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Bridgin connects the outer kinetochore to centromeric chromatin.

ABSTRACT: The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-C^Mif2, CENP-T^Cnn1, and CENP-U^Ame1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein "bridgin" was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.

SUBMITTER: Sridhar S

PROVIDER: S-EPMC7794384 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Bridgin connects the outer kinetochore to centromeric chromatin

Project description:In this project, 3XFlag-tagged protein affinity purifications were performed to identify the protein-protein interactors using Cryptococcus neoformans cell lysate. After fractionation with SDS-PAGE, peptide samples for nanoLC-MS/MS were prepared by in-gel digestion. Raw data from Orbotrap Velos pro were processed with MASCOT via Proteome discoverer2.2. Validation of peptide and protein identifications were performed with Scaffold proteome software.

2020-09-23 | PXD021072 | JPOST Repository

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Orc4 spatiotemporally stabilizes centromeric chromatin.

Project description:The establishment of centromeric chromatin and its propagation by the centromere-specific histone CENPA is mediated by epigenetic mechanisms in most eukaryotes. DNA replication origins, origin binding proteins, and replication timing of centromere DNA are important determinants of centromere function. The epigenetically regulated regional centromeres in the budding yeast Candida albicans have unique DNA sequences that replicate earliest in every chromosome and are clustered throughout the cell cycle. In this study, the genome-wide occupancy of the replication initiation protein Orc4 reveals its abundance at all centromeres in C. albicans Orc4 is associated with four different DNA sequence motifs, one of which coincides with tRNA genes (tDNA) that replicate early and cluster together in space. Hi-C combined with genome-wide replication timing analyses identify that early replicating Orc4-bound regions interact with themselves stronger than with late replicating Orc4-bound regions. We simulate a polymer model of chromosomes of C. albicans and propose that the early replicating and highly enriched Orc4-bound sites preferentially localize around the clustered kinetochores. We also observe that Orc4 is constitutively localized to centromeres, and both Orc4 and the helicase Mcm2 are essential for cell viability and CENPA stability in C. albicans Finally, we show that new molecules of CENPA are recruited to centromeres during late anaphase/telophase, which coincides with the stage at which the CENPA-specific chaperone Scm3 localizes to the kinetochore. We propose that the spatiotemporal localization of Orc4 within the nucleus, in collaboration with Mcm2 and Scm3, maintains centromeric chromatin stability and CENPA recruitment in C. albicans.

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Orc4 spatiotemporally stabilizes centromeric chromatin.

| S-EPMC8015856 | biostudies-literature

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19<sup>CCAN</sup> Complex in Meiotic Kinetochore Assembly.

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Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins.

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