Structural basis for Mis18 complex assembly and its implications for centromere maintenance.

The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

Target Identification and Mechanistic Characterization of Indole Terpenoid Mimics: Proper Spindle Microtubule Assembly Is Essential for Cdh1-Mediated Proteolysis of CENP-A.

Centromere protein A (CENP-A), a centromere-specific histone H3 variant, is crucial for kinetochore positioning and chromosome segregation. However, its regulatory mechanism in human cells remains incompletely understood. A structure-activity relationship (SAR) study of the cell-cycle-arresting indole terpenoid mimic JP18 leads to the discovery of two more potent analogs, (+)-6-Br-JP18 and (+)-6-Cl-JP18. Tubulin is identified as a potential cellular target of these halogenated analogs by using the drug affinity responsive target stability (DARTS) based method. X-ray crystallography analysis reveals that both molecules bind to the colchicine-binding site of ß-tubulin. Treatment of human cells with microtubule-targeting agents (MTAs), including these two compounds, results in CENP-A accumulation by destabilizing Cdh1, a co-activator of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. This study establishes a link between microtubule dynamics and CENP-A accumulation using small-molecule tools and highlights the role of Cdh1 in CENP-A proteolysis.

Vertebrate centromere architecture: from chromatin threads to functional structures.

Centromeres are chromatin structures specialized in sister chromatid cohesion, kinetochore assembly, and microtubule attachment during chromosome segregation. The regional centromere of vertebrates consists of long regions of highly repetitive sequences occupied by the Histone H3 variant CENP-A, and which are flanked by pericentromeres. The three-dimensional organization of centromeric chromatin is paramount for its functionality and its ability to withstand spindle forces. Alongside CENP-A, key contributors to the folding of this structure include components of the Constitutive Centromere-Associated Network (CCAN), the protein CENP-B, and condensin and cohesin complexes. Despite its importance, the intricate architecture of the regional centromere of vertebrates remains largely unknown. Recent advancements in long-read sequencing, super-resolution and cryo-electron microscopy, and chromosome conformation capture techniques have significantly improved our understanding of this structure at various levels, from the linear arrangement of centromeric sequences and their epigenetic landscape to their higher-order compaction. In this review, we discuss the latest insights on centromere organization and place them in the context of recent findings describing a bipartite higher-order organization of the centromere.

Contribution of CENP-F to FOXM1-Mediated Discordant Centromere and Kinetochore Transcriptional Regulation.

Proper chromosome segregation is required to ensure chromosomal stability. The centromere (CEN) is a unique chromatin domain defined by CENP-A and is responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating microtubule spindle attachment and mitotic checkpoint function. Upregulation of many CEN/KT genes is commonly observed in cancer. Here, we show that although FOXM1 occupies promoters of many CEN/KT genes with MYBL2, FOXM1 overexpression alone is insufficient to drive the FOXM1-correlated transcriptional program. CENP-F is canonically an outer kinetochore component; however, it functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in altered chromatin accessibility at G2/M genes and reduced FOXM1-MBB complex formation. We show that coordinated CENP-FFOXM1 transcriptional regulation is a cancer-specific function. We observe a small subset of CEN/KT genes including CENP-C, that are not regulated by FOXM1. Upregulation of CENP-C in the context of CENP-A overexpression leads to increased chromosome missegregation and cell death suggesting that escape of CENP-C from FOXM1 regulation is a cancer survival mechanism. Together, we show that FOXM1 and CENP-F coordinately regulate G2/M genes, and this coordination is specific to a subset of genes to allow for maintenance of chromosome instability levels and subsequent cell survival.

The Chaperone NASP Contributes to de Novo Deposition of the Centromeric Histone Variant CENH3 in Arabidopsis Early Embryogenesis.

The centromere is an essential chromosome region where the kinetochore is formed to control equal chromosome distribution during cell division. The centromere-specific histone H3 variant CENH3 (also called CENP-A) is a prerequisite for the kinetochore formation. Since CENH3 evolves rapidly, associated factors, including histone chaperones mediating the deposition of CENH3 on the centromere, are thought to act through species-specific amino acid sequences. The functions and interaction networks of CENH3 and histone chaperons have been well-characterized in animals and yeasts. However, molecular mechanisms involved in recognition and deposition of CENH3 are still unclear in plants. Here, we used a swapping strategy between domains of CENH3 of Arabidopsis thaliana and the liverwort Marchantia polymorpha to identify specific regions of CENH3 involved in targeting the centromeres and interacting with the general histone H3 chaperone, nuclear autoantigenic sperm protein (NASP). CENH3's LoopN-α1 region was necessary and sufficient for the centromere targeting in cooperation with the α2 region and was involved in interaction with NASP in cooperation with αN, suggesting a species-specific CENH3 recognition. In addition, by generating an Arabidopsis nasp knock-out mutant in the background of a fully fertile GFP-CENH3/cenh3-1 line, we found that NASP was implicated for de novo CENH3 deposition after fertilization and thus for early embryo development. Our results imply that the NASP mediates the supply of CENH3 in the context of the rapidly evolving centromere identity in land plants.

DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of histone supply chains.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

Cdc48 and its co-factor Ufd1 extract CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A determines the identity of the centromere. Because the position and size of the centromere and its number per chromosome must be maintained, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A (Cnp1SP) in fission yeast. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to Cnp1 expressed at a high level and allows mislocalization of Cnp1. The level of Cnp1 in centromeric chromatin is increased in the ufd1-73 mutant even when Cnp1 is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins interact physically with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of Cnp1 from Ch16, leading to an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 remove excess Cnp1 from chromatin, likely in a direct manner. This mechanism may play a role in centromere disassembly, a process to eliminate Cnp1 to inactivate the kinetochore function during development, differentiation, and stress response.

