The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N6-methyladenosine (m6A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m6A RNA level at the irradiated genomic regions. After genome injury, m6A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m6A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m6A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m3G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N1-methyladenosine (m1A) in RNAs. Based on these results, we prefer a model in which m6A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m1A) RNAs and m3G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.

• Keywords
• DNA repair, METTL-like enzymes, RNA methylation, epigenetics, histones,
• MeSH
• Adenosine analogs & derivatives   metabolism   MeSH
• Chromatin metabolism   MeSH
• DNA Demethylation radiation effects   MeSH
• Stress, Physiological radiation effects   MeSH
• Guanosine analogs & derivatives   metabolism   MeSH
• DNA Methylation genetics   radiation effects   MeSH
• Methylation radiation effects   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• RNA, Untranslated metabolism   MeSH
• Genomic Instability radiation effects   MeSH
• DNA Damage MeSH
• RNA metabolism   MeSH
• Ultraviolet Rays * 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
• Adenosine MeSH
• Chromatin MeSH
• Guanosine MeSH
• N-methyladenosine MeSH Browser
• N(2),N(2),7-trimethylguanosine MeSH Browser
• RNA, Untranslated MeSH
• RNA 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.

• Keywords
• DNA damage, H3K9me3, H4K20me1/me2/me3, Suv39h1/h2, epigenetics,
• MeSH
• Tumor Suppressor p53-Binding Protein 1 genetics   metabolism   MeSH
• Cell Nucleus genetics   metabolism   radiation effects   MeSH
• Cell Line MeSH
• Cell Cycle MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Epigenesis, Genetic * radiation effects   MeSH
• Histones metabolism   MeSH
• Chromobox Protein Homolog 5 MeSH
• Humans MeSH
• DNA Methylation * radiation effects   MeSH
• Methylation MeSH
• Mice MeSH
• DNA End-Joining Repair * MeSH
• DNA Damage * MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• Chromatin Assembly and Disassembly MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• CBX1 protein, human MeSH Browser
• Cbx1 protein, mouse MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Histones MeSH
• Chromobox Protein Homolog 5 MeSH
• PCNA protein, human MeSH Browser
• Proliferating Cell Nuclear Antigen MeSH
• TP53BP1 protein, human MeSH Browser
• Trp53bp1 protein, mouse MeSH Browser

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

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus, PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression, splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However, when cell nuclei were microirradiated by UV-A, the mobility of PRMT1 cytoplasmic bodies increased, size was reduced, and disappeared within approximately 20 min. The same response occurred after γ-irradiation of the whole cell population, but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 overexpression. Taken together, we show that PRMT1 concentrates in cytoplasmic bodies, which respond to DNA injury in the cell nucleus, and to treatment with various PRMT1 inhibitors.

• MeSH
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Chromosomal Proteins, Non-Histone genetics   metabolism   MeSH
• Cytoplasm enzymology   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• HeLa Cells MeSH
• Intracellular Signaling Peptides and Proteins genetics   metabolism   MeSH
• Humans MeSH
• Mice MeSH
• DNA Damage * MeSH
• Protein-Arginine N-Methyltransferases antagonists & inhibitors   genetics   metabolism   MeSH
• Repressor Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Ultraviolet Rays * MeSH
• Gamma Rays * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Chromosomal Proteins, Non-Histone MeSH
• DNA-Binding Proteins MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• PRMT1 protein, human MeSH Browser
• Prmt1 protein, mouse MeSH Browser
• Protein-Arginine N-Methyltransferases MeSH
• Repressor Proteins MeSH
• TP53BP1 protein, human MeSH Browser
• Trp53bp1 protein, mouse MeSH Browser

Determining averaged effective diffusion constants from experimental measurements of fluorescent proteins in an inhomogeneous medium in the presence of ligand-receptor interactions poses problems of analytical tractability. Here, we introduced a nonfitting method to evaluate the averaged effective diffusion coefficient of a region of interest (which may include a whole nucleus) by mathematical processing of the entire cellular two-dimensional spatial pattern of recovered fluorescence. Spatially and temporally resolved measurements of protein transport inside cells were obtained using the fluorescence recovery after photobleaching technique. Two-dimensional images of fluorescence patterns were collected by laser-scanning confocal microscopy. The method was demonstrated by applying it to an estimation of the mobility of green fluorescent protein-tagged heterochromatin protein 1 in the nuclei of living mouse embryonic fibroblasts. This approach does not require the mathematical solution of a corresponding system of diffusion-reaction equations that is typical of conventional fluorescence recovery after photobleaching data processing, and is most useful for investigating highly inhomogeneous areas, such as cell nuclei, which contain many protein foci and chromatin domains.

• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Chromosomal Proteins, Non-Histone chemistry   genetics   metabolism   MeSH
• Diffusion MeSH
• Fluorescence MeSH
• Photobleaching MeSH
• Fluorescence Recovery After Photobleaching methods   MeSH
• Chromobox Protein Homolog 5 MeSH
• Microscopy, Confocal methods   MeSH
• Humans MeSH
• Mathematics MeSH
• Models, Molecular * MeSH
• Mice MeSH
• Reproducibility of Results MeSH
• Solutions MeSH
• Green Fluorescent Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• Chromobox Protein Homolog 5 MeSH
• Solutions MeSH
• Green Fluorescent Proteins MeSH

Heterochromatin protein 1 (HP1), which binds to sites of histone H3 lysine 9 (H3K9) methylation, is primarily responsible for gene silencing and the formation of heterochromatin. We observed that HP1 beta is located in both the chromocenters and fibrillarin-positive nucleoli interiors. However, HP1 alpha and HP1 gamma occupied fibrillarin-positive compartments to a lesser extent, corresponding to the distinct levels of HP1 subtypes at the promoter of rDNA genes. Deficiency of histone methyltransferases SUV39h and/or inhibition of histone deacetylases (HDACi) decreased HP1 beta and H3K9 trimethylation at chromocenters, but not in fibrillarin-positive regions that co-localized with RNA polymerase I. Similarly, SUV39h- and HDACi-dependent nucleolar rearrangement and inhibition of rDNA transcription did not affect the association between HP1 beta and fibrillarin. Moreover, the presence of HP1 beta in nucleoli is likely connected with transcription of ribosomal genes and with the role of fibrillarin in nucleolar processes.

• MeSH
• Cell Nucleolus metabolism   MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Fibroblasts metabolism   MeSH
• Cells, Cultured MeSH
• Methyltransferases metabolism   MeSH
• Mice MeSH
• Repressor Proteins metabolism   MeSH
• Protein Binding 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
• fibrillarin MeSH Browser
• Methyltransferases MeSH
• Repressor Proteins MeSH
• Suv39h1 protein, mouse MeSH Browser