Subunit interactions and arrangements in the fission yeast Mis16–Mis18–Mis19 complex

Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1CENP-A. Cell cycle–specific deposition of Cnp1 requires the Mis16–Mis18–Mis19 complex, which is thought to direct recruitment of Scm3-chaperoned Cnp1/histone H4 dimers to DNA. Here, we present the structure of the essential Mis18 partner protein Mis19 and describe its interaction with Mis16, revealing a bipartite-binding site. We provide data on the stoichiometry and overall architecture of the complex and provide detailed insights into the Mis18–Mis19 interface.

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Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin and the associated centromere complex

10.1101/620088 ◽

2019 ◽

Author(s):

Sreyoshi Mitra ◽

Dani L. Bodor ◽

Ana F. David ◽

João F. Mata ◽

Beate Neumann ◽

...

Keyword(s):

Cell Cycle ◽

Genetic Screening ◽

Chromatin Modification ◽

Histone H3 ◽

Centromeric Chromatin ◽

Sumo Protease ◽

Chromatin Regulators ◽

Histone H3 Variant ◽

Key Factor ◽

Labeling Approach

AbstractCentromeres are defined by a unique self-propagating chromatin structure featuring nucleosomes containing the histone H3 variant CENP-A. CENP-A turns over slower than general chromatin and a key question is whether this unusual stability is intrinsic to CENP-A nucleosomes or rather imposed by external factors. We designed a specific genetic screen to identify proteins involved in CENP-A stability based on SNAP-tag pulse chase labeling. Using a double pulse-labeling approach we simultaneously assay for factors with selective roles in CENP-A chromatin assembly. We discover a series of new proteins involved in CENP-A propagation, including proteins with known roles in DNA replication, repair and chromatin modification and transcription, revealing that a broad set of chromatin regulators impacts in CENP-A transmission through the cell cycle. The key factor we find to strongly affect CENP-A stability is SENP6. This SUMO-protease controls not only the levels of chromatin bound CENP-A but is required for the maintenance of virtually the entire centromere and kinetochore, with the exception of CENP-B. Acute depletion of SENP6 protein reveals its requirement for maintaining centromeric CENP-A levels throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex.

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Quantitative single-molecule microscopy reveals that CENP-A Cnp1 deposition occurs during G2 in fission yeast

Open Biology ◽

10.1098/rsob.120078 ◽

2012 ◽

Vol 2 (7) ◽

pp. 120078 ◽

Cited By ~ 110

Author(s):

David Lando ◽

Ulrike Endesfelder ◽

Harald Berger ◽

Lakxmi Subramanian ◽

Paul D. Dunne ◽

...

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Single Molecule ◽

Cell Biology ◽

Histone H3 ◽

Histone Variants ◽

Epigenetic Mechanism ◽

Eukaryotic Cells ◽

Stochastic Variation ◽

Histone H3 Variant

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A Cnp1 with single-molecule sensitivity in fission yeast ( Schizosaccharomyces pombe ). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A Cnp1 levels at fission yeast ( S. pombe ) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A Cnp1 is deposited solely during the G2 phase of the cell cycle.

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Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2004.1614 ◽

2005 ◽

Vol 360 (1455) ◽

pp. 595-607 ◽

Cited By ~ 41

Author(s):

Kohta Takahashi ◽

Yuko Takayama ◽

Fumie Masuda ◽

Yasuyo Kobayashi ◽

Shigeaki Saitoh

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

S Phase ◽

Histone H4 ◽

Gata Factor ◽

Checkpoint Response ◽

Histone H3 Variant ◽

Two Factors ◽

A Cell ◽

G2 Phase

CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast.

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Histone H3 Variants in Trichomonas vaginalis

Eukaryotic Cell ◽

10.1128/ec.00006-12 ◽

2012 ◽

Vol 11 (5) ◽

pp. 654-661 ◽

Cited By ~ 7

Author(s):

Zuzana Zubáčová ◽

Jitka Hostomská ◽

Jan Tachezy

Keyword(s):

Cell Cycle ◽

Nuclear Membrane ◽

Trichomonas Vaginalis ◽

Histone H3 ◽

Direct Connection ◽

Centromeric Chromatin ◽

Content Type ◽

The Third ◽

Histone H3 Variant ◽

Active Transcription

ABSTRACT The parabasalid protist Trichomonas vaginalis is a widespread parasite that affects humans, frequently causing vaginitis in infected women. Trichomonad mitosis is marked by the persistence of the nuclear membrane and the presence of an asymmetric extranuclear spindle with no obvious direct connection to the chromosomes. No centromeric markers have been described in T. vaginalis , which has prevented a detailed analysis of mitotic events in this organism. In other eukaryotes, nucleosomes of centromeric chromatin contain the histone H3 variant CenH3. The principal aim of this work was to identify a CenH3 homolog in T. vaginalis . We performed a screen of the T. vaginalis genome to retrieve sequences of canonical and variant H3 histones. Three variant histone H3 proteins were identified, and the subcellular localization of their epitope-tagged variants was determined. The localization of the variant TVAG_185390 could not be distinguished from that of the canonical H3 histone. The sequence of the variant TVAG_087830 closely resembled that of histone H3. The tagged protein colocalized with sites of active transcription, indicating that the variant TVAG_087830 represented H3.3 in T. vaginalis . The third H3 variant (TVAG_224460) was localized to 6 or 12 distinct spots at the periphery of the nucleus, corresponding to the number of chromosomes in G 1 phase and G 2 phase, respectively. We propose that this variant represents the centromeric marker CenH3 and thus can be employed as a tool to study mitosis in T. vaginalis . Furthermore, we suggest that the peripheral distribution of CenH3 within the nucleus results from the association of centromeres with the nuclear envelope throughout the cell cycle.

