Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1β to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time τ = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to γH2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HPβ to UV-irradiated chromatin.

• MeSH
• acetylace MeSH
• buněčné linie MeSH
• buňky 3T3 MeSH
• časosběrné zobrazování MeSH
• chromatin metabolismus   účinky záření   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• FRAP MeSH
• histony metabolismus   MeSH
• inhibitory histondeacetylas metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kyseliny hydroxamové metabolismus   MeSH
• lidé MeSH
• myši MeSH
• poškození DNA MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

• MeSH
• adenokarcinom genetika   metabolismus   patologie   terapie   MeSH
• lidé MeSH
• mutace genetika   MeSH
• myši knockoutované MeSH
• myši MeSH
• nádory plic genetika   metabolismus   patologie   terapie   MeSH
• PRC1 genetika   metabolismus   MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• Klíčová slova
• geny Bmi1 a Ezh2, proteiny rodiny PcG,
• MeSH
• exprese genu * genetika   imunologie   účinky léků   MeSH
• imunohistochemie metody   trendy   využití   MeSH
• klinické laboratorní techniky metody   využití   MeSH
• lékařská onkologie metody   trendy   MeSH
• lidé MeSH
• nádorové buněčné linie mikrobiologie   účinky léků   MeSH
• neuroektodermové nádory * diagnóza   etiologie   genetika   MeSH
• polycomb proteiny genetika   izolace a purifikace   MeSH
• statistika jako téma MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND INFORMATION: The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. RESULTS: We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. CONCLUSION: We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most striking protein present not only at DNA lesions, but also spreading out in their vicinity. SIGNIFICANCE: Our conclusions highlight the significant role of the spatiotemporal dynamics of DNA repair-related proteins and their specific assembly/disassembly at DNA lesions, which can be cell type- and cell cycle dependent.

• MeSH
• buněčný cyklus genetika   účinky záření   MeSH
• DNA genetika   metabolismus   MeSH
• fosforylace účinky záření   MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny genetika   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• oprava DNA genetika   účinky záření   MeSH
• poškození DNA genetika   účinky záření   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND INFORMATION: The optimal repair of DNA lesions is fundamental for physiological processes. We asked whether the recruitment of HP1β, 53BP1 and BMI1 proteins to ultraviolet (UVA)-induced DNA lesions requires functional A-type lamins. RESULTS: We found that UVA irradiation of nuclear lamina abolished the fluorescence of mCherry-tagged A-type lamins and destroyed the nuclear lamina as also observed by electron microscopy studies. Similarly, an absence of endogenous A- and B-type lamins was found in irradiated regions by UVA. However, irradiation did not affect the recruitment of HP1β, 53BP1 and BMI1 to DNA lesions. The UVA-induced shrinkage of the nuclear lamina, which anchors chromatin, explains why UVA-micro-irradiated chromatin is relaxed. Conversely, additional experiments with γ-irradiation showed that the nuclear lamina remained intact and the genome-wide level of HP1β was stable. Fluorescence intensity of HP1β and BMI1 in UVA-induced DNA lesions and level of HP1β after γ-irradiation were unaffected by deficiency in A-type lamins, whereas those parameters of 53BP1 were changed. CONCLUSIONS: We conclude that only the 53BP1 status in DNA lesions, induced by UVA or γ-rays, is affected by A-type lamin deficiency, which was not observed for heterochromatin-related proteins HP1β and BMI1.

• MeSH
• buňky 3T3 MeSH
• chromozomální proteiny, nehistonové analýza   metabolismus   MeSH
• DNA vazebné proteiny analýza   metabolismus   MeSH
• DNA genetika   MeSH
• lamin typ A analýza   metabolismus   MeSH
• myši MeSH
• poškození DNA účinky záření   MeSH
• PRC1 analýza   metabolismus   MeSH
• protoonkogenní proteiny analýza   metabolismus   MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• acetylace MeSH
• antitumorózní látky fytogenní MeSH
• apoptóza účinky léků   MeSH
• buněčná adheze MeSH
• chromatin účinky léků   genetika   metabolismus   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• epigeneze genetická účinky léků   MeSH
• etoposid MeSH
• fibroblasty cytologie   účinky léků   metabolismus   MeSH
• histony genetika   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• metylace MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• protoonkogenní proteiny c-bcl-2 genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• biologické markery * MeSH
• buněčné dělení MeSH
• epidermální buňky MeSH
• epidermis metabolismus   MeSH
• fluorescenční barviva MeSH
• imunohistochemie MeSH
• indoly MeSH
• inhibitor p16 cyklin-dependentní kinasy genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• kůže * metabolismus   MeSH
• lidé MeSH
• PRC1 MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• regulace genové exprese * fyziologie   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• škára metabolismus   MeSH
• stárnutí buněk * fyziologie   MeSH
• věkové rozložení MeSH
• Check Tag
• lidé 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

BACKGROUND INFORMATION: A Polycomb (PcG) body is an orphan nuclear subcompartment characterised by accumulations of Polycomb repressive complex 1 (PRC1) proteins. However, seemingly contradictory reports have appeared that describe the PcG bodies either as protein-based bodies in the interchromatin compartment or chromatin domains. In this respect, molecular crowding is an important factor for the assembly and stability of nuclear subcompartments. In order to settle this contradiction, crowding experiments, that represent a convenient model distinguishing between interchromatin and chromatin compartments, were carried out. RESULTS: In sucrose-hypertonically induced crowding, we observed in U-2 OS cells that PcG bodies disappeared, but persisted as nuclear domains characterised by accumulations of DNA. This phenomenon was also observed in cells hypertonically treated with sorbitol and NaCl. Importantly, the observed changes were quickly reversible after re-incubation of cells in normal medium. We found that the PcG foci disappearance and the dissociation of PRC1 proteins (BMI1 and RING1a proteins) from chromatin were associated with their hyper-phosphorylation. In addition, under hyper- and hypotonic conditions, the behaviour of the PcG bodies differed from that of the typical nucleoplasmic body. CONCLUSION: PRC1 proteins accumulations do not represent a genuine nuclear subcompartment. The PcG body is a chromosomal domain, rather than a nucleoplasmic body.

• MeSH
• anthrachinony metabolismus   MeSH
• barvení a značení MeSH
• chromatin metabolismus   MeSH
• fluorescence MeSH
• fosforylace účinky léků   MeSH
• genetická transkripce účinky léků   MeSH
• hypertonické roztoky farmakologie   MeSH
• lidé MeSH
• makromolekulární látky metabolismus   MeSH
• nádorové buněčné linie MeSH
• polycomb proteiny metabolismus   MeSH
• PRC1 metabolismus   MeSH
• RNA genetika   metabolismus   MeSH
• sacharosa farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Polycomb group (PcG) proteins of the Polycomb repressive complex 1 (PRC1) are found to be diffusely distributed in nuclei of cells from various species. However they can also be localized in intensely fluorescent foci, whether imaged using GFP fusions to proteins of PRC1 complex, or by conventional immunofluorescence microscopy. Such foci are termed PcG bodies, and are believed to be situated in the nuclear intechromatin compartment. However, an ultrastructural description of the PcG body has not been reported to date. To establish the ultrastructure of PcG bodies in human U-2 OS cells stably expressing recombinant polycomb BMI1-GFP protein, we used correlative light-electron microscopy (CLEM) implemented with high-pressure freezing, cryosubstitution and on-section labeling of BMI1 protein with immunogold. This approach allowed us to clearly identify fluorescent PcG bodies, not as distinct nuclear bodies, but as nuclear domains enriched in separated heterochromatin fascicles. Importantly, high-pressure freezing and cryosubstitution allowed for a high and clear-cut immunogold BMI1 labeling of heterochromatin structures throughout the nucleus. The density of immunogold labeled BMI1 in the heterochromatin fascicles corresponding to fluorescent "PcG bodies" did not differ from the density of labeling of heterochromatin fascicles outside of the "PcG bodies". Accordingly, an appearance of the fluorescent "PcG bodies" seems to reflect a local accumulation of the labeled heterochromatin structures in the investigated cells. The results of this study should allow expansion of the knowledge about the biological relevance of the "PcG bodies" in human cells.

• MeSH
• elektronová mikroskopie MeSH
• heterochromatin metabolismus   MeSH
• imunohistochemie MeSH
• kryoprezervace MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• represorové proteiny chemie   metabolismus   MeSH
• světlo MeSH
• tlak MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH