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Kinetochores assemble on distinct ‘centrochromatin’ containing the histone H3 variant CENP‐A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4–K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine‐specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α‐satellite DNA and to no longer efficiently recruit HJURP, the CENP‐A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP‐A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α‐satellite transcription, maintenance of CENP‐A levels and kinetochore stability. Here, centromeric histone marks on a human artificial chromosome are found to resemble the chromatin landscape in transcribed genes, and selective manipulation shows them to govern the incorporation of the centromere‐specifying CENP‐A histone variant.

The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP‐A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type‐specific barrier for de novo stable CENP‐A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP‐A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri‐methylase (Suv39h1) to the array prevents de novo CENP‐A assembly and kinetochore formation. CENP‐A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells. Establishment of Human Artificial Chromosomes (HACs) depends on an interplay of H3 lysine 9 modifications at centromeres, providing insights into the pathways that control incorporation of the kinetochore‐specificing histone H3 variant CENP‐A.

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation.The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by inhibiting anaphase until all kinetochores are attached to microtubules. Recent studies highlight the dynamic properties of SAC signalling and begin to explain signal integration at mammalian kinetochores, which feature multiple attachment points.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes 1 , 2 , function to connect centromeric chromatin to microtubules of the mitotic spindle 3 , 4 . Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules 5 , 6 . All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-A Nuc ), each of which is perfectly centred on its cognate centromere 7 – 9 . The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN–Cenp-A Nuc ). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-A Nuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-A Nuc are mediated predominantly by a DNA-binding groove in the Cenp-L–Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-A Nuc . Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation. Cryo-electron microscopy structures of the Saccharomyces cerevisiae inner kinetochore complex provide insights into the interdependencies of constituent subcomplexes and the mechanism of centromeric nucleosome recognition.

Key Points The spindle checkpoint signalling cascade prevents anaphase onset until all chromosomes are correctly attached, through their kinetochores, to spindle microtubules. Molecular interactions between kinetochore and spindle checkpoint proteins have been defined and characterized. There have been significant advances in understanding the molecular details of phosphoregulation and checkpoint scaffolding. Monopolar spindle protein 1 (MPS1) has emerged as a direct activating kinase of the checkpoint. The checkpoint response strength is variable and corresponds with the number of unattached kinetochores. Inactivation of cyclin-dependent kinase 1 (CDK1) by cyclin B degradation is a basis for checkpoint inactivation during anaphase. Nuclear pore complexes, in addition to kinetochores, signal the checkpoint. The mechanisms underlying spindle checkpoint signalling at the kinetochore, which ensures faithful chromosome segregation during cell division, are being unravelled. They indicate that the checkpoint response is graded rather than switch-like (completely on or off) as traditionally thought, and provide insights for the treatment of cancers in which the checkpoint is bypassed. The spindle checkpoint ensures proper chromosome segregation during cell division. Unravelling checkpoint signalling has been a long-standing challenge owing to the complexity of the structures and forces that regulate chromosome segregation. New reports have now substantially advanced our understanding of checkpoint signalling mechanisms at the kinetochore, the structure that connects microtubules and chromatin. In contrast to the traditional view of a binary checkpoint response — either completely on or off — new findings indicate that the checkpoint response strength is variable. This revised perspective provides insight into how checkpoint bypass can lead to aneuploidy and informs strategies to exploit these errors for cancer treatments.

Biochemical reconstitution of a synthetic human kinetochore with 21 protein subunits and centromeric nucleosomal DNA unveils fundamental principles of kinetochore organization and function. A reconstituted 21-subunit kinetochore Kinetochores are multisubunit complexes that assemble on centromeres and connect chromosomes with microtubules in the mitotic spindle during cell division. Andrea Musacchio and colleagues have reconstituted a synthetic human kinetochore with 21 protein subunits and centromeric nucleosomal DNA. A seven-subunit subcomplex determines the binding selectivity for the centromeric histone CENP-A and binds directly to the microtubule-binding subcomplex of the outer kinetochore — an architecture reminiscent of that in Saccharomyces cerevisiae . Reconstituting this minimal linkage between centromeric chromatin and the mitotic spindle in vitro should allow further insights into kinetochore structure and function. Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle 1 . Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A) 2 . A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN) 3 , 4 , 5 , 6 , establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore 7 , 8 . Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described 9 , 10 , 11 , but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro . This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD–Zwilch–ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments. Sacristan et al. show that the dynein adaptor Spindly facilitates oligomerisation of the RZZ complex to expand the kinetochore, after which Spindly-associated dynein compacts the kinetochore to allow for faithful chromosome segregation.

Human kinetochores assemble onto centromeric DNA comprising repetitive arrays of the 171 bp α-satellite sequence. To understand the higher-order architecture of the inner kinetochore assembled onto α-satellite arrays, we show cryo-EM structures of CCAN with free DNA, and α-satellite repeat monomers and dimers with CENP-A nucleosomes. CCAN bound to free DNA and a monomeric CENP-A nucleosome engages 70 bp of DNA comprising 30 bp of an upstream α-satellite repeat. This upstream DNA interacts with the histone-fold domain subunits of the CENP-TWSX module in a manner resembling how nucleosomes wrap DNA gyres. A complex of CCAN assembled onto a dimeric α-satellite repeat with two CENP-A nucleosomes shows that CCAN can only be accommodated on the linker DNA by unwrapping DNA from both the CENP-TWSX module and the upstream nucleosome. We discuss the implications of these results for models of CCAN assembly on arrays of α-satellite chromatin containing CENP-A nucleosomes. Based on cryo-EM structures, here the authors propose three models for how the inner kinetochore and CENP-A nucleosomes are organized on repetitive alpha-satellite sequences, providing insights into the higher-order architecture of human centromeres.

Key Points Centromeres are defined epigenetically and require the presence of the centromere-specific histone H3 variant centromere protein A (CENP-A). Although DNA sequences are not strictly required for centromere specification, similarities in the organization of centromere DNA suggest that DNA structures contribute to centromere function. CENP-A nucleosomes contain unique sequence and structural features that allow them to stably mark the centromere and be recognized by kinetochore components. CENP-A propagation requires specialized deposition factors and tight regulatory control. The centromere directs the assembly of the kinetochore via the 16-subunit constitutive centromere-associated network (CCAN). Most eukaryotic centromeres are defined epigenetically and require nucleosomes containing the histone H3 variant centromere protein A (CENP-A). We are now gaining insight into the mechanisms that regulate CENP-A deposition and positioning to specify and propagate centromeres during cell division, and into the function of centromeres in recruiting kinetochores to connect chromosomes to spindle microtubules. The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.

Faithful chromosome segregation during mitosis in eukaryotes requires attachment of the kinetochore, a large protein complex assembled on the centromere of each chromosome, to the spindle microtubules. The kinetochore is a structural interface for the microtubule attachment and provides molecular surveillance mechanisms that monitor and ensure the precise microtubule attachment as well, including error correction and spindle assembly checkpoint. During mitotic progression, the kinetochore undergoes dynamic morphological changes that are observable through electron microscopy as well as through fluorescence microscopy. These structural changes might be associated with the kinetochore function. In this review, we summarize how the dynamics of kinetochore morphology are associated with its functions and discuss recent findings on the switching of protein interaction networks in the kinetochore during cell cycle progression.