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Depletion of KNL2 Results in Altered Expression of Genes Involved in Regulation of the Cell Cycle, Transcription, and Development in Arabidopsis

International Journal of Molecular Sciences ◽

10.3390/ijms20225726 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5726 ◽

Cited By ~ 1

Author(s):

Anastassia Boudichevskaia ◽

Andreas Houben ◽

Anne Fiebig ◽

Klara Prochazkova ◽

Ales Pecinka ◽

...

Keyword(s):

Cell Cycle ◽

Critical Role ◽

Histone H3 ◽

Global Gene Expression ◽

Proper Function ◽

Knockout Mutant ◽

Flower Bud ◽

Altered Expression ◽

Expression Of Genes ◽

Histone H3 Variant

Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.

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Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres

eLife ◽

10.7554/elife.02203 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 59

Author(s):

Jan Wisniewski ◽

Bassam Hajj ◽

Jiji Chen ◽

Gaku Mizuguchi ◽

Hua Xiao ◽

...

Keyword(s):

Cell Cycle ◽

Elevated Temperatures ◽

De Novo ◽

Histone H3 ◽

S Phase ◽

Nuclear Accumulation ◽

Histone H3 Variant ◽

Functionally Impaired ◽

Cell Cycle Dynamics ◽

Cell Lethality

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.

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Ser68 phosphoregulation is essential for CENP-A deposition, centromere function and viability in mice

10.1101/2021.11.23.469796 ◽

2021 ◽

Author(s):

Yuting Liu ◽

Kehui Wang ◽

Li Huang ◽

Jicheng Zhao ◽

Xinpeng Chen ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Histone H3 ◽

Dependent Manner ◽

Centromere Function ◽

Histone H3 Variant ◽

A Cell ◽

Cycle Dependent ◽

Spatiotemporal Control

Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.

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Global Replication-Independent Histone H4 Exchange in Budding Yeast

Eukaryotic Cell ◽

10.1128/ec.00202-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1780-1787 ◽

Cited By ~ 19

Author(s):

Jeffrey Linger ◽

Jessica K. Tyler

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Budding Yeast ◽

Histone H3 ◽

Eukaryotic Genome ◽

Histone H4 ◽

Histone Proteins ◽

Dynamic Exchange ◽

Histone Exchange

ABSTRACT The eukaryotic genome is packaged together with histone proteins into chromatin following DNA replication. Recent studies have shown that histones can also be assembled into chromatin independently of DNA replication and that this dynamic exchange of histones may be biased toward sites undergoing transcription. Here we show that epitope-tagged histone H4 can be incorporated into nucleosomes throughout the budding yeast (Saccharomyces cerevisiae) genome regardless of the phase of the cell cycle, the transcriptional status, or silencing of the region. Direct comparisons reveal that the amount of histone incorporation that occurs in G1-arrested cells is similar to that occurring in cells undergoing DNA replication. Additionally, we show that this histone incorporation is not dependent on the histone H3/H4 chaperones CAF-1, Asf1, and Hir1 individually. This study demonstrates that DNA replication and transcription are not necessary prerequisites for histone exchange in budding yeast, indicating that chromatin is more dynamic than previously thought.

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CENP-A nucleosome—a chromatin-embedded pedestal for the centromere: lessons learned from structural biology

Essays in Biochemistry ◽

10.1042/ebc20190074 ◽

2020 ◽

Vol 64 (2) ◽

pp. 205-221

Author(s):

Ahmad Ali-Ahmad ◽

Nikolina Sekulić

Keyword(s):

Cell Division ◽

Structural Biology ◽

Histone H3 ◽

Lessons Learned ◽

Centromeric Chromatin ◽

Centromere Proteins ◽

Molecular Determinants ◽

Histone H3 Variant ◽

Segregation Of Chromosomes

Abstract The centromere is a chromosome locus that directs equal segregation of chromosomes during cell division. A nucleosome containing the histone H3 variant CENP-A epigenetically defines the centromere. Here, we summarize findings from recent structural biology studies, including several CryoEM structures, that contributed to elucidate specific features of the CENP-A nucleosome and molecular determinants of its interactions with CENP-C and CENP-N, the only two centromere proteins that directly bind to it. Based on those findings, we propose a role of the CENP-A nucleosome in the organization of centromeric chromatin beyond binding centromeric proteins.

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N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

10.1101/176206 ◽

2017 ◽

Cited By ~ 1

Author(s):

Kentaro Ohkuni ◽

Reuben Levy-Myers ◽

Jack Warren ◽

Wei-Chun Au ◽

Yoshimitsu Takahashi ◽

...

Keyword(s):

Genome Stability ◽

Histone H3 ◽

Physiological Role ◽

Centromeric Chromatin ◽

E3 Ligases ◽

Physiological Conditions ◽

Evolutionarily Conserved ◽

Histone H3 Variant

AbstractStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

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