m6A-modified cenRNA stabilizes CENPA to ensure centromere integrity in cancer cells.

m6A modification is best known for its critical role in controlling multiple post-transcriptional processes of the mRNAs. Here, we discovered elevated levels of m6A modification on centromeric RNA (cenRNA) in cancerous cells compared with non-cancerous cells. We then identified CENPA, an H3 variant, as an m6A reader of cenRNA. CENPA is localized at centromeres and is essential in preserving centromere integrity and function during mitosis. The m6A-modified cenRNA stabilizes centromeric localization of CENPA in cancer cells during the S phase of the cell cycle. Mutations of CENPA at the Leu61 and the Arg63 or removal of cenRNA m6A modification lead to loss of centromere-bound CENPA during S phase. This in turn results in compromised centromere integrity and abnormal chromosome separation and hinders cancer cell proliferation and tumor growth. Our findings unveil an m6A reading mechanism by CENPA that epigenetically governs centromere integrity in cancer cells, providing potential targets for cancer therapy.

Human Artificial Chromosomes that Bypass Centromeric DNA.

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.

Centromere/kinetochore is assembled through CENP-C oligomerization.

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

Something's gotta give at the centromeric chromatin foundation of the kinetochore.

Structures of the reconstituted human inner kinetochore complex by Pesenti et al. (2022) and Yatskevich et al. (2022) raise the question of whether it is the CENP-A nucleosome or the CCAN complex itself that provides the foundation for kinetochore assembly.

CENP-B-mediated DNA loops regulate activity and stability of human centromeres.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Genomic and Epigenetic Foundations of Neocentromere Formation.

Centromeres are essential to genome inheritance, serving as the site of kinetochore assembly and coordinating chromosome segregation during cell division. Abnormal centromere function is associated with birth defects, infertility, and cancer. Normally, centromeres are assembled and maintained at the same chromosomal location. However, ectopic centromeres form spontaneously at new genomic locations and contribute to genome instability and developmental defects as well as to acquired and congenital human disease. Studies in model organisms have suggested that certain regions of the genome, including pericentromeres, heterochromatin, and regions of open chromatin or active transcription, support neocentromere activation. However, there is no universal mechanism that explains neocentromere formation. This review focuses on recent technological and intellectual advances in neocentromere research and proposes future areas of study. Understanding neocentromere biology will provide a better perspective on chromosome and genome organization and functional context for information generated from the Human Genome Project, ENCODE, and other large genomics consortia.

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.

Direct observation of coordinated assembly of individual native centromeric nucleosomes.

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.

Argonaute protein CSR-1 restricts localization of holocentromere protein HCP-3, the C. elegans CENP-A homolog.

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.

MLL methyltransferases regulate H3K4 methylation to ensure CENP-A assembly at human centromeres.

The active state of centromeres is epigenetically defined by the presence of CENP-A interspersed with histone H3 nucleosomes. While the importance of dimethylation of H3K4 for centromeric transcription has been highlighted in various studies, the identity of the enzyme(s) depositing these marks on the centromere is still unknown. The MLL (KMT2) family plays a crucial role in RNA polymerase II (Pol II)-mediated gene regulation by methylating H3K4. Here, we report that MLL methyltransferases regulate transcription of human centromeres. CRISPR-mediated down-regulation of MLL causes loss of H3K4me2, resulting in an altered epigenetic chromatin state of the centromeres. Intriguingly, our results reveal that loss of MLL, but not SETD1A, increases co-transcriptional R-loop formation, and Pol II accumulation at the centromeres. Finally, we report that the presence of MLL and SETD1A is crucial for kinetochore maintenance. Altogether, our data reveal a novel molecular framework where both the H3K4 methylation mark and the methyltransferases regulate stability and identity of the centromere.

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.

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.

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.

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.

CENP-I directly targets centromeric DNA to support CENP-A deposition and centromere maintenance.

The enrichment of histone H3 variant CENP-A is the epigenetic mark of centromere and initiates the assembly of the kinetochore at centromere. The kinetochore is a multi-subunit complex that ensures accurate attachment of microtubule centromere and faithful segregation of sister chromatids during mitosis. As a subunit of kinetochore, CENP-I localization at centromere also depends on CENP-A. However, whether and how CENP-I regulates CENP-A deposition and centromere identity remains unclear. Here, we identified that CENP-I directly interacts with the centromeric DNA and preferentially recognizes AT-rich elements of DNA via a consecutive DNA-binding surface formed by conserved charged residues at the end of N-terminal HEAT repeats. The DNA binding-deficient mutants of CENP-I retained the interaction with CENP-H/K and CENP-M, but significantly diminished the centromeric localization of CENP-I and chromosome alignment in mitosis. Moreover, the DNA binding of CENP-I is required for the centromeric loading of newly synthesized CENP-A. CENP-I stabilizes CENP-A nucleosomes upon binding to nucleosomal DNA instead of histones. These findings unveiled the molecular mechanism of how CENP-I promotes and stabilizes CENP-A deposition and would be insightful for understanding the dynamic interplay of centromere and kinetochore during cell cycle.

The ins and outs of CENP-A: Chromatin dynamics of the centromere-specific histone.

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.

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.

The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability.

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 non-centromeric 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 imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) 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 CENP-A 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.

DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide.

Studies of genome regulation routinely use high-throughput DNA sequencing approaches to determine where specific proteins interact with DNA, and they rely on DNA amplification and short-read sequencing, limiting their quantitative application in complex genomic regions. To address these limitations, we developed directed methylation with long-read sequencing (DiMeLo-seq), which uses antibody-tethered enzymes to methylate DNA near a target protein's binding sites in situ. These exogenous methylation marks are then detected simultaneously with endogenous CpG methylation on unamplified DNA using long-read, single-molecule sequencing technologies. We optimized and benchmarked DiMeLo-seq by mapping chromatin-binding proteins and histone modifications across the human genome. Furthermore, we identified where centromere protein A localizes within highly repetitive regions that were unmappable with short sequencing reads, and we estimated the density of centromere protein A molecules along single chromatin fibers. DiMeLo-seq is a versatile method that provides multimodal, genome-wide information for investigating protein-DNA interactions.

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.