Set2 regulates Ccp1 and Swc2 to ensure centromeric stability by retargeting CENP-A.

Precise positioning of the histone-H3 variant, CENP-A, ensures centromere stability and faithful chromosomal segregation. Mislocalization of CENP-A to extra-centromeric loci results in aneuploidy and compromised cell viability associated with formation of ectopic kinetochores. The mechanism that retargets mislocalized CENP-A back to the centromere is unclarified. We show here that the downregulation of the histone H3 lysine 36 (H3K36) methyltransferase Set2 can preserve centromere localization of a temperature-sensitive mutant cnp1-1 Schizosaccharomyces pombe CENP-A (SpCENP-A) protein and reverse aneuploidy by redirecting mislocalized SpCENP-A back to centromere from ribosomal DNA (rDNA) loci, which serves as a sink for the delocalized SpCENP-A. Downregulation of set2 augments Swc2 (SWR1 complex DNA-binding module) expression and releases histone chaperone Ccp1 from the centromeric reservoir. Swc2 and Ccp1 are directed to the rDNA locus to excavate the SpCENP-Acnp1-1, which is relocalized to the centromere in a manner dependent on canonical SpCENP-A loaders, including Mis16, Mis17 and Mis18, thereby conferring cell survival and safeguarding chromosome segregation fidelity. Chromosome missegregation is a severe genetic instability event that compromises cell viability. This mechanism thus promotes CENP-A presence at the centromere to maintain genomic stability.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

DNA satellite and chromatin organization at mouse centromeres and pericentromeres.

BACKGROUND: Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of assembling repetitive genomic regions. RESULTS: Using recently available PacBio long-read sequencing data from the C57BL/6 strain, we find that contrary to the previous reports of their homogeneous nature, both centromeric minor satellites and pericentromeric major satellites exhibit a high degree of variation in sequence and organization within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Using chromatin immunoprecipitation sequencing (ChIP-seq), we find that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence enrichment and homogeneity at these regions. The transposable elements at centromeric regions are not part of functional centromeres as they lack significant CENP-A enrichment. Furthermore, both CENP-A and H3K9me3 nucleosomes occupy minor and major satellites spanning centromeric-pericentric junctions and a low yet significant amount of CENP-A spreads locally at centromere junctions on both pericentric and telocentric sides. Finally, while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays are poorly phased. Interestingly, the homogeneous class of major satellites also phase CENP-A and H3K27me3 nucleosomes, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. CONCLUSIONS: Our findings reveal that mouse centromeres and pericentromeres display a high diversity in satellite sequence, organization, and chromatin structure.

Higher-order protein assembly controls kinetochore formation.

To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.

HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

A role for ß-1,6- and ß-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 ß-1,6- and ß-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 ß-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 ß-1,3-glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular ß-1,6- and ß-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.

The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.

Defining a core configuration for human centromeres during mitosis.

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.

Humanization reveals pervasive incompatibility of yeast and human kinetochore components.

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.

Quantitative Super-Resolution Microscopy Reveals the Relationship between CENP-A Stoichiometry and Centromere Physical Size.

Centromeric chromatin is thought to play a critical role in ensuring the faithful segregation of chromosomes during mitosis. However, our understanding of this role is presently limited by our poor understanding of the structure and composition of this unique chromatin. The nucleosomal variant, CENP-A, localizes to narrow regions within the centromere, where it plays a major role in centromeric function, effectively serving as a platform on which the kinetochore is assembled. Previous work found that, within a given cell, the number of microtubules within kinetochores is essentially unchanged between CENP-A-localized regions of different physical sizes. However, it is unknown if the amount of CENP-A is also unchanged between these regions of different sizes, which would reflect a strict structural correspondence between these two key characteristics of the centromere/kinetochore assembly. Here, we used super-resolution optical microscopy to image and quantify the amount of CENP-A and DNA within human centromere chromatin. We found that the amount of CENP-A within CENP-A domains of different physical sizes is indeed the same. Further, our measurements suggest that the ratio of CENP-A- to H3-containing nucleosomes within these domains is between 8:1 and 11:1. Thus, our results not only identify an unexpectedly strict relationship between CENP-A and microtubules stoichiometries but also that the CENP-A centromeric domain is almost exclusively composed of CENP-A nucleosomes.

A dynamic population of prophase CENP-C is required for meiotic chromosome segregation.

The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.

Centromere innovations within a mouse species.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying centromere protein-A (CENP-A) nucleosomes at the nexus of a satellite repeat that we identified and termed π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 mega-base pairs of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance promotes accumulation of microtubule-binding components of the kinetochore and a microtubule-destabilizing kinesin of the inner centromere. We propose that the balance of pro- and anti-microtubule binding by the new centromere is what permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

Variation in the CENP-A sequence association landscape across diverse inbred mouse strains.

Centromeres are crucial for chromosome segregation, but their underlying sequences evolve rapidly, imposing strong selection for compensatory changes in centromere-associated kinetochore proteins to assure the stability of genome transmission. While this co-evolution is well documented between species, it remains unknown whether population-level centromere diversity leads to functional differences in kinetochore protein association. Mice (Mus musculus) exhibit remarkable variation in centromere size and sequence, but the amino acid sequence of the kinetochore protein CENP-A is conserved. Here, we apply k-mer-based analyses to CENP-A chromatin profiling data from diverse inbred mouse strains to investigate the interplay between centromere variation and kinetochore protein sequence association. We show that centromere sequence diversity is associated with strain-level differences in both CENP-A positioning and sequence preference along the mouse core centromere satellite. Our findings reveal intraspecies sequence-dependent differences in CENP-A/centromere association and open additional perspectives for understanding centromere-mediated variation in genome stability.