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Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

ABSTRACT: CENP-A chromatin the foundation for kinetochore assembly in all eukaryotes. CENP-A is incorporated outside of S phase and depends on its chaperone HJURPScm3, and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURPScm3 to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1KNL2 and RbAp46/48Mis16/Hat2. Mis18BP1KNL2 is critical for the recruitment of Mis18 and HJURPScm3 to vertebrate centromeres. However, no ortholog of Mis18BP1KNL2 has been identified in fission yeast, consequently it remains unknown how the key Cnp1CENP-A loading factor Mis18 is recruited. We identify two novel Mis18-interacting proteins (Eic1 and Eic2) in fission yeast. Our analyses show that Eic1 and Eic2 are components of a Mis18 complex. Eic1 is essential to maintain normal Cnp1CENP-A levels at centromeres and therefore is crucial for kinetochore integrity whereas Eic2 is dispensable. Eic1 also associates with components of the large constitutive CCAN/Sim4/Ctf19 complex. Our findings suggest that Eic1 is the functional counterpart of Mis18BP1KNL2 and acts as a link with constitutive kinetochore components to allow the temporally regulated recruitment of the Mis18 Cnp1CENP-A loader.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE54685 | GEO | 2014/05/19

SECONDARY ACCESSION(S): PRJNA237364

REPOSITORIES: GEO

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Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

Project description:CENP-A chromatin the foundation for kinetochore assembly in all eukaryotes. CENP-A is incorporated outside of S phase and depends on its chaperone HJURPScm3, and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURPScm3 to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1KNL2 and RbAp46/48Mis16/Hat2. Mis18BP1KNL2 is critical for the recruitment of Mis18 and HJURPScm3 to vertebrate centromeres. However, no ortholog of Mis18BP1KNL2 has been identified in fission yeast, consequently it remains unknown how the key Cnp1CENP-A loading factor Mis18 is recruited. We identify two novel Mis18-interacting proteins (Eic1 and Eic2) in fission yeast. Our analyses show that Eic1 and Eic2 are components of a Mis18 complex. Eic1 is essential to maintain normal Cnp1CENP-A levels at centromeres and therefore is crucial for kinetochore integrity whereas Eic2 is dispensable. Eic1 also associates with components of the large constitutive CCAN/Sim4/Ctf19 complex. Our findings suggest that Eic1 is the functional counterpart of Mis18BP1KNL2 and acts as a link with constitutive kinetochore components to allow the temporally regulated recruitment of the Mis18 Cnp1CENP-A loader. 100bp paired end ChIP-Seq analysis of GFP tagged Eic1, Eic2, Scm3, and Mis18 with sonicated whole cell extract input DNA in asynchornous Schizosaccharomyces pombe cells.

2014-05-19 | E-GEOD-54685 | biostudies-arrayexpress

Higher-order assembly is a regulatory switch that promotes outer kinetochore recruitment

Project description:To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated defined oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with other kinetochore proteins, oligomers that mimic the centromeric density of CENP-T trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Thus, the density-dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of CENP-T.

2023-11-09 | PXD042174 | Pride

The N-terminal Tail of CENP-A Confers Epigenetic Stability to Centromeres via the CENP-T Branch of the CCAN in Fission Yeast

Project description:We employ the well-studied fission yeast centromere to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail did not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling, but nevertheless elevated chromosome loss. N-Tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a causal link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres via recruitment of the CENP-T branch of the CCAN. Genome-wide localization of GFP-tagged N-tail Cnp1 variant tailswap versus wt control in cnp1 deletion background

2015-04-16 | E-GEOD-63350 | biostudies-arrayexpress

Sim3, a NASP (N1/N2) related protein, escorts CENP-A for deposition at fission yeast centromeres

