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.
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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.
The authors declare that they have no competing financial interests.
Includes supplementary methods and references (DOC 40 kb)
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)
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Aggarwal, B., Calvi, B. Chromatin regulates origin activity in Drosophila follicle cells. Nature 430, 372–376 (2004). https://doi.org/10.1038/nature02694
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