Constitutive Centromere-associated Network Research Articles

Structure of the human outer kinetochore KMN network complex

Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.

Artificial tethering of constitutive centromere-associated network proteins induces CENP-A deposition without Knl2 in DT40 cells.

The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.

First person – JingHui Cao

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. JingHui Cao is first author on ‘ Artificial tethering of constitutive centromere-associated network proteins induces CENP-A deposition without Knl2 in DT40 cells’, published in JCS. JingHui is a PhD student in the lab of Tatsuo Fukagawa at the Graduate School of Frontier Biosciences, Osaka, Japan, investigating the relationship between cancer and chromosomal instability (CIN).

CENP-A and CENP-B collaborate to create an open centromeric chromatin state

Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.

A role for β-1,6- and β-1,3-glucans in kinetochore function in Saccharomyces cerevisiae.

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A, and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for β-1,6- and β-1,3-glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/ transglycosidase required for β-1,6-glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a constitutive centromere-associated network protein. Similarly, the absence of Fks1, a β-1,3-glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular β-1,6- and β-1,3-glucans, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.

Dynamic phosphorylation of CENP-N by CDK1 guides accurate chromosome segregation in mitosis.

In mitosis, accurate chromosome segregation depends on the kinetochore, a supermolecular machinery that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN) during mitosis remains uncharacterized. Building on our recent cryo-electron microscopic analyses of human CCAN structure, we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.

Centromere/kinetochore is assembled through CENP-C oligomerization

Kinetochore is an essential protein complex required for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin and provides a platform for the kinetochore assembly. The CCAN protein CENP-C is thought to be a central hub for the centromere/kinetochore organization. However, the role of CENP-C in CCAN assembly needs to be elucidated. Here, we demonstrate that both the CCAN-binding domain and the C-terminal region that includes the Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Structural and biochemical analyses reveal self-oligomerization of the Cupin domains of chicken and human CENP-C. We find that the CENP-C Cupin domain oligomerization is vital for CENP-C function, centromeric localization of CCAN, and centromeric chromatin organization. These results suggest that CENP-C facilitates the centromere/kinetochore assembly through its oligomerization.

P97/VCP drives turnover of SUMOylated centromeric CCAN proteins and CENP-A.

The centromere is a unique chromatin domain that links sister chromatids and forms the attachment site for spindle microtubules in mitosis. Centromere inheritance is largely DNA sequence-independent but strongly reliant on a self-propagating chromatin domain featuring nucleosomes containing the H3 variant CENP-A. Unlike other histones, CENP-A is maintained with unusually high stability in chromatin. Previously, we have shown that mitotic maintenance of CENP-A and other constitutive centromere-associated network (CCAN) proteins is controlled by a dynamic SUMO cycle and that the deSUMOylase SENP6 is necessary for stable maintenance of CENP-A at the centromere. Here, we discover that the removal of SENP6 leads to a rapid loss of the CCAN, followed by a delayed loss of centromeric CENP-A, indicating that the CCAN is the primary SUMO target. We found that the ATP-dependent segregase p97/VCP removes centromeric CENP-A in a SUMO-dependent manner and interacts physically with the CCAN and CENP-A chromatin. Our data suggest a direct role of p97 in removing centromeric CENP-A via SUMOylated CCAN proteins, thereby ensuring centromere homeostasis and potentially preventing ectopic CENP-A accumulation.

The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S-CENP-X is involved in regulating the SAC and kinetochore-microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1-Mhf2, the CENP-S-CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1-Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments. This article has an associated First Person interview with the first author of the paper.

The function, structure and dynamic organization of centromeres and kinetochores

It is a fundamental task to ensure the faithful transmission of genetic information across generations for eukaryote species. The centromere is a specialized chromosomal region that is essential for mediating sister chromatid alignment and separation during mitosis. Centromere identity is epigenetically determined by nucleosome-containing centromere protein A (CENP-A). The CENP-A nucleosome provides the foundation for the association of the inner kinetochore and the assembly of the outer kinetochore in mitosis. Here we review centromere identity determination, inner kinetochore function and assembly, and outer kinetochore function and assembly. In particular, we focus on the recent advances in the structure-activity relationship of the constitutive centromere-associated network (CCAN). CCAN structure information sheds new light on our understanding of centromere and kinetochore functions and dynamic organization.

Structural insights into human CCAN complex assembled onto DNA

In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.

Phosphorylation of CENP-R by Aurora B regulates kinetochore-microtubule attachment for accurate chromosome segregation.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis. Although previously proposed to be an adaptor of retinoic acid receptor, here, we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and ensure accurate chromosome segregation in mitosis. We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore, suggesting that phosphorylation may regulate its localization. Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore-microtubule attachment at metaphase. Mechanistically, CENP-R phosphorylation disrupts its binding with CENP-U. Thus, we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore-microtubule attachment in mitosis. As CENP-R is absent from yeast, we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.

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

SummaryCentromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and 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 implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.

