Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines

Abstract

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

Influence of chromatin compaction on simulated early radiation‐induced DNA damage using Geant4‐DNA

“…In terms of DSB/Gy/Gbp, our findings show that more DSBs are produced in the euchromatin than in the heterochromatin but have similar complexities. These results conform to experimental biological observations on the role of chromatin compaction in the protection of DSB induction.…”

“…Conversely, for alphas of 5.27 MeV, more DSB/kbp were observed in the euchromatin than in the heterochromatin, 0.0049 ± 0.0003 and 0.0045 ± 0.0004, respectively. Again, a time of 10 ns seems to be more suitable as it better reproduces the difference between condensed and decondensed chromatin, that is, more DSB damage in the euchromatin than in the heterochromatin as indicated in different studies …”

“…The heterochromatin is tightly packed and is responsible for gene regulation, whereas the euchromatin is less condensed but rich in gene concentration and under active transcription . Among other functions, it is well known that chromatin compaction plays an essential role in DNA repair, and can influence the sensitivity to DNA damage . Indeed, it has been shown in previous studies that γ ‐H2AX foci, which are used as specific markers of DSB signaling, are more often located in euchromatin regions, which could imply that these regions are more susceptible to DNA damage .…”

“…Some present a higher proportion of euchromatin, or even show distinct regions of the two levels of chromatin compaction, rather than a uniform distribution. In the literature, human fibroblast nuclei are described as having a uniform distribution of heterochromatin and euchromatin . As shown by Cremer et al, heterochromatin preferentially resides in the nuclear periphery and euchromatin maps to the nuclear interior in human lymphocyte nuclei.…”

Repair of DNA Double-Strand Breaks in Heterochromatin

“…Protection against unscheduled HR is particularly important in the case of DSBs occurring in the highly repeated sequences in the ribosomal DNA (rDNA) and pericentromeric regions, since such repair could lead to insertions, deletions, and chromosomal rearrangements. The notion of a protective role for HC is supported by a recent study analysing the production and repair of DSBs in human embryonic stem cells (hEScs) where the chromatin has a more open structure than that in differentiated cells [42]. It was observed that the same dose of radiation produced significantly more 53BP1-containing IRIF in hESCs than in normal fibroblasts, from which the authors concluded that DNA within HC may indeed be protected against damage.…”