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Structural basis for the assembly of Mis18a/b core modules

ABSTRACT: 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.

INSTRUMENT(S): Bruker Daltonics timsTOF series

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Chandni Natalia Kumar

LAB HEAD: Dr. Axel

PROVIDER: PXD052964 | Pride | 2024-09-25

REPOSITORIES: Pride

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			Action	DRS
	20240418_Ref9525_MSCK54_01_BB1_1_3719.d.zip	Other
	20240418_Ref9525_MSCK54_02_BB1_1_3720.d.zip	Other
	20240418_Ref9525_MSCK54_03_BB1_1_3721.d.zip	Other
	20240418_Ref9525_MSCK56_01_BB2_1_3723.d.zip	Other
	20240418_Ref9525_MSCK56_02_BB2_1_3724.d.zip	Other

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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

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

Identification of HJURP binding determinants on the Mis18 complex using photo-cross-linking and mass spectrometry

Project description:In the early G1 phase of the cell cycle, human HJURP is recruited to centromeres by the Mis18 complex, which consists of Mis18α, Mis18β and M18BP1. This interaction of HJURP with the Mis18 complex is essential for CENP-A deposition and the preservation of centromere identity. To identify the binding site of HJURP on the Mis18 complex, we used the amber-codon suppression method to incorporate unnatural photo-cross-linking amino acids (Bpa, p-benzoyl-L-phenylalanine; AbK, 3’-azibutyl-N-carbamoyl-lysine) and performed UV-cross-linking and mass spectrometry (MS) experiments.

2019-08-01 | PXD013339 | 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

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

CENP-A chaperone Scm3HJURP contains a cysteine-rich domain required for centromere targeting and kinetochore integrity

Project description:Centromeric chromatin is crucial for supporting kinetochore assembly and chromosome segregation. To specify centromeres, the histone H3 variant CENP-A is loaded by the conserved chaperone Scm3/HJURP. The amino-terminus of Scm3/HJURP interacts with CENP-A, while the carboxyl-terminus contains a small domain that interacts with the Mis18 holocomplex, which facilitates centromere localization. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) with unknown function. In this study, we found that CYS is essential for the localization and function of fission yeast Scm3. Disrupting CYS prevents Scm3 from localizing to the centromere and results in cell lethality. Moreover, introducing point mutations in a predicted zinc-binding motif within CYS hinders Scm3 centromere targeting and kinetochore integrity, consistent with structural evidence that CYS binds zinc. We find that expressing only the Scm3 carboxyl-terminus is harmful as it localizes to centromeres and causes defective kinetochores. Remarkably, mutating the CYS zinc-binding motif in this construct eliminates the observed centromere localization and toxicity. Together, our findings establish this cysteine-rich domain with predicted zinc-binding capability as an essential feature of fungal Scm3 proteins and shed new light on the molecular mechanism for targeting Scm3 to centromeres.

2024-03-06 | GSE231729 | GEO

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.

2016-08-04 | E-GEOD-85166 | biostudies-arrayexpress

Nucleosome Mapping in S. pombe Centromeres

Project description:A key element for defining the centromere identity is the incorporation of a specific histone H3, CENP-A, known as Cnp1p in S. pombe. Previous studies have suggested that functional S. pombe centromeres lack nucleosome arrays and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are in fact positioned in regular intervals in the core of centromere 2, providing the first high resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore proteins cnp1, mis18, mis12, nuf2, mal2, overexpression of Cnp1p, or deletion of ams2. Bioinformatic analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence-bias in nucleosome positioning. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites for the GATA-like protein Ams2p, which participates in CENP-A incorporation. Keywords: Nucleosome Mapping Study Entire cnt regions and histone-related genes were tiled at 1-5 bp spacing using 60-mer probes.

2008-05-30 | E-GEOD-10742 | biostudies-arrayexpress