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Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

During cell division, chromosomes build kinetochores that attach to spindle microtubules. Kinetochores usually form at the centromeres, which contain CENP-A nucleosomes. The outer kinetochore, which is the core attachment site for microtubules, is composed of the KMN network (Knl1c, Mis12c, and Ndc80c complexes) and is recruited downstream of CENP-A and its partner CENP-C. In C. elegans oocytes, kinetochores have been suggested to form independently of CENP-A nucleosomes. Yet kinetochore formation requires CENP-C, which acts in parallel to the nucleoporin MEL-28ELYS. Here, we used a combination of RNAi and Degron-based depletion of CENP-A (or downstream CENP-C) to demonstrate that both proteins are in fact responsible for a portion of outer kinetochore assembly during meiosis I and are essential for accurate chromosome segregation. The remaining part requires the coordinated action of KNL-2 (ortholog of human M18BP1) and of the nucleoporin MEL-28ELYS. Accordingly, co-depletion of CENP-A (or CENP-C) and KNL-2M18BP1 (or MEL-28ELYS) prevented outer kinetochore assembly in oocytes during meiosis I. We further found that KNL-2M18BP1 and MEL-28ELYS are interdependent for kinetochore localization. Using engineered mutants, we demonstrated that KNL-2M18BP1 recruits MEL-28ELYS at meiotic kinetochores through a specific N-terminal domain, independently of its canonical CENP-A loading factor activity. Finally, we found that meiosis II outer kinetochore assembly was solely dependent on the canonical CENP-A/CENP-C pathway. Thus, like in most cells, outer kinetochore assembly in C. elegans oocytes depends on centromeric chromatin. However, during meiosis I, an additional KNL-2M18BP1 and MEL-28ELYS pathway acts in a non-redundant manner and in parallel to canonical centromeric chromatin. [Display omitted] • CENP-A/C are essential for kinetochore assembly and function in meiosis I and II • KNL-2/MEL-28 form a non-canonical kinetochore assembly pathway during meiosis I • CENP-A/C and KNL-2/MEL-28 coordinate kinetochore assembly during meiosis I • Kinetochore assembly is regulated differently between meiosis I and meiosis II Bellutti et al. demonstrate that during meiosis I in C. elegans oocytes, outer kinetochore assembly relies on both the canonical CENP-A/C centromeric pathway and a parallel non-canonical KNL-2/MEL-28 pathway. By contrast, during meiosis II, outer kinetochore assembly depends solely on the CENP-A/C pathway. [ABSTRACT FROM AUTHOR]

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers. [ABSTRACT FROM AUTHOR]

Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, although the specific mechanisms are not fully understood. Here, we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos.We found that depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. Knockdown of CSR-1 does not affect mRNA and protein levels of the centromeric histone H3 variant and CENP-A homolog HCP-3 but does increase the localization of HCP-3 and some kinetochore proteins to the mitotic chromosomes. Such elevation of HCP-3 chromatin localization depends on EGO-1, which is an upstream factor in the CSR-1 RNA interference (RNAi) pathway, and PIWI domain activity of CSR-1. Our results suggest that CSR-1 restricts the level of HCP-3 at the holocentromeres, prevents erroneous kinetochore assembly and thereby promotes accurate chromosome segregation. Our work sheds light on the role of CSR-1 in regulating deposition of HCP-3 on chromatin and centromere function in embryos. [ABSTRACT FROM AUTHOR]

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, Mis18β 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 Mis18β 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 Mis18β, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity. Synopsis: Multiple inter- and intra-molecular interfaces stabilise the overall three-dimensional architecture of the Mis18 complex and ensure centromere localisation and function of the CENP-A loading machinery. Mis18α/β hetero-oligomerisation mediated by the C-terminal triple helical bundle is essential for Mis18 complex assembly. Mis18BP1 binds Mis18α/β via a bipartite mode of interaction involving Mis18α/βYippee heterodimeric interface and Mis18αYippee with Mis18BP1 Cdk1 sites strategically located at the binding interfaces, thus providing a structural rationale for Cdk1 mediated temporal restriction of Mis18 complex assembly. Mis18α can localise to centromeres independently of Mis18β, whilst Mis18β is required for efficient CENP-A loading at centromeres. The N-terminal α-helical region of Mis18α makes extensive contact with the HJURP binding region of Mis18α/β, suggesting a novel regulatory role. Multiple inter- and intra-molecular interfaces stabilise the overall three-dimensional architecture of the Mis18 complex and ensure centromere localisation and function of the CENP-A loading machinery. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function. Results: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes. Conclusions: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant. [ABSTRACT FROM AUTHOR]

