Chromosome Research

, Volume 19, Issue 7, pp 883–899 | Cite as

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

  • Doris M. Seiler
  • Jacques Rouquette
  • Volker J. Schmid
  • Hilmar Strickfaden
  • Christian Ottmann
  • Guido A. Drexler
  • Belinda Mazurek
  • Christoph Greubel
  • Volker Hable
  • Günther Dollinger
  • Thomas Cremer
  • Anna A. Friedl


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.


Chromatin silencing epigenetics DNA damage γ-H2AX 







double-strand break


intensity correlation analysis


intensity correlation quotient


Mega electron volts


product of the differences from the mean



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.

Conflicts of interest

The authors declare no conflict of interest.

Supplementary material

10577_2011_9244_MOESM1_ESM.pdf (5.1 mb)
MOESM 1 (PDF 5231 kb)


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Doris M. Seiler
    • 1
  • Jacques Rouquette
    • 2
    • 3
    • 6
  • Volker J. Schmid
    • 4
  • Hilmar Strickfaden
    • 2
    • 3
    • 7
  • Christian Ottmann
    • 1
  • Guido A. Drexler
    • 1
  • Belinda Mazurek
    • 1
  • Christoph Greubel
    • 5
  • Volker Hable
    • 5
  • Günther Dollinger
    • 5
  • Thomas Cremer
    • 2
    • 3
  • Anna A. Friedl
    • 1
  1. 1.Department of Radiation OncologyUniversity Hospital of MunichMunichGermany
  2. 2.Department Biology IIUniversity of MunichMartinsriedGermany
  3. 3.Munich Center for Integrated Protein ScienceMunichGermany
  4. 4.Department of StatisticsUniversity of MunichMunichGermany
  5. 5.Institute for Applied Physics and Metrology, LRT2University of the Armed ForcesNeubibergGermany
  6. 6.CRT-RIV-ITAVUniversité de Toulouse, UPS, INSA, CNRS, UMS 3039ToulouseFrance
  7. 7.Department of Oncology, Faculty of MedicineUniversity of Alberta and Cross Cancer InstituteEdmontonCanada

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