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

eLife ◽  
2014 ◽  
Vol 3 ◽  
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.

scholarly journals Swr1 mediated H2A.ZPht1 incorporation designates centromere DNA for de novo CENP-ACnp1 assembly

2017 ◽  
Author(s):  
Raghavendran Kulasegaran-Shylini ◽  
Lakxmi Subramanian ◽  
Alastair R. W. Kerr ◽  
Christos Spanos ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
De Novo ◽  
Histone H3 ◽  
S Phase ◽  
Chromatin Integrity

SUMMARYThe underlying hallmark of centromeres is the presence of specialized nucleosomes in which histone H3 is replaced by CENP-A. The events that mediate the installation of CENP-A in place of H3 remain poorly characterized. H2A.Z is linked to transcriptional competence and associates with mammalian centromeres. We find that H2A.ZPht1 and the Swr1 complex are enriched in fission yeast CENP-ACnp1 chromatin. Our analysis shows that Swr1, Msc1 and H2A.ZPht1 are required to maintain CENP-ACnp1 chromatin integrity. Cell cycle analyses demonstrate that H2A.ZPht1 is deposited in S phase, coincident with the deposition of placeholder H3, and prior to CENP-ACnp1 replenishment in G2. Establishment assays reveal that H2A.ZPht1 and Swr1 are required for de novo assembly of CENP-ACnp1 onto naïve centromere DNA. We propose that features akin to promoters within centromere DNA program the incorporation of H2A.ZPht1 via Swr1, and mediate the replacement of resident H3 nucleosomes with CENP-A nucleosomes thereby defining centromeres.

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

2019 ◽  
Vol 20 (22) ◽  
pp. 5726 ◽  
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.

scholarly journals Centromeric DNA destabilizes H3 nucleosomes to promote CENP-A deposition during the cell cycle

2017 ◽  
Author(s):  
Manu Shukla ◽  
Tong Pin ◽  
Sharon A. White ◽  
Puneet P. Singh ◽  
Angus M. Reid ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Chromosomal Location ◽  
Nucleosome Occupancy ◽  
Histone H3 ◽  
S Phase ◽  
Intrinsic Properties ◽  
Centromeric Dna ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Specific Sequences

SummaryActive centromeres are defined by the presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and kinetochores are maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A itself promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences. Intrinsic properties of centromeric DNA may favour the assembly of CENP-A rather than H3 nucleosomes. Here we investigate histone dynamics on centromeric DNA. We show that during S-phase histone H3 is deposited as a placeholder at fission yeast centromeres and is subsequently evicted in G2 when we detect deposition of the majority of new CENP-ACnp1. We also find that centromeric DNA has an innate property of driving high rates of turnover of H3 containing nucleosomes resulting in low nucleosome occupancy. When placed at an ectopic chromosomal location in the absence of any CENP-ACnp1 assembly, centromeric DNA retains its ability to impose S-phase deposition and G2 eviction of H3, suggesting that features within this DNA program H3 dynamics. As RNAPII occupancy on this centromere DNA coincides with H3 eviction in G2, we propose a model in which RNAPII-coupled chromatin remodelling promotes replacement of H3 with CENP-ACnp1 nucleosomes.

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

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.

scholarly journals Interphase Cell Cycle Dynamics of a Late-Replicating, Heterochromatic Homogeneously Staining Region: Precise Choreography of Condensation/Decondensation and Nuclear Positioning

1998 ◽  
Vol 140 (5) ◽  
pp. 975-989 ◽  
Author(s):  
Gang Li ◽  
Gail Sudlow ◽  
Andrew S. Belmont
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Sequences ◽  
Large Scale ◽  
S Phase ◽  
Interphase Cell ◽  
Compact Mass ◽  
Lac Operator ◽  
Cell Cycle Dynamics

Recently we described a new method for in situ localization of specific DNA sequences, based on lac operator/repressor recognition (Robinett, C.C., A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, and A.S. Belmont. 1996. J. Cell Biol. 135:1685–1700). We have applied this methodology to visualize the cell cycle dynamics of an ∼90 Mbp, late-replicating, heterochromatic homogeneously staining region (HSR) in CHO cells, combining immunostaining with direct in vivo observations. Between anaphase and early G1, the HSR extends approximately twofold to a linear, ∼0.3-μm-diam chromatid, and then recondenses to a compact mass adjacent to the nuclear envelope. No further changes in HSR conformation or position are seen through mid-S phase. However, HSR DNA replication is preceded by a decondensation and movement of the HSR into the nuclear interior 4–6 h into S phase. During DNA replication the HSR resolves into linear chromatids and then recondenses into a compact mass; this is followed by a third extension of the HSR during G2/ prophase. Surprisingly, compaction of the HSR is extremely high at all stages of interphase. Preliminary ultrastructural analysis of the HSR suggests at least three levels of large-scale chromatin organization above the 30-nm fiber.

