The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.

• Keywords
• Cell cycle, Chromatin, DNA replication, H3K56ac, Mammalian cells, Nucleosome,
• MeSH
• Cell Cycle genetics   physiology   MeSH
• Chromatin metabolism   MeSH
• G2 Phase genetics   MeSH
• Histones metabolism   MeSH
• HL-60 Cells MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Nucleosomes metabolism   MeSH
• DNA Replication genetics   physiology   MeSH
• S Phase genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   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
• Nucleosomes MeSH
• Saccharomyces cerevisiae 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

Cajal bodies are important nuclear structures containing proteins that preferentially regulate RNA-related metabolism. We investigated the cell-type specific nuclear distribution of Cajal bodies and the level of coilin, a protein of Cajal bodies, in non-irradiated and irradiated human tumor cell lines and embryonic stem (ES) cells. Cajal bodies were localized in different nuclear compartments, including DAPI-poor regions, in the proximity of chromocenters, and adjacent to nucleoli. The number of Cajal bodies per nucleus was cell cycle-dependent, with higher numbers occurring during G2 phase. Human ES cells contained a high coilin level in the nucleoplasm, but coilin-positive Cajal bodies were also identified in nuclei of mouse and human ES cells. Coilin, but not SMN, recognized UVA-induced DNA lesions, which was cell cycle-independent. Treatment with γ-radiation reduced the localized movement of Cajal bodies in many cell types and GFP-coilin fluorescence recovery after photobleaching was very fast in nucleoplasm in comparison with GFP-coilin recovery in DNA lesions. By contrast, nucleolus-localized coilin displayed very slow fluorescence recovery after photobleaching, which indicates very slow rates of protein diffusion, especially in nucleoli of mouse ES cells.

• Keywords
• Cajal bodies, DNA repair, chromatin, coilin, nucleolus, nucleus,
• MeSH
• Cell Nucleus genetics   metabolism   radiation effects   MeSH
• Cell Line MeSH
• K562 Cells MeSH
• Coiled Bodies genetics   metabolism   radiation effects   MeSH
• DNA genetics   radiation effects   MeSH
• G2 Phase genetics   MeSH
• HeLa Cells MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Ultraviolet Rays adverse effects   MeSH
• Gamma Rays adverse effects   MeSH
• Green Fluorescent Proteins genetics   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
• DNA MeSH
• Nuclear Proteins MeSH
• p80-coilin MeSH Browser
• Recombinant Fusion Proteins MeSH
• Green Fluorescent Proteins 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

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

• MeSH
• Ki-67 Antigen metabolism   MeSH
• Cell Differentiation * MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Embryonic Stem Cells cytology   metabolism   MeSH
• Epigenesis, Genetic MeSH
• Fluorescence Recovery After Photobleaching MeSH
• G2 Phase MeSH
• Histones metabolism   MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Microscopy, Confocal MeSH
• Cells, Cultured MeSH
• Lysine metabolism   MeSH
• Methylation MeSH
• Mice MeSH
• Nanog Homeobox Protein MeSH
• Stem Cell Niche MeSH
• Octamer Transcription Factor-3 genetics   metabolism   MeSH
• Pluripotent Stem Cells cytology   metabolism   MeSH
• Proto-Oncogene Proteins c-myc genetics   metabolism   MeSH
• Blotting, Western MeSH
• Green Fluorescent Proteins genetics   metabolism   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
• Ki-67 Antigen MeSH
• Histones MeSH
• Homeodomain Proteins MeSH
• Lysine MeSH
• Myc protein, mouse MeSH Browser
• Nanog protein, mouse MeSH Browser
• Nanog Homeobox Protein MeSH
• Octamer Transcription Factor-3 MeSH
• Proto-Oncogene Proteins c-myc MeSH
• Green Fluorescent Proteins MeSH

Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT), a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.

• MeSH
• Receptors, Androgen metabolism   MeSH
• Androgen Antagonists pharmacology   MeSH
• beta-Galactosidase metabolism   MeSH
• Down-Regulation drug effects   MeSH
• PTEN Phosphohydrolase metabolism   MeSH
• Insulin-Like Growth Factor Binding Protein 3 metabolism   MeSH
• Cathepsin B metabolism   MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms genetics   metabolism   pathology   MeSH
• S-Phase Kinase-Associated Proteins genetics   metabolism   MeSH
• Flow Cytometry MeSH
• RNA Interference MeSH
• Signal Transduction drug effects   MeSH
• Cellular Senescence drug effects   MeSH
• Vimentin metabolism   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Androgen MeSH
• Androgen Antagonists MeSH
• AR protein, human MeSH Browser
• beta-Galactosidase MeSH
• PTEN Phosphohydrolase MeSH
• Insulin-Like Growth Factor Binding Protein 3 MeSH
• Cathepsin B MeSH
• S-Phase Kinase-Associated Proteins MeSH
• Vimentin 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