RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

• Keywords
• DNA demethylation, DNA repair, RNA methylation, base excision repair, epigenetics,
• MeSH
• Chromatin MeSH
• DNA Demethylation * MeSH
• DNA metabolism   MeSH
• DNA Methylation MeSH
• DNA Repair MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * pharmacology   MeSH
• DNA Damage MeSH
• RNA genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * MeSH
• RNA MeSH

The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with β-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are already established in the cytoplasm of the late telophase and at the stage of early cytokinesis. In addition, we observed the occurrence of splicing factors in the nuclear blebs and micronuclei, which are also sites of both transcription and splicing. This conclusion supports the fact that splicing proceeds transcriptionally. According to our data, this process is A-type lamin-dependent. Lamin depletion also reduces the interaction between SC35 and β-catenin in mitotic cells.

• Keywords
• PARP inhibitor, RNA pol II, SC-35, splicing,
• MeSH
• HeLa Cells MeSH
• Lamins metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Poly(ADP-ribose) Polymerase Inhibitors therapeutic use   MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH
• RNA Polymerase II metabolism   MeSH
• RNA Splicing Factors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lamins MeSH
• Poly(ADP-ribose) Polymerase Inhibitors MeSH
• PARP1 protein, human MeSH Browser
• Poly (ADP-Ribose) Polymerase-1 MeSH
• RNA Polymerase II MeSH
• RNA Splicing Factors MeSH

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.

• Keywords
• FLIM-FRET, HP1, epigenetics, irradiation, mass spectrometry, nucleolus, phosphorylation,
• MeSH
• Cell Nucleolus metabolism   MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Phosphorylation MeSH
• HeLa Cells MeSH
• Chromobox Protein Homolog 5 MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Optical Imaging MeSH
• DNA Damage MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Serine metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CBX1 protein, human MeSH Browser
• CBX5 protein, human MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Chromobox Protein Homolog 5 MeSH
• Serine MeSH

Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1-deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro-differentiation was almost suppressed. Neuro-differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia-like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia-like brains that were treated with the cannabinoid receptor-1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co-regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro-differentiation as well as the pathophysiology of a schizophrenia-like phenotype.

• Keywords
• H3K9 acetylation, HDACs, acetylome, mouse neurogenesis, schizophrenia,
• MeSH
• Acetylation MeSH
• Cannabinoid Receptor Antagonists pharmacology   MeSH
• Antipsychotic Agents pharmacology   MeSH
• Time Factors MeSH
• Epigenesis, Genetic MeSH
• Gestational Age MeSH
• Histone Deacetylase 1 antagonists & inhibitors   genetics   metabolism   MeSH
• Histone Deacetylases genetics   metabolism   MeSH
• Histones metabolism   MeSH
• Histone Deacetylase Inhibitors pharmacology   MeSH
• Methylazoxymethanol Acetate MeSH
• Disease Models, Animal MeSH
• Neural Cell Adhesion Molecules genetics   metabolism   MeSH
• Brain drug effects   embryology   enzymology   pathology   MeSH
• Mice, Inbred C57BL MeSH
• Neurogenesis * drug effects   MeSH
• Neurons drug effects   enzymology   pathology   MeSH
• Protein Processing, Post-Translational MeSH
• Rats, Sprague-Dawley MeSH
• Receptor, Cannabinoid, CB1 antagonists & inhibitors   metabolism   MeSH
• Schizophrenia chemically induced   drug therapy   enzymology   genetics   MeSH
• Signal Transduction MeSH
• SOXB1 Transcription Factors genetics   metabolism   MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cannabinoid Receptor Antagonists MeSH
• Antipsychotic Agents MeSH
• Cnr1 protein, rat MeSH Browser
• Hdac1 protein, mouse MeSH Browser
• Hdac1 protein, rat MeSH Browser
• Histone Deacetylase 1 MeSH
• Histone Deacetylases MeSH
• histone deacetylase 3 MeSH Browser
• Histones MeSH
• Histone Deacetylase Inhibitors MeSH
• Methylazoxymethanol Acetate MeSH
• Neural Cell Adhesion Molecules MeSH
• Receptor, Cannabinoid, CB1 MeSH
• Sox2 protein, mouse MeSH Browser
• SOXB1 Transcription Factors MeSH

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

• Keywords
• 53BP1 protein, DNA repair, Histone γH2AX, MDC1 protein, TP53 gene,
• MeSH
• Tumor Suppressor p53-Binding Protein 1 metabolism   MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Down-Regulation MeSH
• Nuclear Proteins deficiency   metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Protein p53 deficiency   genetics   metabolism   MeSH
• DNA Damage * MeSH
• Cell Cycle Proteins MeSH
• Trans-Activators deficiency   metabolism   MeSH
• Ultraviolet Rays * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Nuclear Proteins MeSH
• MDC1 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Cell Cycle Proteins MeSH
• TP53 protein, human MeSH Browser
• TP53BP1 protein, human MeSH Browser
• Trans-Activators MeSH