Chromosome Research

, Volume 22, Issue 4, pp 463–481 | Cite as

Bleomycin-induced γH2AX foci map preferentially to replicating domains in CHO9 interphase nuclei

  • Pablo LiddleEmail author
  • Laura Lafon-Hughes
  • María Vittoria Di Tomaso
  • Ana Laura Reyes-Ábalos
  • Jorge Jara
  • Mauricio Cerda
  • Steffen Härtel
  • Gustavo A. Folle


Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2′-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases. In order to map DNA damage with respect to replicating domains, the distribution of γH2AX foci induced by the radiomimetic agent bleomycin was analyzed in CHO9 interphase nuclei by delineating euchromatic (H3K4me3+) and replicating (EdU+) regions. Quantification of overlapping pixels and 3D inter-object overlap in binary masks revealed colocalization between γH2AX foci and EdU +  domains both in ES and MS/LS nuclei, indicating that primary damage distribution is modulated by DNA synthesis. Further, we verified that EdU incorporation by itself did not influence BLEO-induced γH2AX nuclear patterns. Our results also revealed a repeated localization of γH2AX foci in replicating/nonreplicating interfaces which could reflect short-range chromatin migration following DNA insult.


DNA damage γH2AX foci DNA replication EdU eu-/heterochromatin H3K4me3 



Histone H2AX phosphorylated on serine 139




Chromosomal aberration


Chinese hamster ovary cells






Double-strand break


Euchromatic damage distribution index




Early S


Full width at half maximum


Ionizing radiation


Late S


Mid S


Mid and late S


Point spread function


Replication-related damage distribution index


Regions of increased gene expression


Region of interest


Replication slow zones


Single-strand break


Single-stranded DNA


TNF-related apoptosis-inducing ligand



We are indebted to Thomas Cremer, Marion Cremer and Areli Cárdenas for helpful suggestions on the manuscript as well as to Katrin Pfleghaar and Yolanda Markaki for technical advice. The work was financed by the Alexander von Humboldt Foundation (AvH) and partly by the Programa de Desarrollo en Ciencias Básicas (PEDECIBA, Uruguay) together with the Agencia Nacional de Investigación e Innovación (ANII, Uruguay). PL is a former Fellow of the AvH Förderung Program at the Ludwig-Maximilians-Universität Biozentrum (Munich). Research in SCIAN-Lab is funded by BNI ICM P09-015-F (SH, MC, JJ), FONDECYT 1120579 (SH, MC, JJ), US-LACRN (MC, JJ), FONDEF D07I1019 (SH, JJ), FONDECYT 3140447 (MC), the U. Chile “U-Redes Project: BioMed-HPC” (SH), a CONICYT doctoral fellowship (JJ), and AI•BI (

Conflict of interest

Pablo Liddle, Laura Lafon-Hughes, María Vittoria Di Tomaso, Ana Laura Reyes-Ábalos, Jorge Jara, Mauricio Cerda, Steffen Härtel and Gustavo Folle declare that they have no conflict of interest.

Supplementary material

10577_2014_9433_Fig8_ESM.gif (60 kb)
Supplementary Figure 1

Effect of BLEO exposure on cell cycle dynamics in CHO9 nuclei. Nuclei from EdU, EdU+BLEO40 and EdU+BLEO160 experiments (650 < n < 850) were classified according to cell cycle phase in order to ascertain whether BLEO treatment produced cell cycle delay. (A, D) EdU, (B, E) EdU+BLEO40 and (C, F) EdU+BLEO160 assays, respectively. (AC) Z-stack slices of low-magnification fields (40×) were used to determine the proportion of nuclei in each cell cycle phase. Bar: 20 μm. (D-F) Percentage of G1/G2, ES and MS/LS nuclei in EdU−, EdU+BLEO40- and EdU+BLEO160-treated cells. The proportion of nuclei in each cell cycle phase was not affected by BLEO treatment (chi-square test). (AC) Merged images of DAPI (blue) and (green) signals (GIF 60 kb)

10577_2014_9433_MOESM1_ESM.tif (630 kb)
(TIFF 629 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Pablo Liddle
    • 1
    Email author
  • Laura Lafon-Hughes
    • 1
  • María Vittoria Di Tomaso
    • 1
  • Ana Laura Reyes-Ábalos
    • 1
  • Jorge Jara
    • 3
    • 2
  • Mauricio Cerda
    • 2
  • Steffen Härtel
    • 2
  • Gustavo A. Folle
    • 1
  1. 1.Departamento de GenéticaInstituto de Investigaciones Biológicas Clemente EstableMontevideoUruguay
  2. 2.Laboratory for Scientific Image Analysis (SCIAN-Lab), Programa de Anatomía y Biología del Desarrollo, Biomedical Neuroscience Institute (BNI), Institute of Biomedical Sciences (ICBM), Facultad de MedicinaUniversidad de ChileSantiagoChile
  3. 3.Department of Computer Science, Facultad de Ciencias Físicas y Matemáticas, Universidad de ChileSantiagoChile

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