Project description:A defining feature of centromeres is the presence of the histone H3 variant CENP-ACnp1. It is not known how CENP-ACnp1 is specifically delivered to, and assembled into centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast we identified Sim3. Sim3 is homologous to known histone binding proteins NASPHuman and N1/N2Xenopus and aligns with Hif1S.cerevisiae to define the SHNi-TPR family. Sim3 associates with CENP-ACnp1 but is distributed throughout the nucleoplasm rather than being concentrated at centromeres. Cells defective in Sim3 function have reduced levels of CENP-ACnp1 at centromeres and display chromosome segregation defects. Newly synthesized CENPACnp1 can be deposited at centromeres by a replication-independent mechanism during G2. Sim3 is required to allow this new CENP-ACnp1 to accumulate at centromeres in S and G2-phase arrested cells. We propose that Sim3 acts as an escort which hands off CENP-ACnp1 to chromatin assembly factors, allowing its incorporation into centromeric chromatin. Keywords: Expression of sim3-143 versus wt and sim3-205 versus wt

2007-11-10 | GSE7560 | GEO

The N-terminal Tail of CENP-A Confers Epigenetic Stability to Centromeres via the CENP-T Branch of the CCAN in Fission Yeast

2015-04-16 | GSE63350 | GEO

Direct recruitment of Mis18 to interphase spindle poles promotes CENP-A chromatin assembly.

Project description:CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB LInker of Nucleoskeleton and Cytoskeleton (LINC) complex component Sad1. Mis18 recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1, but is independent of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

2023-09-18 | PXD045191 | Pride

Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq]

Project description:At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A/Cnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation via the RNAi machinery, but also through RNAiindependent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here use transcriptional profiling, gene inactivation experiments, and chromatin immunoprecipitation analyses to demonstrate that the Mediator complex directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator co-localizes with Pol II at centromeres and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired both via the RNAi dependent and independent pathways, resulting in loss of CENP-A/Cnp1 from the core centromere, defect kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ appears to directly block CENP-A/Cnp1 incorporation and inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function. 3 samples examined: wild type chromatin incubated with beads as the non antibody control, wild type chromatin incubated with RNA Polymerase II CTD domain antibody and Protein G beads, and TAP-Med7 cells chromatin incubated with IgG beads.

2012-11-06 | E-GEOD-35524 | biostudies-arrayexpress

Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq]

2012-11-06 | GSE35524 | GEO

The Ino80 complex mediates epigenetic centromere propagation via active removal of histone H3

Project description:Centromere is the chromosomal locus at which kinetochore is assembled to direct chromosome segregation. Histone H3 variant CENP-A epigenetically marks active centromeres; however, the mechanism by which CENP-A propagates at the centromere, replacing histone H3, remains poorly understood. Using fission yeast, we find that CENP-ACnp1 chromatin assembly at the centromere requires the Ino80 ATP-dependent chromatin remodeling complex which removes histone H3-containing nucleosomes from associated chromatin. CENP-ACnp1 chromatin actively recruits the Ino80 complex to centromeres to elicit eviction of histone H3-containing nucleosomes. Artificial targeting of Ino80 subunits to a non-centromeric DNA placed in a native centromere enhances the spreading of CENP-ACnp1 chromatin into the non-centromeric DNA. Based on these results, we propose that CENP-ACnp1 chromatin employs the Ino80 complex to mediate replacement of histone H3 with CENP-ACnp1, and thereby reinforces itself.

2017-07-14 | GSE99589 | GEO

Centromere-Like Regions in Budding Yeast

Project description:Accurate chromosome segregation requires centromeres (CENs), the chromosomal sites where kinetochores form, to bridge DNA and attach to microtubules. In contrast to most eukaryotes, Saccharomyces cerevisiae possesses sequence-defined point centromeres. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of four kinetochore components reveals regions of overlapping, extra-centromeric protein localization upon overproduction of the centromeric histone, Cse4 (CENP-A or CenH3). These identified sequences enhance proper plasmid and chromosome segregation, and are termed Centromere-like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of point and regional centromeres. CLR sequences are conserved among related budding yeasts, suggesting a role in vivo. These studies provide new insights into the origin and evolution of centromeres. ChIP-Seq analysis of the kinetochore components Cse4, Mif2, Ndc10 and Ndc80 in budding yeast strains (Saccharomyces cerevisiae) with normal and elevated levels of Cse4

2013-01-18 | E-GEOD-31466 | biostudies-arrayexpress

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