunstein, M. Histone acetylation regulates the time of replication origin firing. Mol. Cell 10, 1223–1233 (2002)

    CAS  Article  Google Scholar 

  15. 15

    Mottus, R., Sobel, R. E. & Grigliatti, T. A. Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation. Genetics 154, 657–668 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Bosco, G., Du, W. & Orr-Weaver, T. L. DNA replication control through interaction of E2F-RB and the origin recognition complex. Nature Cell Biol. 3, 289–295 (2001)

    CAS  Article  Google Scholar 

  17. 17

    Schwed, G., May, N., Pechersky, Y. & Calvi, B. R. Drosophila minichromosome maintenance 6 is required for chorion gene amplification and genomic replication. Mol. Biol. Cell 13, 607–620 (2002)

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

    Cayirlioglu, P., Ward, W. O., Silver Key, S. C. & Duronio, R. J. Transcriptional repressor functions of Drosophila E2F1 and E2F2 cooperate to inhibit genomic DNA synthesis in ovarian follicle cells. Mol. Cell. Biol 23, 2123–2134 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Kadosh, D. & Struhl, K. Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo. Genes Dev. 12, 797–805 (1998)

    CAS  Article  Google Scholar 

  21. 21

    Iizuka, M. & Stillman, B. Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein. J. Biol. Chem. 274, 23027–23034 (1999)

    CAS  Article  Google Scholar 

  22. 22

    Grienenberger, A. et al. The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression. Curr. Biol. 12, 762–766 (2002)

    CAS  Article  Google Scholar 

  23. 23

    Burke, T. W., Cook, J. G., Asano, M. & Nevins, J. R. Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1. J. Biol. Chem. 276, 15397–15408 (2001)

    CAS  Article  Google Scholar 

  24. 24

    Simon, J. A. & Tamkun, J. W. Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. Curr. Opin. Genet. Dev. 12, 210–218 (2002)

    CAS  Article  Google Scholar 

  25. 25

    Takei, Y. et al. MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3. EMBO Rep. 2, 119–123 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Luo, R. X., Postigo, A. A. & Dean, D. C. Rb interacts with histone deacetylase to repress transcription. Cell 92, 463–473 (1998)

    CAS  Article  Google Scholar 

  27. 27

    Kennedy, B. K., Barbie, D. A., Classon, M., Dyson, N. & Harlow, E. Nuclear organization of DNA replication in primary mammalian cells. Genes Dev. 14, 2855–2868 (2000)

    CAS  Article  Google Scholar 

  28. 28

    Avni, D. et al. Active localization of the retinoblastoma protein in chromatin and its response to S phase DNA damage. Mol. Cell 12, 735–746 (2003)

    CAS  Article  Google Scholar 

  29. 29

    Beall, E. L. et al. Role for a Drosophila Myb-containing protein complex in site-specific DNA replication. Nature 420, 833–837 (2002)

    ADS  CAS  Article  Google Scholar 

Download references


We thank J. Simon and M. O'Connor for FRT Rpd3 flies; D. Buechle for hsp70:Gal4:Pc flies; M. Botchan, L. Beall, T. Orr-Weaver and S. Bell for ORC2 and Dup antibodies; D. Fyodorov and J. Kadonaga for Rpd3 antibody. We thank J. Claycomb for advice about QPCR. Thanks to N. May and M. Thomer for help with injections and flies. We are indebted to J. Bandura, A. K. Bielinsky, and M. Lilly for comments on the manuscript. This work was supported by a PHS grant to B.R.C.

Author information



Corresponding author

Correspondence to Brian R. Calvi.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Information

Includes supplementary methods and references (DOC 40 kb)

Supplementary Figure 1 (JPG 60 kb)

Supplementary Figure 2 (JPG 81 kb)

Supplementary Figure 3 (JPG 18 kb)

Supplementary Figure 4 (JPG 52 kb)

Supplementary Figure 5 (JPG 35 kb)

Supplementary Figure 6 (JPG 38 kb)

Supplementary Figure 7 (JPG 42 kb)

Supplementary Figure 8 (JPG 62 kb)

Supplementary Figure Legends

Figure S1 Developmental time course of BrdU and AcH4 labeling; Figure S2 Quantification of AcH4, ORC2, and TOTO-3 DNA label at the 3rd chorion locus; Figure S3 Acetylated foci are evident despite reduced chorion copy number in the mutant MCM6K1214; Figure S4 Antibodies for other acetylated forms of histone label chorion foci; Figure S5 Antibody labeling of Rpd3 mutant clones with Rpd3 antibody indicates they have reduced Rpd3 protein; Figure S6 Antibodies raised against other modifications of histone H4 indicate Rpd3 clones have widespread hyperacetylation; Figure S7 Quantification of ectopic BrdU in Rpd3 mutant clones; Figure S8 Quantification of ectopic ORC2 in Rpd3 mutant clones. (DOC 30 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Aggarwal, B., Calvi, B. Chromatin regulates origin activity in Drosophila follicle cells. Nature 430, 372–376 (2004).

Download citation

Further reading


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.


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing