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Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX

Nature Structural & Molecular Biology volume 17, pages 299–305 (2010)Cite this article

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Abstract

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication-fork stalling results in phosphorylation of histone H2A yielding γ-H2A (yeast γ-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Here, we describe the genome-wide location analysis of γ-H2A as a strategy to identify loci prone to engaging the Mec1 and Tel1 pathways. Notably, γ-H2A enrichment overlaps with loci prone to replication-fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for γ-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of γ-H2A at these loci. Finally, our work indicates that high-resolution mapping of γ-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.

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Figure 1: Genome-wide location analysis of γ-H2A.
Figure 2: Mutation of the TFIIIB-binding site of a tRNA gene abolishes DNA polymerase and γ-H2A enrichment.
Figure 3: Mapping of the γ-H2A enrichment in cells harboring different combinations of TEL1, MEC1 and SML1 deletions on natural replication-fork barriers.
Figure 4: Mutation of the H2A Ser129 residue increases the frequency of gross chromosomal rearrangements.
Figure 5: Many inactive genes have γ-H2A enrichment.
Figure 6: γ-H2A accumulation at many inactive genes depends on HDAC activities.

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Acknowledgements

We thank the members of the Durocher and Robert laboratories for their help and discussions, N. Sugimoto (Univ. of Medicine and Dentistry of New Jersey), R. Rothstein (Columbia Univ.), D. Lydall (Newcastle Univ.), K. Nasmyth (Univ. Oxford), A. Amon (Massachusetts Institute of Technology), W. Bonner (National Cancer Institute), A. Verreault (Univ. Montréal), K. Labib (Univ. of Manchester), J. Wyrick (Washington State Univ.) and R. Young (Whitehead Institute for Biomedical Research) for gifts of strains, A. Verrault also for the gift of histone H4 antibodies, G. Brown (Univ. of Toronto) for the gift of the Rad52-YFP expression plasmid and P. Pasero (Institute of Human Genetics, Centre Nationale de la Recherche Scientifique, France) for kindly communicating unpublished results. D.D. is the Thomas Kierans Chair in Mechanisms of Cancer Development and holds a Canada Research Chair (Tier II) in Proteomics, Bioinformatics and Functional Genomics. F.R. holds a New Investigator Award from the Canadian Institutes of Health Research. P.-É.J. holds a postdoctoral award from the Institut de recherches cliniques de Montréal training program in cancer research funded by the Canadian Institutes of Health Research. This work was funded by grants from the Canadian Cancer Society Research Institute to D.D. and from the Canadian Institutes of Health Research to F.R. (MOP82891).

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  1. Rachel K Szilard and Pierre-Étienne Jacques: These authors contributed equally to this work.

Authors and Affiliations

  1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Rachel K Szilard, Benjamin Cheng, Sarah Galicia, ManTek Yeung, Megan Mendez & Daniel Durocher

  2. Laboratoire de chromatine et expression du génome, Institut de recherches cliniques de Montréal, Montréal, Québec, Canada

    Pierre-Étienne Jacques, Louise Laramée, Alain R Bataille, Maxime Bergeron & François Robert

  3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    ManTek Yeung & Daniel Durocher

  4. Département de Médecine, Université de Montréal, Montréal, Québec, Canada

    François Robert

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Contributions

R.K.S. generated most yeast strains and performed most ChIP-chip experiments as well as the TEL06R qPCR (assisted by M.M.) and GCR (assisted by S.G.) experiments; P.-É.J. analyzed all ChIP-chip data; L.L. performed the mutant tRNA qPCR experiment; additional ChIP-chip experiments were performed by B.C. (wild-type and h2a-S129A γ-H2A), A.R.B. (MCMs) and M.B. (RNAPII); M.Y. performed focus formation assays; D.D. and F.R., with help from P.-É.J. and R.K.S., conceived the experiments and wrote the manuscript.

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Correspondence to François Robert or Daniel Durocher.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–7 and Supplementary Methods (PDF 7723 kb)

Supplementary File 1

The combined datasets of all the ChIP-chip data used in this study (Supplementary Table 1, along with the γ-H2A sites identified with the two sets of parameters analyzed (Supplementary Table 2). The file, compressed with gzip, is in the BED format compatible with the UCSC Genome Browser (http://genome.ucsc.edu/). (TXT 24746 kb)

Supplementary File 2

The mean log2 calculated on the promoter (defined as half of the upstream intergenic regions and up to 250 bp6) and on the complete length of each annotation of SGD (version Feb. 02 2008) for each ChIP-chip dataset produced in this study. (TXT 4396 kb)

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Szilard, R., Jacques, PÉ., Laramée, L. et al. Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX. Nat Struct Mol Biol 17, 299–305 (2010). https://doi.org/10.1038/nsmb.1754

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