scholarly journals CENP-A nucleosome—a chromatin-embedded pedestal for the centromere: lessons learned from structural biology

2020 ◽  
Vol 64 (2) ◽  
pp. 205-221
Author(s):  
Ahmad Ali-Ahmad ◽  
Nikolina Sekulić
Keyword(s):  
Cell Division ◽  
Structural Biology ◽  
Histone H3 ◽  
Lessons Learned ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Molecular Determinants ◽  
Histone H3 Variant ◽  
Segregation Of Chromosomes

Abstract The centromere is a chromosome locus that directs equal segregation of chromosomes during cell division. A nucleosome containing the histone H3 variant CENP-A epigenetically defines the centromere. Here, we summarize findings from recent structural biology studies, including several CryoEM structures, that contributed to elucidate specific features of the CENP-A nucleosome and molecular determinants of its interactions with CENP-C and CENP-N, the only two centromere proteins that directly bind to it. Based on those findings, we propose a role of the CENP-A nucleosome in the organization of centromeric chromatin beyond binding centromeric proteins.

scholarly journals Epigenetic inheritance of centromere identity in a heterologous system

2019 ◽  
Author(s):  
Virginie Roure ◽  
Bethan Medina-Pritchard ◽  
Eduard Anselm ◽  
A. Arockia Jeyaprakash ◽  
Patrick Heun
Keyword(s):  
Chromosome Segregation ◽  
Chromosomal Region ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Heterologous System ◽  
Histone H3 Variant ◽  
Self Association

SUMMARYThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

scholarly journals Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

2015 ◽  
Vol 208 (5) ◽  
pp. 521-531 ◽  
Author(s):  
Glennis A. Logsdon ◽  
Evelyne J. Barrey ◽  
Emily A. Bassett ◽  
Jamie E. DeNizio ◽  
Lucie Y. Guo ◽  
...  
Keyword(s):  
Cell Division ◽  
Histone H3 ◽  
Lac Repressor ◽  
Chromosomal Locus ◽  
Gain Of Function ◽  
Lac Operator ◽  
Centromere Proteins ◽  
Histone H3 Variant ◽  
Segregation Of Chromosomes

The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity.

scholarly journals N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

2017 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Reuben Levy-Myers ◽  
Jack Warren ◽  
Wei-Chun Au ◽  
Yoshimitsu Takahashi ◽  
...  
Keyword(s):  
Genome Stability ◽  
Histone H3 ◽  
Physiological Role ◽  
Centromeric Chromatin ◽  
E3 Ligases ◽  
Physiological Conditions ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

AbstractStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

scholarly journals Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading

2019 ◽  
Vol 47 (20) ◽  
pp. 10754-10770 ◽  
Author(s):  
Anming Huang ◽  
Leopold Kremser ◽  
Fabian Schuler ◽  
Doris Wilflingseder ◽  
Herbert Lindner ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Posttranslational Modifications ◽  
Centromeric Chromatin ◽  
Phosphorylated Form ◽  
Specific Loading ◽  
Histone H3 Variant ◽  
Proteasome Pathway ◽  
Turn Over ◽  
Fine Tune

Abstract Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

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 Subunit interactions and arrangements in the fission yeast Mis16–Mis18–Mis19 complex

2019 ◽  
Vol 2 (4) ◽  
pp. e201900408 ◽  
Author(s):  
Melanie Korntner-Vetter ◽  
Stéphane Lefèvre ◽  
Xiao-Wen Hu ◽  
Roger George ◽  
Martin R Singleton
Keyword(s):  
Cell Cycle ◽  
Binding Site ◽  
Fission Yeast ◽  
Histone H3 ◽  
Histone H4 ◽  
Subunit Interactions ◽  
Centromeric Chromatin ◽  
Partner Protein ◽  
Histone H3 Variant

Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1CENP-A. Cell cycle–specific deposition of Cnp1 requires the Mis16–Mis18–Mis19 complex, which is thought to direct recruitment of Scm3-chaperoned Cnp1/histone H4 dimers to DNA. Here, we present the structure of the essential Mis18 partner protein Mis19 and describe its interaction with Mis16, revealing a bipartite-binding site. We provide data on the stoichiometry and overall architecture of the complex and provide detailed insights into the Mis18–Mis19 interface.

scholarly journals Incorporation of CENP-A/CID into centromeres during early Drosophila embryogenesis does not require RNA polymerase II–mediated transcription

Chromosoma ◽  
2022 ◽  
Author(s):  
Samadri Ghosh ◽  
Christian F. Lehner
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cultured Cells ◽  
Marginal Effect ◽  
Proliferating Cells ◽  
Centromeric Chromatin ◽  
Embryonic Cleavage ◽  
Histone H3 Variant ◽  
Alpha Amanitin

AbstractIn many species, centromere identity is specified epigenetically by special nucleosomes containing a centromere-specific histone H3 variant, designated as CENP-A in humans and CID in Drosophila melanogaster. After partitioning of centromere-specific nucleosomes onto newly replicated sister centromeres, loading of additional CENP-A/CID into centromeric chromatin is required for centromere maintenance in proliferating cells. Analyses with cultured cells have indicated that transcription of centromeric DNA by RNA polymerase II is required for deposition of new CID into centromere chromatin. However, a dependence of centromeric CID loading on transcription is difficult to reconcile with the notion that the initial embryonic stages appear to proceed in the absence of transcription in Drosophila, as also in many other animal species. To address the role of RNA polymerase II–mediated transcription for CID loading in early Drosophila embryos, we have quantified the effects of alpha-amanitin and triptolide on centromeric CID-EGFP levels. Our analyses demonstrate that microinjection of these two potent inhibitors of RNA polymerase II–mediated transcription has at most a marginal effect on centromeric CID deposition during progression through the early embryonic cleavage cycles. Thus, we conclude that at least during early Drosophila embryogenesis, incorporation of CID into centromeres does not depend on RNA polymerase II–mediated transcription.

scholarly journals A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

Genetics ◽  
2013 ◽  
Vol 194 (2) ◽  
pp. 513-518 ◽  
Author(s):  
Wei Chun Au ◽  
Anthony R. Dawson ◽  
David W. Rawson ◽  
Sara B. Taylor ◽  
Richard E. Baker ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Histone H3 ◽  
N Terminus ◽  
Histone H3 Variant

scholarly journals N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin

2018 ◽  
Vol 8 (4) ◽  
pp. 1215-1223 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Reuben Levy-Myers ◽  
Jack Warren ◽  
Wei-Chun Au ◽  
Yoshimitsu Takahashi ◽  
...  
Keyword(s):  
Histone H3 ◽  
Centromeric Chromatin ◽  
Histone H3 Variant
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