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Abstract Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph). On an individual cellular level, H3S10ph was low in highly γH2AX-positive UV laser-induced DNA lesions, and this nuclear distribution pattern was not changed by HDAC1 depletion. Despite this fact, spontaneous γH2AX-positive DNA lesions colocalized with large H3S10ph-positive nuclear bodies that appear in the G2 phase of the cell cycle. Similarly, by FLIM-FRET analysis, we observed an interaction between H3S10ph and γH2AX in the G2 phase. However, this interaction was reduced when cells were exposed to γ-rays. A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. Together, our data show that despite the fact that H3S10ph is not directly involved in DNA repair, a decrease in H3S10 phosphorylation and weakened interaction between H3S10ph and γH2AX is a result of radiation-induced damage of the genome. In this case, γ-irradiation also decreased the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX. Graphical abstract (A) Irradiation by γ-rays (red line) reduced H3S10ph levels. However, (B) H3S10ph was intact in the γH2AX-positive microirradiated genomic region. (C) Despite the fact that H3S10ph- and γH2AX-positive foci colocalized (interacted) in the G2 phase of the cell cycle, radiation-induced H3S10ph dephosphorylation was not directly linked to DNA repair processes, but instead (D) H3S10ph dephosphorylation was caused by a radiation-induced cell number decrease in the G1 phase. G1 phase was characterized by no interaction between H3S10ph and γH2AX (mentioned in panel C). Together, H3S10ph contributes to the function of γH2AX in DNA repair taking place in G2 phase. Image 1 Highlights • Irradiation by γ-rays increased γH2AX but reduced the level of H3S10ph. • A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. • An interaction between γH2AX and H3S10ph was found in the G2 phase. • H3S10ph contributes to the function of γH2AX in homologous recombination repair. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have shown that histone code dictates the type and structure of chromatin. Bearing in mind the importance of A-type lamins for chromatin arrangement, we studied the effect of trichostatin A (TSA)-induced histone hyperacetylation in lamin A/C-deficient (LMNA−/−) fibroblasts. Lamin A/C deficiency caused condensation of chromosome territories and the nuclear reorganization of centromeric heterochromatin, which was accompanied by the appearance of a chain-like morphology of HP1β foci. Conversely, histone deacetylase (HDAC) inhibition induced de-condensation of chromosome territories, which compensated the effect of lamin A/C deficiency on chromosome regions. The amount of heterochromatin in the area associated with the nuclear membrane was significantly reduced in LMNA−/− cells when compared with lamin A/C-positive (LMNA+/+) fibroblasts. TSA also decreased the amount of peripheral heterochromatin, similarly as lamin A/C deficiency. In both LMNA+/+ and LMNA−/− cells, physically larger chromosomes were positioned more peripherally as compared with the smaller ones, even after TSA treatment. Our observations indicate that lamin A/C deficiency causes not only reorganization of chromatin and some chromatin-associated domains, but also has an impact on the extent of chromosome condensation. As HDAC inhibition can compensate the lamin A/C-dependent chromatin changes, the interaction between lamins and specifically modified histones may play an important role in higher-order chromatin organization, which influences transcriptional activity. [Copyright &y& Elsevier]