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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Schizosaccharomyces Pombe 927

TISSUE(S): Cell Suspension Culture, Cell Culture

SUBMITTER: Nitobe London

LAB HEAD: Robin Allshire

PROVIDER: PXD045191 | Pride | 2023-09-18

REPOSITORIES: Pride

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			Action	DRS
	London_etal_2023_CEN_SPB_Interaction_3.7z	Other
	London_etal_2023_CEN_SPB_interaction_1.7z	Other
	London_etal_2023_CEN_SPB_interaction_2.7z	Other
	London_etal_2023_Sad1_3V5_Rep1.raw	Raw
	London_etal_2023_Sad1_3V5_Rep2.raw	Raw

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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

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

2014-05-19 | GSE54685 | GEO

Hap2-Ino80 mediated transcription facilitates de novo establishment of CENP-A chromatin

Project description:Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily-conserved histone H3 variant, which directs kinetochore assembly and hence, centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity selected solubilised fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 is required for transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilises H3 nucleosomes on centromere DNA through transcription-mediated histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.

2020-05-27 | PXD016602 | Pride

Structural basis for the assembly of Mis18a/b core modules

Project description:The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18a, Mis18b and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18a, 2 Mis18b and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18a can associate with centromeres and deposit CENP-A independently of Mis18b, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

2024-05-15 | PXD047345 | Pride

Structural basis for the assembly of Mis18a/b core modules

2024-09-25 | PXD052964 | Pride

PLK1-Mediated Phosphorylation Cascade Activates the Mis18 Complex to Ensure Centromere Inheritance

Project description:Accurate chromosome segregation requires the attachment of spindle microtubules to 20 centromeres, which are epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, existing CENP-A nucleosomes undergo dilution as they get redistributed among the two DNA strands. To preserve centromere identity, CENP-A levels must be restored in a cellcycle controlled manner orchestrated by the Mis18 complex. Here we provide a comprehensive mechanistic basis for PLK1-mediated licensing of CENP-A loading. We demonstrate that PLK1 25 interacts with Mis18α and Mis18BP1 subunits of the Mis18 complex by recognising self-primed phosphorylations of Mis18α (S54) and Mis18BP1 (T78 and S93) through its Polo-box binding domain. Disrupting these PLK1 phosphorylations perturbed the centromere recruitment of HJURP and new CENP-A loading. Biochemical and functional analyses show that phosphorylation of Mis18α and subsequent PLK1 binding is required to activate the Mis18α/β complex for robust 30 Mis18α/β-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance.

2024-09-09 | PXD053141 | 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

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.

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

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Ccp1 homodimer mediates chromatin integrity by antagonizing CENP-A loading

Project description:CENP-A is a centromere-specific histone 3 variant essential for centromere specification. CENP-A partially replaces canonical histone H3 at the centromeres. How the particular CENP-A/H3 ratio at centromeres is precisely maintained is unknown. It also remains unclear how CENP-A is excluded from non-centromeric chromatin. Here we identify Ccp1, an uncharacterized NAP family protein in fission yeast that antagonizes CENP-A loading at both centromeric and non-centromeric regions. Like the CENP-A loading factor HJURP, Ccp1 interacts with CENP-A, and is recruited to centromeres at the end of mitosis in a Mis16-dependent manner. These data indicate that factors with opposing CENP-A loading activities are recruited to centromeres. Furthermore, Ccp1 also cooperates with H2A.Z to evict CENP-A assembled in euchromatin. Structural analyses indicate that Ccp1 forms a homodimer that is required for its anti-CENP-A loading activity. Our study establishes mechanisms for maintenance of CENP-A homeostasis at centromeres and the prevention of ectopic assembly of centromeres. Examination of cnp1 distribution in one wild type (wt) and two ccp1 mutants.

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