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

BACKGROUND: Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and apoptosis. Heterochromatinization, as the main feature of these processes, is also associated with pronounced trimethylation of histones H3 at the lysine 9 position (H3K9me3). METHODS: By confocal microscopy, we analyzed cell cycle-dependent levels and distribution of phosphorylated histone H1 (H1ph) and H3K9me3. By mass spectrometry, we studied post-translational modifications of linker histones. RESULTS: Phosphorylated histone H1, similarly to H3K9me3, has a comparable level in the G1, S, and G2 phases of the cell cycle. A high density of phosphorylated H1 was inside nucleoli of mouse embryonic stem cells (ESCs). H1ph was also abundant in prophase and prometaphase, while H1ph was absent in anaphase and telophase. H3K9me3 surrounded chromosomal DNA in telophase. This histone modification was barely detectable in the early phases of mitosis. Mass spectrometry revealed several ESC-specific phosphorylation sites of H1. HDAC1 depletion did not change H1 acetylation but potentiated phosphorylation of H1.2/H1.3 and H1.4 at serine 38 positions. CONCLUSIONS: Differences in the level and distribution of H1ph and H3K9me3 were revealed during mitotic phases. ESC-specific phosphorylation sites were identified in a linker histone.

• Keywords
• chromatin, epigenetic, histone H1, histone H3, mass spectrometry, nucleolus,
• Publication type
• Journal Article 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

The epigenetic modification of histones dictates the formation of euchromatin and heterochromatin domains. We studied the effects of a deficiency of histone methyltransferase, SUV39h, and trichostatin A-dependent hyperacetylation on the structural stability of centromeric clusters, called chromocentres. We did not observe the expected disintegration of chromocentres, but both SUV39h deficiency and hyperacetylation in SUV39h+/+ cells induced the re-positioning of chromocentres closer to the nuclear periphery. Conversely, TSA treatment of SUV39h-/- cells re-established normal nuclear radial positioning of chromocentres. This structural re-arrangement was likely caused by several epigenetic events at centromeric heterochromatin. In particular, reciprocal exchanges between H3K9me1, H3K9me2, H3K9me3, DNA methylation, and HP1 protein levels influenced chromocentre nuclear composition. For example, H3K9me1 likely substituted for the function of H3K9me3 in chromocentre nuclear arrangement and compaction. Our results illustrate the important and interchangeable roles of epigenetic marks for chromocentre integrity. Therefore, we propose a model for epigenetic regulation of nuclear stability of centromeric heterochromatin in the mouse genome.

• MeSH
• Cell Line MeSH
• Epigenesis, Genetic genetics   MeSH
• Heterochromatin metabolism   MeSH
• Histone-Lysine N-Methyltransferase genetics   physiology   MeSH
• Histone Methyltransferases MeSH
• Histones metabolism   MeSH
• Methylation 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
• Heterochromatin MeSH
• Histone-Lysine N-Methyltransferase MeSH
• Histone Methyltransferases MeSH
• Histones MeSH

Epigenetic histone (H3) modification patterns and the nuclear radial arrangement of select genetic elements were compared in human embryonic stem cells (hESCs) before and after differentiation. H3K9 acetylation, H3K9 trimethylation, and H3K79 monomethylation were reduced at the nuclear periphery of differentiated hESCs. Differentiation coincided with centromere redistribution, as evidenced by perinucleolar accumulation of the centromeric markers CENP-A and H3K9me3, central repositioning of centromeres 1, 5, 19, and rearrangement of other centromeres at the nuclear periphery. The radial positions of PML, RARalpha genes, and human chromosomes 10, 12, 15, 17, and 19 remained relatively stable as hESCs differentiated. However, the female inactive H3K27-trimethylated X chromosome occupied a more peripheral nuclear position in differentiated cells. Thus, pluripotent and differentiated hESCs have distinct nuclear patterns of heterochromatic structures (centromeres and inactive X chromosome) and epigenetic marks (H3K9me3, and H3K27me3), while relatively conserved gene density-related radial chromatin distributions are already largely established in undifferentiated hES cells.

