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

METTL16 methyltransferase is responsible for the methylation of N6-adenosine (m6A) in several RNAs. In mouse cells, we showed that the nuclear distribution of METTL16 is cell cycle-specific. In the G1/S phases, METTL16 accumulates to the nucleolus, while in the G2 phase, the level of METTL16 increases in the nucleoplasm. In metaphase and anaphase, there is a very low pool of the METTL16 protein, but in telophase, residual METTL16 appears to be associated with the newly formed nuclear lamina. In A-type lamin-depleted cells, we observed a reduction of METTL16 when compared with the wild-type counterpart. However, METTL16 does not interact with A-type and B-type lamins, but interacts with Lamin B Receptor (LBR) and Lap2α. Additionally, Lap2α depletion caused METTL16 downregulation in the nuclear pool. Furthermore, METTL16 interacted with DDB2, a key protein of the nucleotide excision repair (NER), and also with nucleolar proteins, including TCOF, NOLC1, and UBF1/2, but not fibrillarin. From this view, the METTL16 protein may also regulate the transcription of ribosomal genes because we observed that the high level of m6A in 18S rRNA appeared in cells with upregulated METTL16.

• Keywords
• METTL16, cell cycle, epitranscriptome, nucleolus, rDNA,
• Publication type
• Journal Article 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

Repair of ribosomal DNA (rDNA) is a very important nuclear process due to the most active transcription of ribosomal genes. Proper repair of rDNA is required for physiological biogenesis of ribosomes. Here, we analyzed the epigenetics of the DNA damage response in a nucleolar compartment, thus in the ribosomal genes studied in nonirradiated and UVA-irradiated mouse embryonic fibroblasts (MEFs). We found that the promoter of ribosomal genes is not abundant on H4K20me2, but it is densely occupied by H4K20me3. Ribosomal genes, regulated via UBF1/2 proteins, were characterized by an interaction between UBF1/2 and H4K20me2/me3. This interaction was strengthened by UVA irradiation that additionally causes a focal accumulation of H4K20me3 in the nucleolus. No interaction has been found between UBF1/2 and H3K9me3. Interestingly, UVA irradiation decreases the levels of H3K9me3 and H4K20me3 at 28S rDNA. Altogether, the UVA light affects the epigenetic status of ribosomal genes at 28S rDNA and strengthens an interaction between UBF1/2 proteins and H4K20me2/me3.

• Keywords
• DNA damage response, DNA repair, Nucleolus, UBF, UVA irradiation,
• MeSH
• Cell Nucleolus metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin Immunoprecipitation MeSH
• DNA-Binding Proteins MeSH
• Epigenesis, Genetic radiation effects   MeSH
• Fluorescent Antibody Technique MeSH
• Histones metabolism   MeSH
• Methylation MeSH
• Mice MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation radiation effects   MeSH
• DNA, Ribosomal genetics   MeSH
• Pol1 Transcription Initiation Complex Proteins metabolism   MeSH
• Ultraviolet Rays * MeSH
• Protein Binding MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• Histones MeSH
• DNA, Ribosomal MeSH
• transcription factor UBF MeSH Browser
• Pol1 Transcription Initiation Complex Proteins MeSH

We studied the histone signature of embryonic and adult brains to strengthen existing evidence of the importance of the histone code in mouse brain development. We analyzed the levels and distribution patterns of H3K9me1, H3K9me2, H3K9me3, and HP1β in both embryonic and adult brains. Western blotting showed that during mouse brain development, the levels of H3K9me1, H3K9me2, and HP1β exhibited almost identical trends, with the highest protein levels occurring at E15 stage. These trends differed from the relatively stable level of H3K9me3 at developmental stages E8, E13, E15, and E18. Compared with embryonic brains, adult brains were characterized by very low levels of H3K9me1/me2/me3 and HP1β. Manipulation of the embryonic epigenome through histone deacetylase inhibitor treatment did not affect the distribution patterns of the studied histone markers in embryonic ventricular ependyma. Similarly, Hdac3 depletion in adult animals had no effect on histone methylation in the adult hippocampus. Our results indicate that the distribution of HP1β in the embryonic mouse brain is related to that of H3K9me1/me2 but not to that of H3K9me3. The unique status of H3K9me3 in the brain was confirmed by its pronounced accumulation in the granular layer of the adult olfactory bulb. Moreover, among the studied proteins, H3K9me3 was the only posttranslational histone modification that was highly abundant at clusters of centromeric heterochromatin, called chromocenters. When we focused on the hippocampus, we found this region to be rich in H3K9me1 and H3K9me3, whereas H3K9me2 and HP1β were present at a very low level or even absent in the hippocampal blade. Taken together, these results revealed differences in the epigenome of the embryonic and adult mouse brain and showed that the adult hippocampus, the granular layer of the adult olfactory bulb, and the ventricular ependyma of the embryonic brain are colonized by specific epigenetic marks.

• Keywords
• Brain sections, Epigenetics, Hippocampus, Histone methylation, Histones, Olfactory bulb,
• MeSH
• Chromosomal Proteins, Non-Histone analysis   metabolism   MeSH
• Microscopy, Fluorescence MeSH
• Histone-Lysine N-Methyltransferase metabolism   MeSH
• Immunohistochemistry MeSH
• Brain embryology   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cbx1 protein, mouse MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Histone-Lysine N-Methyltransferase MeSH

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

• Keywords
• DNA damage, UBF1, live cells, nucleolus, nuncleoli tracking,
• MeSH
• Apoptosis radiation effects   MeSH
• Cell Nucleolus radiation effects   MeSH
• Cell Line MeSH
• Cell Cycle radiation effects   MeSH
• G2 Phase radiation effects   MeSH
• Transcription, Genetic MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• DNA Damage radiation effects   MeSH
• Pol1 Transcription Initiation Complex Proteins genetics   metabolism   MeSH
• Ultraviolet Rays MeSH
• Computational Biology MeSH
• Gamma Rays adverse effects   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• transcription factor UBF MeSH Browser
• Pol1 Transcription Initiation Complex Proteins MeSH

BACKGROUND: The repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways. RESULTS: We analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1β (HP1β) level. Moreover, Förster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1β that was dependent on a functional chromo shadow domain of HP1β. Thus, overexpression of HP1β with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent. CONCLUSIONS: We propose that the simultaneous presence and interaction of UBF1 and HP1β at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1β. This might have functional significance for nucleotide excision repair.

• Keywords
• DNA repair, DNA-damage response, Irradiation, Live-cell studies, Nucleolus, UBF1,
• Publication type
• Journal Article MeSH

The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson's disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells.

• Keywords
• Chromatin, Differentiation, Embryonic stem cells, Epigenetics, Nucleus, Pluripotency,
• Publication type
• Journal Article MeSH

BACKGROUND: Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D. RESULTS: We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. CONCLUSION: Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.

• Publication type
• Journal Article MeSH