Investigating the Transition of the Core Centromeric Nucleosome Complex from Interphase to Mitosis using Chemical Biology Tools

Centromeres are the location on each chromosome where kinetochores form in mitosis to connect to spindle microtubules that physically separate sister chromatids to the daughter cells. A 16‐subunit complex termed the constitutive centromere associated network (CCAN) is bound to nucleosomes harboring the histone variant, CENP‐A, and is essential for assembling the mitotic kinetochore. The core centromere nucleosome complex (CCNC) consists of CENP‐A nucleosomes and CCAN subunits, CENP‐C and CENP‐N. In prior studies, we proposed that the CCNC undergoes an alteration at mitotic onset where one of two copies of CENP‐N is lost. This model indicates that there would remain only one CENP‐A nucleosome surface with both CENP‐N and CENP‐C that is competent to recruit the CCAN and, in turn, assemble the kinetochore. We hypothesize that the asymmetric surfaces on CENP‐A nucleosomes provide a level of organization of centromeric chromatin that is important for chromosome segregation. We are developing a system to study these functional relationships in cells using chemical biology approaches. We have created a cell line with complementary SNAP and HALO tags on the CCNC components and employed the SNAP‐HALO cross linker, HaXS8, to tether CCNC components (CENP‐A with CENP‐N) irreversibly on nucleosomes. Initial experiments indicate that this system is able to retain CENP‐N at high levels on mitotic centromeres in an HaXS8 concentration‐dependent fashion. This provides additional support for the model of a centromeric chromatin transition as cells enter mitosis, and our system holds promise to test predictions that arise from it.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome.

Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.

A comprehensive investigation on pan-cancer impacts of constitutive centromere associated network gene family by integrating multi-omics data: A CONSORT-compliant article.

Background:The constitutive centromere associated network (CCAN) complex played a critical role in connecting the centromere with the mitotic spindle during mitosis and meiosis. Many studies have indicated that CCAN is related to the tumorigenesis and cancer development. Nonetheless, the overview of CCAN gene family in pan-cancer remain incompletely understood.Methods:We performed a comprehensive investigation on pan-cancer impacts of CCAN by integrating multi-omics data. We comprehensively investigated the expression profile, kyoto encyclopedia of genes and genomes (kegg) pathway, mutation, copy number variation, tumor microenvironment, immune cells infiltration, and drug sensitivity of CCAN in pan-cancer. MRNA expression profiles were collected from the cancer genome atlas, oncomine and ccle, the differential expression and various relevance analysis were performed with R or Perl.Results:The results showed that the expression of CCAN was different in 33 tumors. Intriguingly, the poor survival in adrenocortical carcinoma, cholangiocarcinoma, kidney chromophobe, mesothelioma, kidney renal clear cell carcinoma, brain lower grade glioma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, thyroid carcinoma, uveal melanoma was most likely related to the kegg single transduction pathway including one carbon pool by folate, proteasome, arachidonic acid metabolism and so on. CENPC, ITGB3BP, APITD1, CENPU, and CENPW were more involved in tumor microenvironment, which more likely related to NK cells resting, T cells follicular helper, T cells CD8, neutrophils, macrophages M0, T cells CD4 memory activated. The relationship of CCAN expression with drug sensitivity showed that chelerythrine, nelarabine, and hydroxyurea maybe be potential drugs.Conclusions:This multidimensional study provides a valuable resource to assist mechanism research and clinical utility about CCAN.

Bridgin connects the outer kinetochore to centromeric chromatin

The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-CMif2, CENP-TCnn1, and CENP-UAme1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein “bridgin” was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.

Loss of inner kinetochore genes is associated with the transition to an unconventional point centromere in budding yeast.

BackgroundThe genomic sequences of centromeres, as well as the set of proteins that recognize and interact with centromeres, are known to quickly diverge between lineages potentially contributing to post-zygotic reproductive isolation. However, the actual sequence of events and processes involved in the divergence of the kinetochore machinery is not known. The patterns of gene loss that occur during evolution concomitant with phenotypic changes have been used to understand the timing and order of molecular changes.MethodsI screened the high-quality genomes of twenty budding yeast species for the presence of well-studied kinetochore genes. Based on the conserved gene order and complete genome assemblies, I identified gene loss events. Subsequently, I searched the intergenic regions to identify any un-annotated genes or gene remnants to obtain additional evidence of gene loss.ResultsMy analysis identified the loss of four genes (NKP1, NKP2, CENPL/IML3 and CENPN/CHL4) of the inner kinetochore constitutive centromere-associated network (CCAN/also known as CTF19 complex in yeast) in both the Naumovozyma species for which genome assemblies are available. Surprisingly, this collective loss of four genes of the CCAN/CTF19 complex coincides with the emergence of unconventional centromeres in N. castellii and N. dairenensis. My study suggests a tentative link between the emergence of unconventional point centromeres and the turnover of kinetochore genes in budding yeast.

Crystal structure of the Cenp-HIKHead-TW sub-module of the inner kinetochore CCAN complex.

Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14–16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead–Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN–Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA.

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism.