The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

• MeSH
• Cell Differentiation radiation effects   MeSH
• Cell Line MeSH
• Genetic Loci * MeSH
• Hypoxanthine Phosphoribosyltransferase genetics   metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Humans MeSH
• Mutation Rate * MeSH
• Gamma Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hypoxanthine Phosphoribosyltransferase 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

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: Oct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

• MeSH
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Adenosine Triphosphate metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin metabolism   MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• DNA-Binding Proteins metabolism   MeSH
• Embryonic Stem Cells cytology   MeSH
• Fibroblasts metabolism   MeSH
• Phosphorylation MeSH
• Transcription, Genetic MeSH
• Histones chemistry   MeSH
• Kinetics MeSH
• Mice MeSH
• Octamer Transcription Factor-3 metabolism   MeSH
• DNA Damage MeSH
• Regenerative Medicine methods   MeSH
• Gene Expression Regulation * 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
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Adenosine Triphosphate MeSH
• Chromatin MeSH
• Chromosomal Proteins, Non-Histone MeSH
• DNA-Binding Proteins MeSH
• Histones MeSH
• Octamer Transcription Factor-3 MeSH
• Pou5f1 protein, mouse MeSH Browser
• Trp53bp1 protein, mouse MeSH Browser