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. Synopsis: Centromeric localization of the histone H3 variant CENP-A is critical for faithful chromosome segregation. This study reveals that the histone H3 chaperone DNAJC9 restricts mislocalization of CENP-A to non-centromeric regions, and thereby prevents chromosome instability (CIN). Loss of DNAJC9 leads to enrichment of CENP-A at the non-centromeric regions genome-wide. Mislocalization of CENP-A enhances the CIN phenotypes of DNAJC9-depleted cells. DNA replication-associated H3 chaperone MCM2 contributes to CENP-A mislocalization upon DNAJC9 depletion. Genome-wide RNAi screens reveal the importance of proper histone H3-H4 folding/supply for restricting CENP-A localization to centromeres. [ABSTRACT FROM AUTHOR]

Abstract 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.

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids.

Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human centromere is the primary constriction within mitotic chromosomes made up of repetitive alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. Notably, centromeric DNA is faithfully inherited as intact haplotypes from the parents to the offspring without intervening recombination, yet, outside of meiosis, centromeres are akin to common fragile sites (CFSs), manifesting crossing-overs and ongoing sequence instability. Consequences of DNA changes within the centromere are just starting to emerge, with unclear effects on intra- and inter-generational inheritance driven by centromere's essential role in kinetochore assembly. Here, we review evidence of meiotic selection operating to mitigate centromere drive, as well as recent reports on centromere damage, recombination and repair during the mitotic cell division. We propose an antagonistic pleiotropy interpretation to reconcile centromere DNA instability as both driver of aneuploidy that underlies degenerative diseases, while also potentially necessary for the maintenance of homogenized HORs for centromere function. We attempt to provide a framework for this conceptual leap taking into consideration the structural interface of centromere-kinetochore interaction and present case scenarios for its malfunctioning. Finally, we offer an integrated working model to connect DNA instability, chromatin, and structural changes with functional consequences on chromosome integrity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Centromeres play essential roles in the faithful segregation of chromosomes. CENP-A, the centromere-specific histone H3 variant, and heterochromatin characterized by di- or tri-methylation of histone H3 9th lysine (H3K9) are the hallmarks of centromere chromatin. Contrary to the epigenetic marks, DNA sequences underlying the centromere region of chromosomes are not well conserved through evolution. However, centromeres consist of repetitive sequences in many eukaryotes, including animals, plants, and a subset of fungi, including fission yeast. Advances in long-read sequencing techniques have uncovered the complete sequence of human centromeres containing more than thousands of alpha satellite repeats and other types of repetitive sequences. Not only tandem but also inverted repeats are present at a centromere. DNA recombination between centromere repeats can result in gross chromosomal rearrangement (GCR), such as translocation and isochromosome formation. CENP-A chromatin and heterochromatin suppress the centromeric GCR. The key player of homologous recombination, Rad51, safeguards centromere integrity through conservative noncrossover recombination between centromere repeats. In contrast to Rad51-dependent recombination, Rad52-mediated single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) lead to centromeric GCR. This review summarizes recent findings on the role of centromere and recombination proteins in maintaining centromere integrity and discusses how GCR occurs at centromeres. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Shugoshin-1 (Sgo1) is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis. It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120. However, it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis. Here, we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis. Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A. Interestingly, the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere, resulting in aberrant chromosome segregation and premature chromatid separation. Taken together, these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1–CENP-A interaction guides accurate cell division. [ABSTRACT FROM AUTHOR]

Creating true-breeding lines is a critical step in plant breeding. Novel, completely homozygous true-breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere-specific histone 3 variant (CENH3), including chimeric proteins, expression of non-native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild-type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS-inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9-mediated in-frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild-type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non-transgenic approaches to the generation of haploid inducers.

The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation.

