scholarly journals DNA replication-mediated error correction of ectopic CENP-A deposition maintains centromere identity

2018 ◽  
Author(s):  
Yael Nechemia-Arbely ◽  
Karen H. Miga ◽  
Ofer Shoshani ◽  
Aaron Aslanian ◽  
Moira A. McMahon ◽  
...  
Keyword(s):  
Dna Replication ◽  
Error Correction ◽  
Active Sites ◽  
Histone H3 ◽  
Satellite Dnas ◽  
Reference Models ◽  
Human Centromere ◽  
Genome Wide ◽  
Error Correction Mechanism ◽  
Histone H3 Variant

AbstractChromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A is shown in early G1 to be assembled into nucleosomes within megabase, repetitive α-satellite DNAs at each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. Here we identify that DNA replication acts as an error correction mechanism to sustain centromere identity through the removal of the sites of CENP-A loading on the chromosome arms, while maintaining centromere-bound CENP-A with the same DNA sequence preferences as in its initial loading.

scholarly journals Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (6) ◽  
pp. 2057-2068 ◽  
Author(s):  
Jessica R. Eisenstatt ◽  
Lars Boeckmann ◽  
Wei-Chun Au ◽  
Valerie Garcia ◽  
Levi Bursch ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Initiation ◽  
Centromeric Chromatin ◽  
Dna Replication Initiation ◽  
Link Type ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

scholarly journals Reduced Gene Dosage of Histone H4 Prevents CENP-A Mislocalization in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Jessica R. Eisenstatt ◽  
Kentaro Ohkuni ◽  
Wei-Chun Au ◽  
Olivia Preston ◽  
Evelyn Suva ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosomal Instability ◽  
Budding Yeast ◽  
Gene Dosage ◽  
Histone H3 ◽  
Histone H4 ◽  
Genome Wide ◽  
A Genome ◽  
Mutant Strains ◽  
Histone H3 Variant

ABSTRACTMislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to non-centromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A has been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) leads to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCFMet30, SCFCdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of GALcse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 contributes to defects in sumoylation and reduced mislocalization of overexpressed Cse4. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to non-centromeric regions, contributing to SDL when Cse4 is overexpressed in mutant strains.

scholarly journals DNA replication acts as an error correction mechanism to maintain centromere identity by restricting CENP-A to centromeres

2019 ◽  
Vol 21 (6) ◽  
pp. 743-754 ◽  
Author(s):  
Yael Nechemia-Arbely ◽  
Karen H. Miga ◽  
Ofer Shoshani ◽  
Aaron Aslanian ◽  
Moira A. McMahon ◽  
...  
Keyword(s):  
Dna Replication ◽  
Error Correction ◽  
Error Correction Mechanism

scholarly journals Reduced Gene Dosage of Histone H4 Prevents CENP-A Mislocalization and Chromosomal Instability in Saccharomyces cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Jessica R Eisenstatt ◽  
Kentaro Ohkuni ◽  
Wei-Chun Au ◽  
Olivia Preston ◽  
Loran Gliford ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosomal Instability ◽  
Budding Yeast ◽  
Gene Dosage ◽  
Histone H3 ◽  
Human Cells ◽  
Histone H4 ◽  
Genome Wide ◽  
A Genome ◽  
Histone H3 Variant

Abstract Mislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to non-centromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A has been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in proteolysis of overexpressed Cse4 (GALCSE4) leads to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCFMet30, SCFCdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of overexpressed cse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 leads to defects in sumoylation and reduced mislocalization of overexpressed Cse4, which contributes to suppression of CIN when Cse4 is overexpressed. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to non-centromeric regions, leading to CIN when Cse4 is overexpressed.

scholarly journals Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

2022 ◽  
Author(s):  
Marion E. Pesenti ◽  
Tobias Raisch ◽  
Duccio Conti ◽  
Ingrid Hoffmann ◽  
Dorothee Vogt ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Chromosome Segregation ◽  
Histone H3 ◽  
Large Protein ◽  
Centromeric Chromatin ◽  
Human Centromere ◽  
Cryo Electron Microscopy ◽  
Large Protein Complex ◽  
Histone H3 Variant ◽  
Specificity Determinants

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. The organization of the interface between the kinetochore and the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, remains incompletely understood. A 16-subunit complex, the constitutive centromere associated network (CCAN), bridges CENP-A to the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has also important implications for the mechanism of specific recognition of the CENP-A nucleosome. A supported model depicts the interaction as fuzzy and identifies the disordered CCAN subunit CENP-C as only determinant of specificity. A more speculative model identifies both CENP-C and CENP-N as specificity determinants, but implies CENP-A may be in a hemisome rather than in a classical octamer.

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