BACKGROUND: Chemical modifications in mRNAs, tRNAs, rRNAs, and non-coding RNAs stabilize these nucleic acids and regulate their function. In addition to regulating the translation of genetic information from mRNA to proteins, it has been revealed that modifications in RNAs regulate repair processes in the genome. METHODS: Using local laser microirradiation, confocal microscopy, dot blots, and mass spectrometry we studied the role of N7-methylguanosine (m7G), which is co-transcriptionally installed in RNA. RESULTS: Here, we show that after UVC and UVA irradiation, the level of m7G RNA is increased initially in the cytoplasm, and after local laser microirradiation, m7G RNA is highly abundant in UVA-damaged chromatin. This process is poly(ADP-ribose) polymerase (PARP)-dependent, but not accompanied by changes in the level of m7G-writers, including methyltransferases RNMT, METTL1, and WBSCR22. We also observed that METTL1 deficiency does not affect the recruitment of m7G RNA to microirradiated chromatin. Analyzing the levels of mRNA, let-7e, and miR-203a in both the cytoplasm and the cell nucleus, we revealed that UVC irradiation changed the level of mRNA, and significantly increased the pool of both let-7e and miR-203a, which correlated with radiation-induced m7G RNA increase in the cytoplasm. CONCLUSIONS: Irradiation by UV light increases the m7G RNA pool in the cytoplasm and in the microirradiated genome. Thus, epigenetically modified RNAslikely contribute to DNA damage responses or m7G signals the presence of RNA damage.
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články 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.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- chromatinová imunoprecipitace MeSH
- DNA vazebné proteiny MeSH
- epigeneze genetická účinky záření MeSH
- fluorescenční protilátková technika MeSH
- histony metabolismus MeSH
- metylace MeSH
- myši MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese účinky záření MeSH
- ribozomální DNA genetika MeSH
- transkripční iniciační komplex Pol1 - proteiny metabolismus MeSH
- ultrafialové záření * MeSH
- vazba proteinů MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- Publikační typ
- časopisecké články MeSH
Every day, genomes are affected by genotoxic factors that create multiple DNA lesions. Several DNA repair systems have evolved to counteract the deleterious effects of DNA damage. These systems include a set of DNA repair mechanisms, damage tolerance processes, and activation of cell-cycle checkpoints. This study describes selected confocal microscopy techniques that investigate DNA damage-related nuclear events after UVA- and γ-irradiation and compare the DNA damage response (DDR) induced by the two experimental approaches. In both cases, we observed induction of the nucleotide excision repair (NER) pathway and formation of localized double-strand breaks (DSBs). This was confirmed by analysis of cyclobutane pyrimidine dimers (CPDs) in the DNA lesions and by increased levels of γH2AX and 53BP1 proteins in the irradiated genome. DNA damage by UVA-lasers was potentiated by either BrdU or Hoechst 33342 pre-sensitization and compared to non-photosensitized cells. DSBs were also induced without BrdU or Hoechst 33342 pre-treatment. Interestingly, no cyclobutane pyrimidine dimers (CPDs) were detected after 405 nm UVA laser micro-irradiation in non-photosensitized cells. The effects of UVA and γ-irradiation were also studied by silver staining of nucleolar organizer regions (AgNORs). This experimental approach revealed changes in the morphology of nucleoli after genome injury. Additionally, to precisely characterize DDR in locally induced DNA lesions, we analysed the kinetics of the 53BP1 protein involved in DDR by fluorescence recovery after photobleaching (FRAP).
- MeSH
- 53BP1 MeSH
- antigeny jaderné MeSH
- buněčné jadérko účinky záření MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- histony metabolismus MeSH
- kinetika MeSH
- luminescentní proteiny metabolismus MeSH
- mikroskopie metody MeSH
- myši MeSH
- poškození DNA * MeSH
- pyrimidinové dimery metabolismus MeSH
- ultrafialové záření * MeSH
- záření gama * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND INFORMATION: Promyelocytic leukaemia (PML) bodies are specific nuclear structures with functional significance for acute promyelocytic leukaemia. In this study, we analysed the trajectories of PML bodies using single-particle tracking. RESULTS: We observed that the recovery of PML protein after photobleaching was ATP dependent in both wild-type (wt) and A-type lamin-deficient cells. The movement of PML bodies was faster and the nuclear area occupied by particular PML bodies was larger in A-type lamin-deficient fibroblasts compared with their wt counterparts. Moreover, dysfunction of the LMNA gene increased the frequency of mutual interactions between individual PML bodies and influenced the morphology of these domains at the ultrastructural level. As a consequence of A-type lamin deficiency, PML protein accumulated in nuclear blebs and frequently appeared at the nuclear periphery. CONCLUSIONS: We suggest that the physiological function of lamin A proteins is important for events that occur in the compartment of PML bodies. This observation was confirmed in other experimental models characterised by lamin changes, including apoptosis or the differentiation of mouse embryonic stem cells.
- MeSH
- akutní promyelocytární leukemie metabolismus MeSH
- apoptóza MeSH
- embryo savčí cytologie MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- fibroblasty cytologie metabolismus ultrastruktura MeSH
- FRAP MeSH
- intranukleární inkluzní tělíska metabolismus ultrastruktura MeSH
- kinetika MeSH
- lamin typ A nedostatek metabolismus MeSH
- myši MeSH
- reprodukovatelnost výsledků MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- Publikační typ
- časopisecké články MeSH
The treatment of human promyelocytic leukemia cell lines HL-60, and to some extent NB-4, with 1alpha,25-dihydroxyvitamin D(3) (VD3) induces differentiation toward the monocytic/macrophage lineage, demonstrated by the increased expression of CD11b and CD14, and the production of opsonized zymosan particles (OZP)-stimulated reactive oxygen species (ROS). Moreover, in more sensitive HL-60 cells, increased expression of 5-lipoxygenase (5-LPO), Mcl-1, IkappaB, and c-Jun, accompanied by the activation of p38 MAPK, was detected. These VD3 effects on HL-60 cell differentiation were significantly potentiated by 5-LPO inhibitors MK-886 and AA-861 and were inverted by SB202190 (SB), a p38 MAPK inhibitor. The inhibition of differentiation by SB was demonstrated by a reduction of CD14 expression and by a decrease in OZP-activated ROS production. These results indicated that p38 MAPK pathway is involved in 5-LPO inhibitors-dependent potentiation of VD3-induced monocytic differentiation.
- MeSH
- arachidonát-5-lipoxygenasa genetika MeSH
- benzochinony farmakologie MeSH
- buněčná diferenciace účinky léků MeSH
- HL-60 buňky MeSH
- indoly farmakologie MeSH
- inhibitory lipoxygenas farmakologie MeSH
- lidé MeSH
- mitogenem aktivované proteinkinasy p38 metabolismus MeSH
- monocyty cytologie MeSH
- vitamin D analogy a deriváty farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
