Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
- MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- bezbuněčný systém MeSH
- chromozomální proteiny, nehistonové fyziologie MeSH
- DNA bakterií metabolismus MeSH
- DNA footprinting MeSH
- fungální proteiny fyziologie MeSH
- histony genetika metabolismus MeSH
- mikroskopie atomárních sil MeSH
- multiproteinové komplexy fyziologie MeSH
- nukleozomy fyziologie ultrastruktura MeSH
- plazmidy chemie MeSH
- proteiny vázající RNA fyziologie MeSH
- rekombinantní proteiny metabolismus MeSH
- restrikční endonukleasy typu II MeSH
- restrukturace chromatinu genetika fyziologie MeSH
- Saccharomyces cerevisiae metabolismus ultrastruktura MeSH
- Xenopus laevis genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis.
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- aktiny metabolismus fyziologie MeSH
- buněčné jadérko metabolismus fyziologie MeSH
- chromozomální proteiny, nehistonové metabolismus fyziologie MeSH
- genetická transkripce fyziologie MeSH
- glukosa metabolismus MeSH
- HeLa buňky MeSH
- lidé MeSH
- ribozomální DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis.
- MeSH
- adenokarcinom patologie MeSH
- aparát dělícího vřeténka metabolismus ultrastruktura MeSH
- autoantigeny chemie fyziologie MeSH
- biologický transport MeSH
- centromera metabolismus MeSH
- chromozomální proteiny, nehistonové chemie fyziologie MeSH
- cytokineze fyziologie MeSH
- fluorescenční mikroskopie MeSH
- fosforylace MeSH
- fosfoserin metabolismus MeSH
- fosfothreonin metabolismus MeSH
- histony metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- MFC-7 buňky cytologie metabolismus MeSH
- mitóza fyziologie MeSH
- nádorové proteiny chemie fyziologie MeSH
- nádory prsu patologie MeSH
- posttranslační úpravy proteinů MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In monocentric organisms with asymmetric meiosis, the kinetochore proteins, such as CENH3 and CENP-C, evolve adaptively to counterbalance the deleterious effects of centromere drive, which is caused by the expansion of centromeric satellite repeats. The selection regimes that act on CENH3 and CENP-C genes have not been analyzed in organisms with holocentric chromosomes, although holocentrism is speculated to have evolved to suppress centromere drive. We tested both CENH3 and CENP-C for positive selection in several species of the holocentric genus Caenorhabditis using the maximum likelihood approach and sliding-window analysis. Although CENP-C did not show any signs of positive selection, positive selection has been detected in the case of CENH3. These results support the hypothesis that centromere drive occurs in Nematoda, at least in the telokinetic meiosis of Caenorhabditis.
- MeSH
- Caenorhabditis elegans genetika metabolismus MeSH
- centromera metabolismus fyziologie MeSH
- chromozomální proteiny, nehistonové genetika metabolismus fyziologie MeSH
- chromozomy metabolismus MeSH
- geneticky modifikované organismy MeSH
- histony genetika fyziologie MeSH
- meióza genetika fyziologie MeSH
- pohyb fyziologie MeSH
- segregace chromozomů genetika fyziologie MeSH
- selekce (genetika) MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.
- MeSH
- acetylace MeSH
- buněčné jádro metabolismus ultrastruktura MeSH
- chromatin ultrastruktura MeSH
- chromozomální proteiny, nehistonové fyziologie MeSH
- epigeneze genetická MeSH
- exprese genu MeSH
- financování organizované MeSH
- histony genetika metabolismus MeSH
- interfáze MeSH
- lidé MeSH
- lidské chromozomy X metabolismus MeSH
- metylace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.
- MeSH
- buněčné linie MeSH
- chromatin fyziologie MeSH
- chromozomální proteiny, nehistonové fyziologie MeSH
- DNA * metabolismus účinky záření MeSH
- fosforylace MeSH
- genom * MeSH
- jaderné proteiny fyziologie MeSH
- kultivované buňky MeSH
- lasery MeSH
- lidé MeSH
- oprava DNA * MeSH
- poškození DNA * MeSH
- protein BRCA1 fyziologie MeSH
- proteinkinasy fyziologie MeSH
- proteiny buněčného cyklu * fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Movement of labelled plasmid DNA relative to heterochromatin foci in nuclei, visualized with HP1-GFP, was studied using live-cell imaging and object tracking. In addition to Brownian motion of plasmid DNA we found a pronounced, non-random movement of plasmid DNA towards the nearest HP1 focus, while time-lapse microscopy showed that HP1 foci are relatively immobile and positionally stable. The movement of plasmid DNA was much faster than that of the HP1 foci. Contact of transgene DNA with an HP1 focus usually resulted in cessation of the directional motion. Moreover, the motion of plasmid DNA inside the heterochromatin compartment was more restricted (limited to 0.25 microm) than when the plasmid DNA was outside heterochromatin (R = 0.7 microm). Three days after transfection most of the foreign labelled DNA colocalized with centromeric heterochromatin.
- MeSH
- biologický transport fyziologie genetika MeSH
- buněčné jádro fyziologie MeSH
- chromozomální proteiny, nehistonové fyziologie genetika MeSH
- DNA fyziologie genetika MeSH
- financování organizované MeSH
- heterochromatin MeSH
- lidé MeSH
- mikroskopie MeSH
- nádorové buněčné linie MeSH
- plazmidy fyziologie genetika MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
