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

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.

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Epigenetic inheritance of centromere identity in a heterologous system

10.1101/560391 ◽

2019 ◽

Cited By ~ 2

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.

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Centromeric chromatin gets loaded

The Journal of Cell Biology ◽

10.1083/jcb.200702020 ◽

2007 ◽

Vol 176 (6) ◽

pp. 735-736 ◽

Cited By ~ 3

Author(s):

Christopher W. Carroll ◽

Aaron F. Straight

Keyword(s):

Chromosome Segregation ◽

Molecular Mechanisms ◽

Protein A ◽

Histone H3 ◽

Chromatin Assembly ◽

Centromeric Chromatin ◽

Kinetochore Assembly ◽

Centromere Protein A ◽

Specific Loading ◽

Histone H3 Variant

Centromeric nucleosomes contain a histone H3 variant called centromere protein A (CENP-A) that is required for kinetochore assembly and chromosome segregation. Two new studies, Jansen et al. (see p. 795 of this issue) and Maddox et al. (see p. 757 of this issue), address when CENP-A is deposited at centromeres during the cell division cycle and identify an evolutionally conserved protein required for CENP-A deposition. Together, these studies advance our understanding of centromeric chromatin assembly and provide a framework for investigating the molecular mechanisms that underlie the centromere-specific loading of CENP-A.

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

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401131 ◽

2020 ◽

Vol 10 (6) ◽

pp. 2057-2068 ◽

Cited By ~ 3

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.

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

Essays in Biochemistry ◽

10.1042/ebc20190074 ◽

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.

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

10.1101/176206 ◽

2017 ◽

Cited By ~ 1

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.

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

Chromosoma ◽

10.1007/s00412-022-00767-2 ◽

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.

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Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species

10.20944/preprints202101.0320.v1 ◽

2021 ◽

Author(s):

Evgeny Bakin ◽

Fatih Sezer ◽

Irem Kilic ◽

Aslıhan Özbilen ◽

Mike Rayko ◽

...

Keyword(s):

Chromosome Segregation ◽

Genetic Regulation ◽

Single Copy ◽

Egg Cell ◽

Expression Levels ◽

Histone H3 Variant ◽

Before And After ◽

Spindle Microtubules ◽

Mitosis And Meiosis

Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO (Aspartate Glutamate Aspartate Aspartate histidine exonuclease) is the only described gene associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Also, in this research we characterize the structure of CENH3, study expression levels of CENH3 and APOLLO in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of before and after fertilization. At the premeiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, it decreased in both species by the time of meiosis and increased after fertilization. By 1 DAP CENH3 expression started dropping in sexual B. stricta siliques and kept increasing in apomictic B. divaricarpa ones. That might indicate to a role of CENH3 in apomictic development in Boechera species. The expression levels of APOLLO also sharply decreased by the time of meiosis in gynoecium of both species; however, by 3 DAP, the level of APOLLO expression in siliques of apomicts was almost 1.5 times higher than that of the sexuals. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes.

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Centromere repositioning induced by inner kinetochore impairment generates a meiosis barrier

10.1101/401257 ◽

2018 ◽

Author(s):

Min Lu ◽

Xiangwei He

Keyword(s):

Meiotic Chromosome ◽

Centromeric Chromatin ◽

Kinetochore Assembly ◽

Eukaryotic Species ◽

Histone H3 Variant ◽

Single Deletion ◽

Centromere Inactivation ◽

Evolutionary New Centromeres ◽

Two Populations

AbstractCentromeres dictate the sites for kinetochore assembly on chromosomes, while their own position on each chromosome is determined epigenetically by a specific histone H3 variant CENP-A. For all eukaryotic species, the chromosomal position of each centromere is distinct and inherited with high fidelity, although the mechanisms underlying the epigenetic stability and its functional significance remain largely unknown. Here in the fission yeast Schizosaccharomyces pombe, we show that mutations in inner kinetochore components influence centromeric chromatin organization to various levels. In extreme cases, a single deletion of wip1, mhf1 and mhf2 (the conserved CENP-T-W-S-X complex subunits) or double deletions of cnp3 (a homologue of mammalian CENP-C) and fta6 (a pombe specific component) induce centromere repositioning - inactivation of the original centromere and formation of a neocentromere - in one of the three chromosomes at random. Neocentromeres tend to locate in pericentromeric heterochromatin regions, although heterochromatin is not required for centromere inactivation. Cells carrying a neocentromere are competent in mitosis and in meiosis of homozygotes. However, when these cells are crossed to cells carrying the original centromere, the progeny suffers severe lethality due to defects in meiotic chromosome segregation. These results recapitulate a meiosis barrier that could initiate genetic divergence between two populations with mismatched centromeres, documenting a potential role of the Evolutionary New Centromeres (ENCs) in speciation.Significance StatementIn eukaryotes, centromeres are chromosomal regions where kinetochores are assembled and the positions of centromeres are accurately inherited. While the centromere and kinetochore assembly are extensively studied, the mechanisms that each centromere maintain its identity on chromosomes are still not well understood. In this study, we demonstrated that the inner kinetochore is required for the normal centromere identity as single depletion of the inner kinetochore CENP-T-W-S-X complex or double deletions of cnp3/CENP-C and fta6 induce centromere repositioning. We further showed cells carrying a neocentromere are reproductively isolated from the wildtype population carrying the original centromere. Taken together, these results suggest that induced centromere repositioning mimics the evolutionary new centromeres and is sufficient to cause reproductive isolation.

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Guarding the Genome: CENP-A-Chromatin in Health and Cancer

Genes ◽

10.3390/genes11070810 ◽

2020 ◽

Vol 11 (7) ◽

pp. 810 ◽

Cited By ~ 2

Author(s):

Megan A. Mahlke ◽

Yael Nechemia-Arbely

Keyword(s):

Chromosome Segregation ◽

Posttranslational Modifications ◽

Current Knowledge ◽

Protein A ◽

Ctcf Binding ◽

Centromere Protein A ◽

Structure Specification ◽

Binding Factor ◽

Histone H3 Variant ◽

Recognition Protein

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

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The ATAD2/ANCCA homolog Yta7 cooperates with Scm3HJURP to deposit Cse4CENP-A at the centromere in yeast

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917814117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5386-5393 ◽

Cited By ~ 2

Author(s):

Sara Shahnejat-Bushehri ◽

Ann E. Ehrenhofer-Murray

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Histone H3 ◽

Nucleosome Assembly ◽

Direct Role ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Assembly Factor

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP. The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.

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