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Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4

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Abstract

Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.

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Abbreviations

2D:

two-dimensional

3D:

three-dimensional

DSB:

double-strand break

ICA:

intensity correlation analysis

ICQ:

intensity correlation quotient

MeV:

Mega electron volts

PDM:

product of the differences from the mean

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Acknowledgments

We thank the staff of the Maier-Leibnitz-Laboratorium for operating the accelerator facility; K. Pfleghaar and K. Schneider for their help with laser microirradiation; B. Scholz for the help with RT-PCR; H. Hofmann, D. Wieland, and S. Lindemaier for excellent technical assistance, and S. Auer for managing the beam time. Anti-polymerase II Ser5-P and Ser2-P monoclonal antibodies as well as antibody Pol3/3 were a kind gift of D. Eick, anti-JARID1A a kind gift of E. Benevolenskaya, and anti-H3K27me3 a kind gift of T.E. Schmid. Parts of this work were supported by grants of the Bundesministerium für Bildung und Forschung (02S8457 and 03NUK007), the DFG Cluster of Excellence Munich Centre for Advanced Photonics, and the LMU innovative project Biomed-S.

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The authors declare no conflict of interest.

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Author notes

  1. Jacques Rouquette

    Present address: CRT-RIV-ITAV, Université de Toulouse, UPS, INSA, CNRS, UMS 3039, 1, place Pierre Potier, entrée B, BP 50624, 31106, Toulouse, Cedex 1, France

  2. Hilmar Strickfaden

    Present address: Department of Oncology, Faculty of Medicine, University of Alberta and Cross Cancer Institute, Edmonton, Canada

Authors and Affiliations

  1. Department of Radiation Oncology, University Hospital of Munich, Schillerstr. 42, 80336, Munich, Germany

    Doris M. Seiler, Christian Ottmann, Guido A. Drexler, Belinda Mazurek & Anna A. Friedl

  2. Department Biology II, University of Munich, Grosshadernerstr. 2, 82152, Martinsried, Germany

    Jacques Rouquette, Hilmar Strickfaden & Thomas Cremer

  3. Munich Center for Integrated Protein Science, Butenandtstr. 5-13, 81377, Munich, Germany

    Jacques Rouquette, Hilmar Strickfaden & Thomas Cremer

  4. Department of Statistics, University of Munich, Ludwigstr. 33, 80539, Munich, Germany

    Volker J. Schmid

  5. Institute for Applied Physics and Metrology, LRT2, University of the Armed Forces, Werner-Heisenberg-Weg 39, 85577, Neubiberg, Germany

    Christoph Greubel, Volker Hable & Günther Dollinger

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  1. Doris M. Seiler
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Correspondence to Anna A. Friedl.

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Seiler, D.M., Rouquette, J., Schmid, V.J. et al. Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4. Chromosome Res 19, 883–899 (2011). https://doi.org/10.1007/s10577-011-9244-1

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