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to noncentromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imagingbased high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP--CENPA) in HeLa cells. Among the top five candidates in the screen -- the depletion of which showed increased nuclear YFP--CENP-A fluorescence -- were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENPA mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN. [ABSTRACT FROM AUTHOR]

Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 methylation and Cse4-K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components. [ABSTRACT FROM AUTHOR]

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

Centromeres are unique chromosomal loci that form the anchorage point for the mitotic spindle during mitosis and meiosis. Their position and function are specified by a unique chromatin domain featuring the histone H3 variant CENP-A. While typically formed on centromeric satellite arrays, CENP-A nucleosomes are maintained and assembled by a strong self-templated feedback mechanism that can propagate centromeres even at non-canonical sites. Central to the epigenetic chromatin-based transmission of centromeres is the stable inheritance of CENP-A nucleosomes. While long-lived at centromeres, CENP-A can turn over rapidly at non-centromeric sites and even erode from centromeres in non-dividing cells. Recently, SUMO modification of the centromere complex has come to the forefront as a mediator of centromere complex stability, including CENP-A chromatin. We review evidence from different models and discuss the emerging view that limited SUMOylation appears to play a constructive role in centromere complex formation, while polySUMOylation drives complex turnover. The deSUMOylase SENP6/Ulp2 and the proteins segregase p97/Cdc48 constitute the dominant opposing forces that balance CENP-A chromatin stability. This balance may be key to ensuring proper kinetochore strength at the centromere while preventing ectopic centromere formation.

Centromere protein A (CENP‐A) nucleosomes containing the centromere‐specific histone H3 variant CENP‐A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP‐A deposition via the CENP‐A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP‐C‐like motif, and our previous study suggested that ggKNL2 directly binds to the CENP‐A nucleosome to recruit HJURP/CENP‐A to the centromere. However, the molecular basis for CENP‐A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo‐EM structure of the chicken CENP‐A nucleosome in complex with a ggKNL2 fragment containing the CENP‐C‐like motif. Chicken KNL2 distinguishes between CENP‐A and histone H3 in the nucleosome using the CENP‐C‐like motif and its downstream region. Both the C‐terminal tail and the RG‐loop of CENP‐A are simultaneously recognized as CENP‐A characteristics. The CENP‐A nucleosome–ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression. Synopsis: Cryo‐EM demonstrates the direct interaction of CENP‐A nucleosome of and licensing factor KNL2, and provides an insight into dynamic regulation of centromere localization of KNL2 during cell cycle progression. Mis‐localized KNL2 mutants caused reduction of CENP‐A levels at centromeres in chicken DT40 cells. Chicken KNL2 recognizes the centromeric nucleosome containing CENP‐A via a conserved CENP‐C‐like motif.Chicken KNL2 shares the CENP‐A nucleosome binding mode with kinetochore protein CENP‐C.CENP‐A nucleosome binding of chicken KNL2 is essential for its centromere localization in interphase cells but not mitotic cells.Mitotic centromere localization of chicken KNL2 depends on CENP‐C.Centromere localization of chicken KNL2 is dynamically regulated during the cell cycle. [ABSTRACT FROM AUTHOR]

Centromeres are highly specialised chromosome domains defined by the presence of an epigenetic mark, the specific histone H3 variant called CENP-A (centromere protein A). They constitute the genomic regions on which kinetochores form and when defective cause segregation defects that can lead to aneuploidy and cancer. Here, we discuss how CENP-A is established and maintained to propagate centromere identity while subjected to dynamic chromatin remodelling during essential cellular processes like DNA repair, replication, and transcription. We highlight parallels and identify conserved mechanisms between different model organism with a particular focus on 1) the establishment of CENP-A at centromeres, 2) CENP-A maintenance during transcription and replication, and 3) the mechanisms that help preventing CENP-A localization at non-centromeric sites. We then give examples of how timely loading of new CENP-A to the centromere, maintenance of old CENP-A during S-phase and transcription, and removal of CENP-A at non-centromeric sites are coordinated and controlled by an intricate network of factors whose identity is slowly being unravelled. [ABSTRACT FROM AUTHOR]

