Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae
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PROVIDER: PRJNA622995 | ENA |
REPOSITORIES: ENA
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Project description:Restricting the localization of the centromeric histone H3 variant CENP-A to centromeres is essential to prevent chromosomal instability (CIN). Mislocalization of overexpressed CENP-A contributes to CIN in yeast, fly, and human cells. CENP-A is overexpressed in many cancers. Therefore, defining mechanisms that prevent CENP-A mislocalization will help us understand how CENP-A overexpression contributes to CIN in cancer. A genome-wide screen to characterize essential genes required for growth when CENP-A is overexpressed identified the replication initiation Dbf4-Dependent Kinase (DDK) complex. We show that DDK regulates ubiquitin-mediated proteolysis of Cse4 and prevents mislocalization of Cse4 independently of its role in DNA replication.
2020-04-04 | GSE148068 | GEO
Project description:The centromeric histone H3 variant CENP-A is overexpressed in many cancers. Mislocalization of CENP-A to non-centromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. Despite these observations, pathways that promote and prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen to identify regulators of CENP-A localization. We identified DNAJC9, a J-domain protein as a lead candidate from the screen and showed that cells depleted for DNAJC9 exhibit mislocalization of CENP-A, enrichment of CENP-A in chromatin, and CIN phenotypes. Global interactome analysis showed an enhanced interaction of CENP-A with the replication-associated H3-H4 chaperone MCM2 in DNAJC9-depleted cells and co-depletion of MCM2 and DNAJC9 suppressed CENP-A mislocalization. Furthermore, we showed that cells ablated for the ability of DNAJC9 to promote the proper folding of H3–H4, exhibit CENP-A mislocalization. CUT&RUN Sequencing analysis of genome-wide CENP-A occupancy in DNAJC9-depleted cells identified 16,603 sites of non-centromeric localization, that broadly overlapped with open chromatin regions. Our comprehensive analysis has identified factors that prevent mislocalization of CENP-A and has defined DNAJC9 as an important safeguard that prevents CENP-A mislocalization and CIN.
2024-03-26 | GSE253387 | GEO
Project description:Mislocalization of CENP-A to non-centromeric regions contributes to chromosomal instability (CIN). Here, we defined a role for the histone H3/H4 chaperone CHAF1B in preventing mislocalization of CENP-A and CIN.
2023-05-05 | GSE227373 | GEO
Project description:The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3–H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3–H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3–H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.
2024-04-11 | PXD043961 | Pride
Project description:Precise localization of the histone H3 variant CENP-A(Cse4) to centromeres is essential for accurate chromosome segregation. In budding yeast, CENP-A(Cse4) is regulated by ubiquitin-mediated proteolysis to ensure its exclusive localization to the centromere. Overexpression of CENP-A(Cse4) is lethal when the CENP-A(Cse4) E3 ubiquitin ligase, Psh1, is deleted. To identify the genomic sites of CENP-A(Cse4) mislocalization in this condition, we investigated the genome-wide mislocalization pattern of CENP-A(Cse4) by ChIP-seq.
2016-03-08 | GSE69695 | GEO
Project description:The centromere specific histone H3 variant CENP-A/CENH3 specifies where the kinetochore is formed in most eukaryotes. Despite tight regulation of CENP-A levels in normal cells, overexpression of CENP-A is a feature shared by various types of solid tumors and results in its mislocalization to non-centromeric DNA. How CENP-A is assembled ectopically and the consequences of this mislocalization remain topics of high interest. Here, we report that in human colon cancer cells, the H3.3 chaperones HIRA and DAXX promote ectopic CENP-A deposition. Moreover, the correct balance between levels of the centromeric chaperone HJURP and CENP-A is essential to preclude ectopic assembly by H3.3 chaperones. In addition, we find that ectopic localization can recruit kinetochore components, and correlates with mitotic defects and DNA damage in G1 phase. Finally, CENP-A occupancy at the 8q24 locus is also correlated with amplification and overexpression of the MYC gene within that locus. Overall, these data provide insights into the causes and consequences of histone variant mislocalization in human cancer cells.
2018-10-10 | GSE120230 | GEO
Project description: Not available
| S-EPMC7263675 | biostudies-literature
Project description:Centromeric localization of the evolutionarily conserved histone H3 variant, CENP-A. is essential for chromosomal stability. CENP-A overexpression (OE) causesd its mislocalization to non-centromeric regions resulting in chromosomal instability (CIN) in yeast, flies and human cells. CENP-A OE and mislocalization have been observed in cancers and correlates with poor prognosis. However, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we overexpressed YFP-CENP-A in a pseudodiploid DLD1 cell line and showed that CENP-A OE leads to its mislocalization and CIN due to defects in kinetochore integrity and kinetochore-microtubule attachments. CENP-A OE also leads to its mislocalization and CIN in a xenograft mouse model. Under these conditions, it contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our studies provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A overexpressing cancers.
2020-07-16 | GSE154460 | GEO
Project description:Precise localization of the histone H3 variant CENP-A(Cse4) to centromeres is essential for accurate chromosome segregation. In budding yeast, CENP-A(Cse4) is regulated by ubiquitin-mediated proteolysis to ensure its exclusive localization to the centromere. Overexpression of CENP-A(Cse4) is lethal when the CENP-A(Cse4) E3 ubiquitin ligase, Psh1, is deleted. CENP-A(Cse4) mislocalizes to promoters in this condition, so we investigated if there was an effect on gene expression of downstream genes using RNA-seq.
2016-03-08 | GSE77596 | GEO
Project description:Centromeric localization of CENP-A (Cse4 in S. cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Overexpression of CENP-A leads to its mislocalization and contributes to aneuploidy in yeast, flies, humans and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and non-centromeric regions, respectively. Ubiquitin ligases such as Psh1 and Slx5 and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to non-centromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C-terminus of Cse4 and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A (K changed to arginine (R) or alanine (A)) and reduced interaction of mutant Cse4 K215/215R/A with Scm3 and CAF-1 when compared to wild type Cse4. Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction at centromeric and non-centromeric regions were reduced. Furthermore, in contrast to GAL-CSE4 which exhibits Synthetic Dosage Lethality (SDL) in psh1Æ, slx5Æ, and hir2Æ strains, GAL-cse4 K215/216R does not exhibit SDL in these strains. Taken together, our results identify and define a role for C-terminal sumoylation of Cse4 for its incorporation into chromatin.
2020-02-21 | GSE145602 | GEO