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

2019 ◽  
Vol 2 (4) ◽  
pp. e201900408 ◽  
Author(s):  
Melanie Korntner-Vetter ◽  
Stéphane Lefèvre ◽  
Xiao-Wen Hu ◽  
Roger George ◽  
Martin R Singleton
Keyword(s):  
Cell Cycle ◽  
Binding Site ◽  
Fission Yeast ◽  
Histone H3 ◽  
Histone H4 ◽  
Subunit Interactions ◽  
Centromeric Chromatin ◽  
Partner Protein ◽  
Histone H3 Variant

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.

scholarly journals Post-mitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal transcriptome, identity, and connectivity

2021 ◽  
Author(s):  
Owen H Funk ◽  
Yaman Qalieh ◽  
Daniel Z Doyle ◽  
Mandy M Lam ◽  
Kenneth Y Kwan
Keyword(s):  
Cortical Neurons ◽  
Neural Progenitor Cells ◽  
De Novo ◽  
Histone H3 ◽  
Histone Variants ◽  
Total Histone ◽  
Critical Window ◽  
Histone H3 Variant ◽  
Transcriptomic Changes ◽  
Encoding Genes

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide replacement histones in terminally post-mitotic cells, including neurons. Histone variants can also serve active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover at regulatory regions. Here, we find that newborn cortical excitatory neurons substantially accumulate the histone H3 variant H3.3 immediately post-mitosis. Co-deletion of H3.3-encoding genes H3f3a and H3f3b from new neurons abrogates this accumulation, and causes widespread disruptions in the developmental establishment of the neuronal transcriptome. These broad transcriptomic changes coincide with neuronal maturation phenotypes in acquisition of distinct neuronal identities and formation of axon tracts. Stage-dependent deletion of H3f3a and H3f3b from (1) cycling neural progenitor cells, (2) neurons immediately after terminal mitosis, or (3) several days later, reveals the first post-mitotic days as a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, co-deletion of H3f3a and H3f3b from neurons causes progressive H3.3 depletion over several months without widespread transcriptional disruptions. Our study thus uncovers a key role for H3.3 in establishing neuronal transcriptome, identity, and connectivity immediately post-mitosis that is distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.

scholarly journals Induction of spontaneous human neocentromere formation and long-term maturation

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Marina Murillo-Pineda ◽  
Luis P. Valente ◽  
Marie Dumont ◽  
João F. Mata ◽  
Daniele Fachinetti ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
De Novo ◽  
Histone H3 ◽  
Engineering System ◽  
Chromosome Engineering ◽  
Histone H3 Variant ◽  
Chromosomal Passenger ◽  
Long Read ◽  
Functional Kinetochore

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

Deoxyribonucleotide Biosynthesis in Green Algae. S Phase-Specific Thymidylate Kinase and Unspecific Nucleoside Diphosphate Kinase in Scenedesmus obliquus

1988 ◽  
Vol 43 (5-6) ◽  
pp. 377-385 ◽  
Author(s):  
Beate Klein ◽  
Hartmut Follmann
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Green Algae ◽  
De Novo ◽  
Scenedesmus Obliquus ◽  
Nucleoside Diphosphate Kinase ◽  
S Phase ◽  
Ndp Kinase ◽  
Thymidylate Kinase

NDP kinase and thymidylate kinase are essential for DNA precursor formation in that they phosphorylate the products of de novo deoxyribonucleotide biosynthesis, deoxyribonucleoside 5′-diphosphates and thymidine 5′-monophosphate to the corresponding triphosphates which then serve as DNA polymerase substrates. The two enzymes have been measured in synchronous cultures of the green algae, S. obliquus. Thymidylate kinase exhibits an activity peak at the 11 -12th hour of the 24-hour cell cycle, coinciding with DNA synthesis. Enzyme activity is markedly stimulated in presence of fluorodeoxyuridine in the culture medium. This behaviour of dTMP kinase is very similar to that of three other S phase-specific peak enzymes previously analyzed in synchronous algae, viz. ribonucleotide reductase, thymidylate synthase, and dihydrofolate reductase. In contrast, NDP kinase exhibits high and constant activity through the entire cell cycle. The two kinases have been isolated from cell-free extracts, and separated from each other by chromatography on Blue Sepharose. The peak enzyme, dTMP kinase, has been purified to near homogeneity and its catalytic properties are described; the molecular weight is 56,000. NDP kinase activity is separable into two enzyme fractions, both of molecular weight 100,000 (or higher), which are unspecific with respect to ribonucleotide and deoxyribonucleotide substrates. Characterization and purification of the whole series of deoxyribonucleotide-synthesizing enzymes from one organism provides a basis for in vitro experiments towards reconstitution of an S phase-specific DNA precursor/DNA replication multienzyme aggregate.

Inheritance of chromatin modifications through the cell cycle

2019 ◽  
pp. 184-190
Author(s):  
John C. Lucchesi
Keyword(s):  
Cell Cycle ◽  
Histone H3 ◽  
Parent Cell ◽  
Dna Repeats ◽  
Cpg Dinucleotides ◽  
Daughter Cells ◽  
Histone H3 Variant ◽  
Dna Strands ◽  
Non Coding Rnas ◽  
Transcriptional Landscape

Following mitosis, the particular transcriptional landscape of the parent cell must be faithfully transmitted to daughter cells. Although transcription ceases, not all transcription factors are displaced. DNA methylation has been implicated in the inheritance of chromatin characteristics because maintenance DNA methyl transferases methylate CpG dinucleotides on the newly replicated strand if the corresponding GpC on the parent strand is methylated. Nucleosomes that are deposited on the newly synthesized DNA strands are made up of old and new histones, and some marks present on the old histones are maintained. The proper distribution of nucleosomes and the topological organization of the genome into topologically associating domains (TADs) must be transmitted to daughter cells. Following DNA replication, centromeres must be specified on the daughter chromatids. In most eukaryotes, centromeres are identified by the presence of nucleosomes bearing the histone H3 variant CENP-A. An additional number of proteins and non-coding RNAs originating from centric and pericentromeric DNA repeats associate with centromeres and appear to play a role in centromere function.

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