Centromeres play essential roles in the faithful segregation of chromosomes. CENP-A, the centromere-specific histone H3 variant, and heterochromatin characterized by di- or tri-methylation of histone H3 9th lysine (H3K9) are the hallmarks of centromere chromatin. Contrary to the epigenetic marks, DNA sequences underlying the centromere region of chromosomes are not well conserved through evolution. However, centromeres consist of repetitive sequences in many eukaryotes, including animals, plants, and a subset of fungi, including fission yeast. Advances in long-read sequencing techniques have uncovered the complete sequence of human centromeres containing more than thousands of alpha satellite repeats and other types of repetitive sequences. Not only tandem but also inverted repeats are present at a centromere. DNA recombination between centromere repeats can result in gross chromosomal rearrangement (GCR), such as translocation and isochromosome formation. CENP-A chromatin and heterochromatin suppress the centromeric GCR. The key player of homologous recombination, Rad51, safeguards centromere integrity through conservative noncrossover recombination between centromere repeats. In contrast to Rad51-dependent recombination, Rad52-mediated single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) lead to centromeric GCR. This review summarizes recent findings on the role of centromere and recombination proteins in maintaining centromere integrity and discusses how GCR occurs at centromeres.

Oncogenes are increasingly recognized as important factors in the development and progression of cancer. Holliday Junction Recognition Protein (HJURP) is a highly specialized mitogenic protein that is a chaperone protein of histone H3. The HJURP gene is located on chromosome 2q37.1 and is involved in nucleosome composition in the mitotic region, forming a three-dimensional crystal structure with Centromere Protein A (CENP-A) and the histone 4 complex. HJURP is involved in the recruitment and assembly of centromere and kinetochore and plays a key role in stabilizing the chromosome structure of tumor cells, and its dysfunction may contribute to tumorigenesis. In the available studies HJURP is upregulated in a variety of cancer tissues and cancer cell lines and is involved in tumor proliferation, invasion, metastasis and immune response. In an in vivo model, overexpression of HJURP in most cancer cell lines promotes cell proliferation and invasiveness, reduces susceptibility to apoptosis, and promotes tumor growth. In addition, upregulation of HJURP was associated with poorer prognosis in a variety of cancers. These properties suggest that HJURP may be a possible target for the treatment of certain cancers. Various studies targeting HJURP as a prognostic and therapeutic target for cancer are gradually attracting interest and attention. This paper reviews the functional and molecular mechanisms of HJURP in a variety of tumor types with the aim of providing new targets for future cancer therapy.

The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation. [ABSTRACT FROM AUTHOR]

The centromere is a specialized DNA locus that ensures the faithful segregation of chromosomes during cell division. It does so by directing the assembly of an essential proteinaceous structure called the kinetochore. The centromere identity is primarily epigenetically defined by a nucleosome containing an H3 variant called CENP-A as well as by the interplay of several factors such as differential chromatin organization driven by CENP-A and H2A.Z, centromere-associated proteins, and post-translational modifications. At the centromere, CENP-A is not just a driving force for kinetochore assembly but also modifies the structural and dynamic properties of the centromeric chromatin, resulting in a distinctive chromatin organization. An additional level of regulation of the centromeric chromatin conformation is provided by post-translational modifications of the histones in the CENP-A nucleosomes. Further, H2A.Z is present in the regions flanking the centromere for heterochromatinization. In this review, we focus on the above-mentioned factors to describe how they contribute to the organization of the centromeric chromatin: CENP-A at the core centromere, post-translational modifications that decorate CENP-A, and the variant H2A.Z. [ABSTRACT FROM AUTHOR]

Precise chromosome segregation is essential for maintaining genomic stability, and its proper execution centers on the centromere, a chromosomal locus that mounts the kinetochore complex to mediate attachment of chromosomes to the spindle microtubules. The location of the centromere is epigenetically determined by a centromere-specific histone H3 variant, CENP-A. Many human cancers exhibit overexpression of CENP-A, which correlates with occurrence of aneuploidy in these malignancies. Centromeric targeting of CENP-A depends on its histone fold, but recent studies showed that the N-terminal tail domain (NTD) also plays essential roles. Here, we investigated implications of NTD in conferring aneuploidy formation when CENP-A is overexpressed in fission yeast. A series of mutant genes progressively lacking one amino acid of the NTD have been constructed for overexpression in wild-type cells using the intermediate strength nmt41 promoter. Constructs hosting disrupted GRANT (Genomic stability-Regulating site within CENP-A N-Terminus) motif in NTD results in growth retardation, aneuploidy, increased localization to the centromere, upregulated RNA polymerase II accessibility and transcriptional derepression of the repressive centromeric chromatin, suggesting that GRANT residues fine-tune centromeric CENP-A incorporation and restrict RNA polymerase II accessibility. This work highlighted the importance of CENP-A NTD, particularly the GRANT motif, in aneuploidy formation of overexpressed CENP-A in fission yeast. [ABSTRACT FROM AUTHOR]

Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability. [ABSTRACT FROM AUTHOR]

Background: Centromeric protein A (CENP-A), an essential protein involved in chromosomal segregation during cell division, is associated with several cancer types. However, its role in gliomas remains unclear. This study examined the clinical and prognostic significance of CENP-A in gliomas. Methods: Data of patients with glioma were collected from the Cancer Genome Atlas. Logistic regression, the Kruskal--Wallis test, and the Wilcoxon signed-rank test were performed to assess the relationship between CENP-A expression and clinicopathological parameters. The Cox regression model and Kaplan--Meier curve were used to analyze the association between CENP-A and survival outcomes. A prognostic nomogram was constructed based on Cox multivariate analysis. Gene set enrichment analysis (GSEA) was conducted to identify key CENP-A-related pathways and biological processes. Results: CENP-A was upregulated in glioma samples. Increased CENP-A levels were significantly associated with the world health organization (WHO) grade [Odds ratio (OR) = 49.88 (23.52-129.06) for grade 4 vs. grades 2 and 3], primary therapy outcome [OR = 2.44 (1.64--3.68) for progressive disease (PD) and stable disease (SD) vs. partial response (PR) and complete response (CR)], isocitrate dehydrogenase (IDH) status [OR = 13.76 (9.25-20.96) for wild-type vs. mutant], 1p/19q co-deletion [OR = 5.91 (3.95-9.06) for no codeletion vs. co-deletion], and age [OR = 4.02 (2.68--6.18) for > 60 vs. ≤ 60]. Elevated CENP-A expression was correlated with shorter overall survival in both univariate [hazard ratio (HR): 5.422; 95% confidence interval (CI): 4.044-7.271; p < 0.001] and multivariate analyses (HR: 1.967; 95% CI: 1.280-3.025; p < 0.002). GSEA showed enrichment of numerous cell cycle-and tumor-related pathways in the CENP-A high expression phenotype. The calibration plot and C-index indicated the favorable performance of our nomogram for prognostic prediction in patients with glioma. Conclusion: We propose a role for CENP-A in glioma progression and its potential as a biomarker for glioma diagnosis and prognosis. [ABSTRACT FROM AUTHOR]

Background: Recent advances demonstrate the role of chromatin regulators, including histone variants and histone chaperones, in cancer initiation and progression. Methods: Histone H3K4me3, histone variant centromere protein (CENP-A) and histone chaperones Holliday junction recognition protein (HJURP) as well as DAXX expression were examined immunohistochemically in 95 thymic epithelial tumor (TET) specimens. Our results were compared with the expression profile of DAXX, HJURP and CENP-A in gene expression profiling interactive analysis (GEPIA2). Results: The lymphocyte-poor B3- and C-type TETs were more frequently DAXX negative (p = 0.043). B3 and C-Type TETs showed higher cytoplasmic and nuclear CENP-A (p = 0.007 and p = 0.002) and higher cytoplasmic HJURP H-score (p < 0.001). Higher nuclear CENP-A and cytoplasmic HJURP expression was associated with advanced Masaoka–Koga stage (p = 0.048 and p < 0.001). A positive correlation between HJURP and CENP-A was also observed. The presence of cytoplasmic CENP-A expression was correlated with a favorable overall survival (p = 0.03). CENP-A overexpression in survival analysis of TCGA TETs showed similar results. H3K4me3 expression was not associated with any clinicopathological parameters. Conclusions: Our results suggest a significant interaction between CENP-A and HJURP in TETs. Moreover, we confirmed the presence of a cytoplasmic CENP-A immunolocalization, suggesting also a possible favorable prognostic value of this specific immunostaining pattern. [ABSTRACT FROM AUTHOR]

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position. [ABSTRACT FROM AUTHOR]

Chromosome instability (CIN) contributes to the development of many cancer. In this paper, we summarize our recent finding that a novel pathway by which FBW7 loss promotes Centromere Protein A (CENP-A) phosphorylation on Serine 18 through Cyclin E1/CDK2, therefore promoting CIN and tumorigenesis. Our finding demonstrates the importance of CENP-A post-translational modification on modulating centromere and mitotic functions in cancer.