• MeSH
• Cell Differentiation * drug effects   MeSH
• Cell Line MeSH
• Chromatin genetics   MeSH
• Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• Epigenesis, Genetic genetics   MeSH
• Histones metabolism   MeSH
• Humans MeSH
• Tretinoin pharmacology   MeSH
• Cell Shape MeSH
• Gene Expression Regulation, Developmental MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH
• Tretinoin MeSH

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (∼40%) recovery of GFP-HP1α and GFP-HP1β after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic.

• MeSH
• Acetylation MeSH
• Antineoplastic Agents, Phytogenic MeSH
• Apoptosis drug effects   MeSH
• Cell Adhesion MeSH
• Chromatin drug effects   genetics   metabolism   MeSH
• Chromosomal Proteins, Non-Histone genetics   metabolism   MeSH
• Epigenesis, Genetic drug effects   MeSH
• Etoposide MeSH
• Fibroblasts cytology   drug effects   metabolism   MeSH
• Histones genetics   metabolism   MeSH
• Chromobox Protein Homolog 5 MeSH
• Kinetics MeSH
• Humans MeSH
• Methylation MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Protein Processing, Post-Translational drug effects   MeSH
• Proto-Oncogene Proteins c-bcl-2 genetics   metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Signal Transduction drug effects   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
• Antineoplastic Agents, Phytogenic MeSH
• CBX1 protein, human MeSH Browser
• Cbx1 protein, mouse MeSH Browser
• CBX5 protein, human MeSH Browser
• Chromatin MeSH
• Chromosomal Proteins, Non-Histone MeSH
• Etoposide MeSH
• Histones MeSH
• Chromobox Protein Homolog 5 MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-bcl-2 MeSH
• Recombinant Fusion Proteins MeSH

We have studied JMJD2b histone demethylase, which antagonizes H3K9me3 in the pericentromeric heterochromatin. In cells with a deficiency in the histone methyltransferase SUV39h, the level of full-length JMJD2b (JMJD2b-GFP-1086) at chromocenters was reduced, corresponding to a global decrease in JMJD2b and H3K9me3. In wild-type fibroblasts, the chromatin of ribosomal genes, which is dense with H3K9 methylation, lacked JMJD2b-GFP-1086, while mutant and truncated forms of JMJD2b densely occupied the nucleolar compartment. This implies that the PHD Zn-fingers and Tudor domains, which were removed in truncated JMJD2b, are responsible for the aberrant JMJD2b function. Intriguingly, the JMJD2b-GFP-1086 level was significantly higher in tumor cell nucleoli. The kinetic properties of JMJD2b-GFP-1086 in the nucleoli and nucleoplasm of normal and tumor cells were similar; ∼50% recovery of prebleached intensity was reached after <1 s. However, the mobile fraction of JMJD2b-GFP-1086 was increased in SUV39h-deficient cells. Similarly, the mobile fractions of mutant JMJD2b(1-424)H189A-GFP and truncated JMJD2b(1-424)GFP were greater than that measured for the full-length protein. We suggest that nucleoli are the site of an aberrant function of JMJD2b, the kinetic properties of which can be influenced by a mutant genetic background.

• MeSH
• Cell Nucleolus genetics   metabolism   MeSH
• Cell Line MeSH
• Chromatin metabolism   MeSH
• Chromatin Immunoprecipitation MeSH
• Fibroblasts metabolism   MeSH
• Gene Rearrangement MeSH
• Histone-Lysine N-Methyltransferase genetics   MeSH
• Histone Methyltransferases MeSH
• Jumonji Domain-Containing Histone Demethylases genetics   metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Mutation MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• DNA, Ribosomal metabolism   MeSH
• Ribosomes metabolism   MeSH
• Zinc Fingers 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
• Chromatin MeSH
• Histone-Lysine N-Methyltransferase MeSH
• Histone Methyltransferases MeSH
• Jumonji Domain-Containing Histone Demethylases MeSH
• DNA, Ribosomal MeSH

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.

