The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that gamma-radiation reduced the number of HP1 alpha-positive foci, but not HP1 beta and HP1 gamma foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that gamma-radiation has the ability to significantly decrease the level of HP1 alpha in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by gamma-rays enhanced the HP1 beta serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1 beta, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to gamma-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and gamma-radiation increased the level of HP1 alpha and HP1 gamma. The level of HP1 beta remained identical before and after the HDACi/gamma-rays treatment, but HDACi strengthened HP1 beta interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1 gamma did not interact with KAP1, although approximately 40% of HP1 gamma foci co-localized with accumulated KAP1. Especially HP1 gamma foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, gamma-irradiation-induced hyperphosphorylation of the HP1 beta protein; thus, HP1 beta-S88ph could be considered as an important marker of DNA damage.