CENP-A Dotaz Zobrazit nápovědu
The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
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
The centromere drive model explaining rapid evolution of eukaryotic centromeres predicts higher frequency of positive selection acting on centromeric histone H3 (CenH3) in clades with asymmetric meiosis compared to the clades with only symmetric meiosis. However, despite the impression one might get from the literature, this key prediction of the centromere drive model has not only never been confirmed, but it has never been tested, because all the previous studies dealt only with the presence or absence instead of the frequency of positive selection. To provide evidence for or against different frequencies of positively selected CenH3 in asymmetrics and symmetrics, we have inferred the selective pressures acting on CenH3 in seventeen eukaryotic clades, including plants, animals, fungi, ciliates and apicomplexa, using codon-substitution models, and compared the inferred frequencies between asymmetrics and symmetrics in a quantitative manner. We have found that CenH3 has been evolving adaptively much more frequently in clades with asymmetric meiosis compared with clades displaying only symmetric meiosis which confirms the prediction of centromere drive model. Our findings indicate that the evolution of asymmetric meiosis required CenH3 to evolve adaptively more often to counterbalance the negative consequences of centromere drive.
- MeSH
- centromera genetika MeSH
- histony genetika MeSH
- houby genetika MeSH
- kodon genetika MeSH
- lidé MeSH
- meióza genetika MeSH
- molekulární evoluce * MeSH
- protein CENP-A genetika MeSH
- rostlinné proteiny genetika MeSH
- sekvenční seřazení MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alterations in the gene encoding the E3 ubiquitin ligase substrate receptor FBXO38 have been associated with several diseases, including early-onset motor neuronopathy. However, the cellular processes affected by the enzymatic action of FBXO38 are not yet known. Here, we identify the zinc finger proteins ZXDA/B as its interaction partners. FBXO38 controls the stability of ZXDA/B proteins via ubiquitination and proteasome-dependent degradation. We show that ZXDA/B proteins associate with the centromeric protein CENP-B and that the interaction between ZXDA/B and FBXO38 or CENP-B is mutually exclusive. Functionally, ZXDA/B factors control the protein level of chromatin-associated CENP-B. Furthermore, their inappropriate stabilization leads to upregulation of CENP-A and CENP-B positive centromeric chromatin. Thus we demonstrate a previously unknown role of cullin-dependent protein degradation in the control of centromeric chromatin integrity.
- Publikační typ
- časopisecké články MeSH
SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCFFBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCFFBXO38 and PD-1 proteolysis. Our data do not support the notion that SCFFBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.
- MeSH
- antigeny CD279 * metabolismus genetika MeSH
- F-box proteiny * metabolismus genetika MeSH
- lidé MeSH
- myši MeSH
- proteinligasy komplexu SCF metabolismus genetika MeSH
- proteolýza MeSH
- T-lymfocyty metabolismus MeSH
- ubikvitinace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Epigenetic histone (H3) modification patterns and the nuclear radial arrangement of select genetic elements were compared in human embryonic stem cells (hESCs) before and after differentiation. H3K9 acetylation, H3K9 trimethylation, and H3K79 monomethylation were reduced at the nuclear periphery of differentiated hESCs. Differentiation coincided with centromere redistribution, as evidenced by perinucleolar accumulation of the centromeric markers CENP-A and H3K9me3, central repositioning of centromeres 1, 5, 19, and rearrangement of other centromeres at the nuclear periphery. The radial positions of PML, RARalpha genes, and human chromosomes 10, 12, 15, 17, and 19 remained relatively stable as hESCs differentiated. However, the female inactive H3K27-trimethylated X chromosome occupied a more peripheral nuclear position in differentiated cells. Thus, pluripotent and differentiated hESCs have distinct nuclear patterns of heterochromatic structures (centromeres and inactive X chromosome) and epigenetic marks (H3K9me3, and H3K27me3), while relatively conserved gene density-related radial chromatin distributions are already largely established in undifferentiated hES cells. (c) 2008 Wiley-Liss, Inc.
- MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné linie MeSH
- chromatin MeSH
- embryonální kmenové buňky cytologie metabolismus účinky léků MeSH
- epigeneze genetická genetika MeSH
- histony metabolismus MeSH
- lidé MeSH
- tretinoin farmakologie MeSH
- tvar buňky MeSH
- vývojová regulace genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Centromeric and pericentromeric chromosome regions are occupied by satellite DNA. Satellite DNAs play essential roles in chromosome segregation, and, thanks to their extensive sequence variability, to some extent, they can also be used as phylogenetic markers. In this paper, we isolated and sequenced satellite DNA I-IV in 11 species of Cervidae. The obtained satellite DNA sequences and their chromosomal distribution were compared among the analysed representatives of cervid subfamilies Cervinae and Capreolinae. Only satI and satII sequences are probably present in all analysed species with high abundance. On the other hand, fluorescence in situ hybridisation (FISH) with satIII and satIV probes showed signals only in a part of the analysed species, indicating interspecies copy number variations. Several indices, including FISH patterns, the high guanine and cytosine (GC) content, and the presence of centromere protein B (CENP-B) binding motif, suggest that the satII DNA may represent the most important satellite DNA family that might be involved in the centromeric function in Cervidae. The absence or low intensity of satellite DNA FISH signals on biarmed chromosomes probably reflects the evolutionary reduction of heterochromatin following the formation of chromosome fusions. The phylogenetic trees constructed on the basis of the satellite I-IV DNA relationships generally support the present cervid taxonomy.
- MeSH
- centromera genetika MeSH
- heterochromatin genetika MeSH
- hybridizace in situ fluorescenční metody MeSH
- lidé MeSH
- přežvýkavci genetika MeSH
- repetitivní sekvence nukleových kyselin genetika MeSH
- satelitní DNA genetika MeSH
- variabilita počtu kopií segmentů DNA genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.
- MeSH
- anafázi podporující komplex MeSH
- buněčný cyklus * MeSH
- cyklin E metabolismus MeSH
- cyklin-dependentní kinasa 2 MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- DNA vazebné proteiny * MeSH
- fluorescenční protilátková technika MeSH
- interfáze účinky léků MeSH
- kinasy CDC2-CDC28 * MeSH
- komplexy ubikvitinligas * MeSH
- lidé MeSH
- ligasy * metabolismus MeSH
- makromolekulární látky MeSH
- mitóza * MeSH
- nádorové buňky kultivované MeSH
- protein Cdc20 MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- proteiny Drosophily * MeSH
- protilátky farmakologie MeSH
- průtoková cytometrie MeSH
- replikace DNA * MeSH
- Saccharomyces cerevisiae - proteiny * MeSH
- trans-aktivátory * MeSH
- transkripční faktor DP1 MeSH
- transkripční faktory E2F MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny * MeSH
- ubikvitinligasy MeSH
- vazba proteinů MeSH
- vazebný protein 1 retinoblastomu MeSH
- western blotting MeSH
- Check Tag
- lidé 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
UNLABELLED: Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLKKKT10/19, PpSYCP2L1KKT17/18, and PpINCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system. IMPORTANCE: A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.
