scholarly journals Sim4

2003 ◽  
Vol 161 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Alison L. Pidoux ◽  
William Richardson ◽  
Robin C. Allshire
Keyword(s):  
Chromatin Structure ◽  
Coiled Coil ◽  
Central Core ◽  
Centromeric Heterochromatin ◽  
Kinetochore Assembly ◽  
Powerful Approach ◽  
Histone H3 Variant ◽  
Specific Proteins ◽  
Domain Functional ◽  
Functional Kinetochore

Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

scholarly journals Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A

2001 ◽  
Vol 114 (19) ◽  
pp. 3529-3542 ◽  
Author(s):  
Aaron A. Van Hooser ◽  
Ilia I. Ouspenski ◽  
Heather C. Gregson ◽  
Daniel A. Starr ◽  
Tim J. Yen ◽  
...  
Keyword(s):  
Dna Binding Protein ◽  
Centromeric Heterochromatin ◽  
Additional Mechanism ◽  
Centromeric Chromatin ◽  
Microtubule Associated Proteins ◽  
Kinetochore Assembly ◽  
Double Minute ◽  
Histone H3 Variant ◽  
Associated Proteins ◽  
Double Minute Chromosomes

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

scholarly journals The role of heterochromatin in centromere function

2005 ◽  
Vol 360 (1455) ◽  
pp. 569-579 ◽  
Author(s):  
Alison L Pidoux ◽  
Robin C Allshire
Keyword(s):  
Fission Yeast ◽  
Histone H3 ◽  
Centromeric Heterochromatin ◽  
Chromatin Domain ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Centromere Function ◽  
Kinetochore Assembly ◽  
Histone H3 Variant

Chromatin at centromeres is distinct from the chromatin in which the remainder of the genome is assembled. Two features consistently distinguish centromeres: the presence of the histone H3 variant CENP-A and, in most organisms, the presence of heterochromatin. In fission yeast, domains of silent ‘heterochromatin’ flank the CENP-A chromatin domain that forms a platform upon which the kinetochore is assembled. Thus, fission yeast centromeres resemble their metazoan counterparts where the kinetochore is embedded in centromeric heterochromatin. The centromeric outer repeat chromatin is underacetylated on histones H3 and H4, and methylated on lysine 9 of histone H3, which provides a binding site for the chromodomain protein Swi6 (orthologue of Heterochromatin Protein 1, HP1). The remarkable demonstration that the assembly of repressive heterochromatin is dependent on the RNA interference machinery provokes many questions about the mechanisms of this process that may be tractable in fission yeast. Heterochromatin ensures that a high density of cohesin is recruited to centromeric regions, but it could have additional roles in centromere architecture and the prevention of merotely, and it might also act as a trigger for kinetochore assembly. In addition, we discuss an epigenetic model for ensuring that CENP-A is targeted and replenished at the kinetochore domain.

scholarly journals Coiled-coil networking shapes cell molecular machinery

2012 ◽  
Vol 23 (19) ◽  
pp. 3911-3922 ◽  
Author(s):  
Yongqiang Wang ◽  
Xinlei Zhang ◽  
Hong Zhang ◽  
Yi Lu ◽  
Haolong Huang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Protein Protein Interactions ◽  
Full Spectrum ◽  
Kinetochore Assembly ◽  
Genome Wide ◽  
Molecular Machinery ◽  
Systematic Understanding

The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.

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 Measuring the Stoichiometry and Physical Interactions between Components Elucidates the Architecture of the Vertebrate Kinetochore

2005 ◽  
Vol 16 (10) ◽  
pp. 4882-4892 ◽  
Author(s):  
Michael J. Emanuele ◽  
Mark L. McCleland ◽  
David L. Satinover ◽  
P. Todd Stukenberg
Keyword(s):  
Hydrodynamic Analysis ◽  
Centromeric Chromatin ◽  
Outer Plate ◽  
Kinetochore Assembly ◽  
Assembly Pathway ◽  
Ndc80 Complex ◽  
Specific Proteins ◽  
Physical Interactions ◽  
Monomeric Proteins ◽  
Structural Architecture

Vertebrate kinetochores contain over 50 different proteins organized into three distinct regions: the inner plate, outer plate, and fibrous corona. The present study characterizes numerous precursors of kinetochore assembly in a system free of centromeric chromatin, Xenopus extracts. Hydrodynamic analysis suggests there are a minimum of two monomeric proteins and six preassembled complexes that accumulate on centromeres to form the kinetochore. The inner and outer kinetochore assemble from at least two distinct kinetochore complexes containing the proteins Mis12, Zwint, and Ndc80, all of which interact by immunoprecipitation. There is also a network of interactions between the fibrous corona proteins that is dissociated by microtubules. We quantify the number of molecules of specific proteins assembled into a single kinetochore. There are between 800 and 1200 molecules of the measured inner and outer kinetochore proteins, demonstrating that the components in these regions are in similar stoichiometry. In contrast, the measured fibrous corona proteins are present at 250-300 molecules per kinetochore. Zwint, but not Mis12, requires the Ndc80 complex for assembly into the kinetochore. Further, Ndc80 requires Zwint for assembly, indicating a codependency for these two proteins. Our data provide a model for the structural architecture and assembly pathway of the vertebrate kinetochore.

scholarly journals Scm3 Is Essential to Recruit the Histone H3 Variant Cse4 to Centromeres and to Maintain a Functional Kinetochore

2007 ◽  
Vol 26 (6) ◽  
pp. 853-865 ◽  
Author(s):  
Raymond Camahort ◽  
Bing Li ◽  
Laurence Florens ◽  
Selene K. Swanson ◽  
Michael P. Washburn ◽  
...  
Keyword(s):  
Histone H3 ◽  
Histone H3 Variant ◽  
Functional Kinetochore

scholarly journals Mcp4, a Meiotic Coiled-Coil Protein, Plays a Role in F-Actin Positioning during Schizosaccharomyces pombe Meiosis

2007 ◽  
Vol 6 (6) ◽  
pp. 971-983 ◽  
Author(s):  
Ayami Ohtaka ◽  
Daisuke Okuzaki ◽  
Takamune T. Saito ◽  
Hiroshi Nojima
Keyword(s):  
Subcellular Localization ◽  
Schizosaccharomyces Pombe ◽  
Coiled Coil ◽  
Microscopic Analysis ◽  
Specific Protein ◽  
Membrane Formation ◽  
Meiotic Progression ◽  
Specific Proteins ◽  
Fluorescence Microscopic Analysis

ABSTRACT Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Δ cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Δ cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis.

scholarly journals Decoding the centromeric nucleosome through CENP-N

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Satyakrishna Pentakota ◽  
Keda Zhou ◽  
Charlotte Smith ◽  
Stefano Maffini ◽  
Arsen Petrovic ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Double Helix ◽  
Structural Basis ◽  
Binding Motif ◽  
Kinetochore Assembly ◽  
Nucleosomal Dna ◽  
Chromosome Domains ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Dna Double Helix

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

scholarly journals Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin

2007 ◽  
Vol 176 (6) ◽  
pp. 757-763 ◽  
Author(s):  
Paul S. Maddox ◽  
Francie Hyndman ◽  
Joost Monen ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Functional Genomics ◽  
Protein A ◽  
Common Mechanism ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Close Proximity ◽  
Single Protein ◽  
Histone H3 Variant ◽  
New Protein

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

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.

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