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 γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ 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, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage.

• Klíčová slova
• FLIM-FRET, HP1, epigenetics, irradiation, mass spectrometry, nucleolus, phosphorylation,
• MeSH
• buněčné jadérko metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• homolog proteinu s chromoboxem 5 MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• optické zobrazování MeSH
• poškození DNA MeSH
• rezonanční přenos fluorescenční energie MeSH
• serin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CBX1 protein, human MeSH Prohlížeč
• CBX5 protein, human MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• homolog proteinu s chromoboxem 5 MeSH
• serin MeSH

Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

• Klíčová slova
• 53BP1, BRCA1, DNA damage, epigenetics, histone modifications,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 53BP1 MeSH

Methylation of histones H4 at lysine 20 position (H4K20me), which is functional in DNA repair, represents a binding site for the 53BP1 protein. Here, we show a radiation-induced increase in the level of H4K20me3 while the levels of H4K20me1 and H4K20me2 remained intact. H4K20me3 was significantly pronounced at DNA lesions in only the G1 phase of the cycle, while this histone mark was reduced in very late S and G2 phases when PCNA was recruited to locally micro-irradiated chromatin. H4K20me3 was diminished in locally irradiated Suv39h1/h2 double knockout (dn) fibroblasts, and the same phenomenon was observed for H3K9me3 and its binding partner, the HP1β protein. Immunoprecipitation showed the existence of an interaction between H3K9me3-53BP1 and H4K20me3-53BP1; however, HP1β did not interact with 53BP1. Together, H3K9me3 and H4K20me3 represent epigenetic markers that are important for the function of the 53BP1 protein in non-homologous end joining (NHEJ) repair. The very late S phase represents the cell cycle breakpoint when a DDR function of the H4K20me3-53BP1 complex is abrogated due to recruitment of the PCNA protein and other DNA repair factors of homologous recombination to DNA lesions.

• Klíčová slova
• DNA damage, H3K9me3, H4K20me1/me2/me3, Suv39h1/h2, epigenetics,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   účinky záření   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• epigeneze genetická * účinky záření   MeSH
• histony metabolismus   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• lidé MeSH
• metylace DNA * účinky záření   MeSH
• metylace MeSH
• myši MeSH
• oprava DNA spojením konců * MeSH
• poškození DNA * MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• restrukturace chromatinu MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• CBX1 protein, human MeSH Prohlížeč
• Cbx1 protein, mouse MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• histony MeSH
• homolog proteinu s chromoboxem 5 MeSH
• PCNA protein, human MeSH Prohlížeč
• proliferační antigen buněčného jádra MeSH
• TP53BP1 protein, human MeSH Prohlížeč
• Trp53bp1 protein, mouse MeSH Prohlížeč

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

• Klíčová slova
• HDAC1, cardiomyocytes, embryonic stem cells, epigenetics, histones H3 and H4,
• MeSH
• acetylace MeSH
• buněčná diferenciace účinky léků   MeSH
• delece genu * MeSH
• embryo savčí cytologie   MeSH
• embryoidní tělíska účinky léků   metabolismus   MeSH
• histony metabolismus   MeSH
• inhibitory histondeacetylas farmakologie   MeSH
• kardiomyocyty cytologie   účinky léků   metabolismus   MeSH
• metylace MeSH
• myší embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• organogeneze * účinky léků   MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histony MeSH
• inhibitory histondeacetylas MeSH