scholarly journals Decoding the centromeric nucleosome through CENP-N

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Satyakrishna Pentakota ◽  
Keda Zhou ◽  
Charlotte Smith ◽  
Stefano Maffini ◽  
Arsen Petrovic ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Double Helix ◽  
Structural Basis ◽  
Binding Motif ◽  
Kinetochore Assembly ◽  
Nucleosomal Dna ◽  
Chromosome Domains ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Dna Double Helix

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

scholarly journals Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

2001 ◽  
Vol 153 (6) ◽  
pp. 1209-1226 ◽  
Author(s):  
Karen Oegema ◽  
Arshad Desai ◽  
Sonja Rybina ◽  
Matthew Kirkham ◽  
Anthony A. Hyman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Cell Stage ◽  
Kinetochore Assembly ◽  
Chromosomal Passenger ◽  
Complete Failure ◽  
Spindle Microtubules ◽  
The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

scholarly journals Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

2005 ◽  
Vol 187 (5) ◽  
pp. 1833-1844 ◽  
Author(s):  
Martin K. Safo ◽  
Qixun Zhao ◽  
Tzu-Ping Ko ◽  
Faik N. Musayev ◽  
Howard Robinson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Crystal Structures ◽  
Double Helix ◽  
Free Form ◽  
Binding Motif ◽  
Dimerization Domain ◽  
Gene Encoding ◽  
Dna Binding Sites ◽  
Dna Double Helix

ABSTRACT The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding β-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to β-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when β-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix α3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.

scholarly journals Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Open Biology ◽  
2016 ◽  
Vol 6 (2) ◽  
pp. 150236 ◽  
Author(s):  
Yahui Liu ◽  
Arsen Petrovic ◽  
Pascaline Rombaut ◽  
Shyamal Mosalaganti ◽  
Jenny Keller ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Chromosome Segregation ◽  
Functional Organization ◽  
Histone H3 Variant ◽  
Centromeric Protein ◽  
Spindle Microtubules ◽  
Specialized Region ◽  
Structural Methods ◽  
Mitosis And Meiosis ◽  
Shed Light

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.

scholarly journals ConnectingGCN5’s centromeric SAGA to the mitotic tension-sensing checkpoint

2018 ◽  
Vol 29 (18) ◽  
pp. 2201-2212 ◽  
Author(s):  
Emily L. Petty ◽  
Masha Evpak ◽  
Lorraine Pillus
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Spindle Assembly Checkpoint ◽  
Binding Function ◽  
Chromatid Cohesion ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Low Tension ◽  
Centromere Histone H3 ◽  
Segregation Of Chromosomes

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome, whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of protein phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1’s Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand the understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

scholarly journals The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

2006 ◽  
Vol 173 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Susan L. Kline ◽  
Iain M. Cheeseman ◽  
Tetsuya Hori ◽  
Tatsuo Fukagawa ◽  
Arshad Desai
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Essential Role ◽  
Stable Complex ◽  
Wild Type ◽  
Outer Plate ◽  
Checkpoint Protein ◽  
Kinetochore Assembly ◽  
Attachment Sites ◽  
Spindle Microtubules

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.

scholarly journals CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

2019 ◽  
Author(s):  
N Cortes-Silva ◽  
J Ulmer ◽  
T Kiuchi ◽  
E Hsieh ◽  
G Cornilleau ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Gene Editing ◽  
Mitotic Progression ◽  
Critical Function ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Essential Function ◽  
Necessary And Sufficient ◽  
Assembly Pathways

AbstractAccurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

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

2021 ◽  
Author(s):  
Evgeny Bakin ◽  
Fatih Sezer ◽  
Aslıhan Özbilen ◽  
Irem Kilic ◽  
Buket Uner ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Genetic Regulation ◽  
Single Copy ◽  
Expression Level ◽  
Egg Cell ◽  
Copy Gene ◽  
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 one of the very few described genes 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, we characterize the structure of CENH3, study expression levels of APOLLO and CENH3 in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of before and after fertilization. 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. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes.

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

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.

scholarly journals Phosphorylation of CENP-C by Aurora B facilitates kinetochore attachment error correction in mitosis

2017 ◽  
Vol 114 (50) ◽  
pp. E10667-E10676 ◽  
Author(s):  
Xing Zhou ◽  
Fan Zheng ◽  
Chengliang Wang ◽  
Minhao Wu ◽  
Xiaozhen Zhang ◽  
...  
Keyword(s):  
Error Correction ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Chromosome ◽  
Regulatory Mechanism ◽  
Dynamic Interaction ◽  
Aurora B ◽  
Kinetochore Assembly ◽  
Important Pathway ◽  
Spindle Microtubules

Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12–Ndc80–Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12–Nnf1 complex and showed that N-terminal residues 26–50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C–Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.

scholarly journals CENP-C is a blueprint for constitutive centromere–associated network assembly within human kinetochores

2015 ◽  
Vol 210 (1) ◽  
pp. 11-22 ◽  
Author(s):  
Kerstin Klare ◽  
John R. Weir ◽  
Federica Basilico ◽  
Tomasz Zimniak ◽  
Lucia Massimiliano ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
The Other ◽  
Microtubule Binding ◽  
Kinetochore Assembly ◽  
Binding Interface ◽  
Other Hand ◽  
Spindle Microtubules ◽  
Data Point

Kinetochores are multisubunit complexes that assemble on centromeres to bind spindle microtubules and promote faithful chromosome segregation during cell division. A 16-subunit complex named the constitutive centromere–associated network (CCAN) creates the centromere–kinetochore interface. CENP-C, a CCAN subunit, is crucial for kinetochore assembly because it links centromeres with the microtubule-binding interface of kinetochores. The role of CENP-C in CCAN organization, on the other hand, had been incompletely understood. In this paper, we combined biochemical reconstitution and cellular investigations to unveil how CENP-C promotes kinetochore targeting of other CCAN subunits. The so-called PEST domain in the N-terminal half of CENP-C interacted directly with the four-subunit CCAN subcomplex CENP-HIKM. We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex. When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.

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