A mutual balance between specific histone markers is essential for optimal DNA repair. For example, the epigenetic factors HDAC1 and HDAC2 play a crucial regulating role in the DNA damage response [1]. To show how HDAC1 depletion in mouse embryonic stem cells (mESCs) can affect the histone signatures after being exposed to γ-rays, we used mass spectrometry, western blotting, immunohistochemistry, and advanced confocal microscopy. We mainly compared the epigenetic profiles of histones H3 and H4 in wild-type (wt) and HDAC1 double-knockout (dn) ES cells exposed to γ-rays. We observed that a decrease in H3S10 phosphorylation is a hallmark of γ-irradiated mESCs. However, radiation-induced changes in H3S10 phosphorylation (H3S10ph) were not affected by HDAC1 depletion. We found, as well a decrease in H3K9me3 induced by γ-irradiation but no difference at the level of HP1β. These results fit well with the work published by [2] showing that all HP1 isoforms are recruited to UV-induced DNA lesions independently of H3K9me3. Changes in H3S10ph were likely caused by a γ-radiation-induced decrease in the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX. However, an interaction of such modified histones we found in the G2 phase of the cell cycle. Together, our data show that even though H3S10ph is not directly involved in DNA repair, radiation-induced changes in the cell cycle can affect the function of H3S10ph. This work was supported by the Czech Science Foundation (grant number: 18-07384S). The work was also supported by Strategie AV21, program Qualitas, from the Center of Epigenetics (ICO: 68081707). The CIISB research infrastructure project LM2015043 funded by MEYS CR is gratefully acknowledged for the financial support of the LC-MS/MS measurements at the Proteomics Core Facility. The work was co-funded by the MEYS project CEITEC 2020 (LQ1601). [1] K.M. Miller, J.V. Tjeertes, J. Coates, G., et al.: Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining, Nat. Struct. Mol. Biol. 17 (2010) 1144e1151. [2] M.S. Luijsterburg, C. Dinant, H. Lans, et al.: Heterochromatin protein 1 is recruited to various types of DNA damage, J. Cell Biol. 185 (2009) 577e586.