• MeSH
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Chromatin genetics   MeSH
• Chromosomal Proteins, Non-Histone metabolism   radiation effects   MeSH
• DNA-Binding Proteins radiation effects   MeSH
• Fibroblasts radiation effects   MeSH
• Chromosome Fragile Sites genetics   MeSH
• Histones radiation effects   MeSH
• Chromobox Protein Homolog 5 MeSH
• Nuclear Proteins metabolism   radiation effects   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Proteins metabolism   radiation effects   MeSH
• Genomic Instability MeSH
• DNA Repair genetics   MeSH
• Osteosarcoma MeSH
• DNA Damage radiation effects   MeSH
• Promyelocytic Leukemia Protein MeSH
• Ribosomes genetics   MeSH
• Transcription Factors metabolism   radiation effects   MeSH
• Gamma Rays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• CBX1 protein, human MeSH Browser
• Chromatin MeSH
• Chromosomal Proteins, Non-Histone MeSH
• DNA-Binding Proteins MeSH
• Histones MeSH
• Chromobox Protein Homolog 5 MeSH
• Nuclear Proteins MeSH
• Tumor Suppressor Proteins MeSH
• Pml protein, mouse MeSH Browser
• Promyelocytic Leukemia Protein MeSH
• Transcription Factors MeSH
• Trp53bp1 protein, mouse MeSH Browser

Small non-coding RNAs control normal development and differentiation in the embryo. These regulatory molecules play a key role in the development of human diseases and are used often today for researching new treatments for different pathologies. In this study, CaCo2 colorectal adenocarcinoma cells were initially epigenetically reprogrammed and transformed into CD4+ cells with nano-sized complexes of amphiphilic poly-(N-vinylpyrrolidone) (PVP) with miRNA-152 and piRNA-30074. The transformation of cells was confirmed by morphological and genetic changes in the dynamic of reprogramming. CD4+ lymphocytes marker was detected using immunofluorescence. Amphiphilic poly-(N-vinylpyrrolidone)/small non-coding RNAs complexes were investigated for transfection efficiency and duration of transfection of CaCo2 colorectal adenocarcinoma cells using fluorescence.

• Keywords
• AGO2, argonaute 2, Amphiphilic poly-(N-vinylpyrrolidone), BACH1, BTB domain and CNC homolog 1, CD, cluster of differentiation, CaCo2 colorectal adenocarcinoma, DICER1, ribonuclease III, DNMT1, DNA methyltransferase 1, DTT, dithyothreitol, ERK1/2, extracellular signal regulated kinase ½, FGF2, fibroblast growth factor 2, GITR3A, glucocorticoid-induced TNFR-related protein, H3K9me3, tri-methyl lysine 9 of histone H3, HILI, human piwi, HMOX1, heme oxygenase 1, HOXA10, homebox A10, ICOS1B, inducible T-cell co-stimulator, IL, interleukin, KIR1DL2, CD158b, expressed on natural killer cells and a subset of T cells, MKI-67, marker of proliferation ki-67, OCT4, octamer-binding transcription factor 4, PIWIL1, piwi-like protein 1, PNVP, poly-(N-vinylpyrrolidone), Polymer carriers, RB1, retinoblastoma 1, Reprogramming, SncRNAs, small non-coding RNAs, TE, transposon elements, TGFBR2, transforming growth factor beta receptor 2, TNFRS6B, TNF receptor superfamily 6B, TSS, transcriptional start sites, VMAF, musculoaponeurotic fibrosarcoma, Wnt-1, wingless type MMTV integration site family, member 1, iPS, induced pluripotent stem cells, mTOR, mechanistic target of rapamycin, miR, micro-RNA, miRNA-152, piR, piwi-interacting RNA, P-element induced wimpy testis interacting RNA, piRNA-30074,
• Publication type
• Journal Article MeSH