CENP-A is a histone H3 variant key to epigenetic specification of mammalian centromeres. Using transient overexpression of CENP-A mutants, two recent reports in Developmental Cell proposed essential centromere functions for post-translational modifications of human CENP-A. Phosphorylation at Ser68 was proposed to have an essential role in CENP-A deposition at centromeres. Blockage of ubiquitination at Lys124 was proposed to abrogate localization of CENP-A to the centromere. Following gene inactivation and replacement in human cells, we demonstrate that CENP-A mutants that cannot be phosphorylated at Ser68 or ubiquitinated at Lys124 assemble efficiently at centromeres during G1, mediate early events in centromere establishment at an ectopic chromosomal locus, and maintain centromere function indefinitely. Thus, neither Ser68 nor Lys124 post-translational modification is essential for long-term centromere identity, propagation, cell-cycle-dependent deposition, maintenance, function, or mediation of early steps in centromere establishment.

Abstract RSF1, remodelling and spacing factor 1, is an important interphase centromere protein and is overexpressed in many types of cancers and correlated with poor overall survival. RSF1 has functions mainly in maintaining chromosome stability, facilitating DNA repair, maintaining the protein homeostasis of RSF1 and suppressing the transcription of some oncogenes when RSF1 protein is expressed at an optimal level; however, RSF1 overexpression facilitates drug resistance and cell cycle checkpoint inhibition to prompt cancer proliferation and survival. The RSF1 expression level and gene background are crucial for RSF1 functions, which may explain why RSF1 has different functions in different cancer types. This review summarizes the functional domains of RSF1, the overexpression status of RSF1 and SNF2H in cancer based on the TCGA and GTEX databases, the cancer-related functions of RSF1 in interacting with H2Aub, HDAC1, CENP-A, PLK1, ATM, CENP-S, SNF2H, HBX, BubR1, cyclin E1, CBP and NF-κB and the potential clinical value of RSF1, which will lay a theoretical foundation for the structural biology study of RSF1 and application of RSF1 inhibitors, truncated RSF1 proteins and SNF2H inhibitors in the treatment of RSF1-overexpressing tumours.

Abstract Background Mammalian centromeres are satellite-rich chromatin domains that execute conserved roles in kinetochore assembly and chromosome segregation. Centromere satellites evolve rapidly between species, but little is known about population-level diversity across these loci. Results We developed a k-mer based method to quantify centromere copy number and sequence variation from whole genome sequencing data. We applied this method to diverse inbred and wild house mouse (Mus musculus) genomes to profile diversity across the core centromere (minor) satellite and the pericentromeric (major) satellite repeat. We show that minor satellite copy number varies more than 10-fold among inbred mouse strains, whereas major satellite copy numbers span a 3-fold range. In contrast to widely held assumptions about the homogeneity of mouse centromere repeats, we uncover marked satellite sequence heterogeneity within single genomes, with diversity levels across the minor satellite exceeding those at the major satellite. Analyses in wild-caught mice implicate subspecies and population origin as significant determinants of variation in satellite copy number and satellite heterogeneity. Intriguingly, we also find that wild-caught mice harbor dramatically reduced minor satellite copy number and elevated satellite sequence heterogeneity compared to inbred strains, suggesting that inbreeding may reshape centromere architecture in pronounced ways. Conclusion Taken together, our results highlight the power of k-mer based approaches for probing variation across repetitive regions, provide an initial portrait of centromere variation across Mus musculus, and lay the groundwork for future functional studies on the consequences of natural genetic variation at these essential chromatin domains.

Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin. [ABSTRACT FROM AUTHOR]

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology. [ABSTRACT FROM AUTHOR]

Eukaryotes segregate their chromosomes during mitosis and meiosis by attaching chromosomes to the microtubules of the spindle so that they can be distributed into daughter cells. The complexity of centromeres ranges from the point centromeres of yeast that attach to a single microtubule to the more complex regional centromeres found in many metazoans or holocentric centromeres of some nematodes, arthropods and plants, that bind to dozens of microtubules per kinetochore. In vertebrates, the centromere is defined by a centromere specific histone variant termed Centromere Protein A (CENP-A) that replaces histone H3 in a subset of centromeric nucleosomes. These CENP-A nucleosomes are distributed on long stretches of highly repetitive DNA and interspersed with histone H3 containing nucleosomes. The mechanisms by which cells control the number and position of CENP-A nucleosomes is unknown but likely important for the organization of centromeric chromatin in mitosis so that the kinetochore is properly oriented for microtubule capture. CENP-A chromatin is epigenetically determined thus cells must correct errors in CENP-A organization to prevent centromere dysfunction and chromosome loss. Recent improvements in sequencing complex centromeres have paved the way for defining the organization of CENP-A nucleosomes in centromeres. Here we discuss the importance and challenges in understanding CENP-A organization and highlight new discoveries and advances enabled by recent improvements in the human genome assembly.

Human centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation. Without the continuing presence of CENP-A, CENP-B binding to alphoid DNA sequences becomes essential to preserve anchoring of CENP-C and the kinetochore to each centromere. Thus, there is a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA sequences bound by CENP-B in the maintenance of human chromosome segregation.

Background: The centromere is the special region on a chromosome, which serves as the site for assembly of kinetochore complex and is essential for maintaining genomic integrity. Neocentromeres are new centromeres that form on the non-centromeric regions of the chromosome when the natural centromere is disrupted or inactivated. Although neocentromeres lack the typical features found in centromeres, cells with neocentromeres divide normally during mitosis and meiosis. Neocentromeres not only arise naturally but their formation can also be induced experimentally. Therefore, neocentromeres are a great tool for studying functions and formation of centromeres. Objective: To study neocentromeres and use that knowledge to gain insights into the epigenetic regulation of canonical centromeres. Discussion: Here, we review the characteristics of naturally occurring centromeres and neocentromeres and those of experimentally induced neocentromeres. We also discuss the mechanism of centromere formation and epigenetic regulation of centromere function, which we learned from studying the neocentromeres. Although neocentromeres lack main features of centromeres, such as presence of repetitive ⍺-satellite DNA and pericentric heterochromatin, they behave quite similar to the canonical centromere, indicating the epigenetic nature of the centromere. Still, further investigation will help to understand the formation and maintenance of the centromere, and the correlation to human diseases. Conclusion: Neocentromeres helped us to understand the formation of canonical centromeres. Also, since neocentromeres are associated with certain cancer types, knowledge about them could be helpful to treat cancer. [ABSTRACT FROM AUTHOR]

Bi-oriented attachment of microtubules to the centromere is a pre-requisite for faithful chromosome segregation during mitosis. Budding yeast have point centromeres containing the cis -element proteins CDE-I, –II, and –III, which interact with trans -acting factors such as Cbf1, Cse4, and Ndc10. Our previous genetic screens, using a comprehensive library of histone point mutants, revealed that the TBS-I, –II, and –III regions of nucleosomes are required for faithful chromosome segregation. In TBS-III deficient cells, peri-centromeric nucleosomes containing the H2A.Z homolog Htz1 are lacking, however, it is unclear why chromosome segregation is defective in these cells. Here, we show that, in cells lacking TBS-III, both chromatin binding at the centromere and the total amount of some of the centromere proteins are reduced, and transcription through the centromere is up-regulated during M-phase. Moreover, the chromatin binding of Cse4, Mif2, Cbf1, Ndc10, and Scm3 was reduced upon ectopic transcription through the centromere in wild-type cells. These results suggest that transcription through the centromere displaces key centromere proteins and, consequently, destabilizes the interaction between centromeres and microtubules, leading to defective chromosome segregation. The identification of new roles for histone binding residues in TBS-III will shed new light on nucleosome function during chromosome segregation. [Display omitted] • Transcription through the centromere displaces key centromeric proteins. • Unbound centromeric proteins are degraded. • Histone chaperone CIA/Asf1 deposits Cse4 (CENP-A) at the centromere. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.