The centromeric histone H3 variant centromeric protein A (CENP-A), whose sequence is the least conserved among all histone variants, is responsible for specifying the location of the centromere. Here, we present a comprehensive study of CENP-A nucleosome arrays by cryo-electron tomography. We see that CENP-A arrays have different biophysical properties than canonical ones under low ionic conditions, as they are more condensed with a 20% smaller average nearest-neighbor distance and a 30% higher nucleosome density. We find that CENP-A nucleosomes have a predominantly crossed DNA entry/exit site that is narrowed on average by 8°, and they have a propensity to stack face to face. We therefore propose that CENP-A induces geometric constraints at the nucleosome DNA entry/exit site to bring neighboring nucleosomes into close proximity. This specific property of CENP-A may be responsible for generating a fundamental process that contributes to increased chromatin fiber compaction that is propagated under physiological conditions to form centromeric chromatin.

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

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.

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171-base-pair monomers at normal centromeres and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA-wrapping behavior, as do octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on conventional nucleosomes containing canonical histone H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centriole duplication occurs once per cell cycle and is controlled by Polo-like kinase 4 (Plk4). We showed previously that Plk4 phosphorylates itself to promote its degradation by the proteasome. Here we demonstrate that this autoregulated instability controls the abundance of endogenous Plk4. Preventing Plk4 autoregulation causes centrosome amplification, stabilization of p53, and loss of cell proliferation; moreover, suppression of p53 allows growth of cells carrying amplified centrosomes. Plk4 autoregulation thus guards against genome instability by limiting centrosome duplication to once per cell cycle.

Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat-containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1-containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2-4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases.

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.

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.

The histone H3 variant CENP-A is incorporated into nucleosomes that mark centromere location. We have recently reported that CENP-A nucleosomes, compared with their H3 counterparts, confer an altered nucleosome shape. Here, using a single-molecule fluorescence resonance energy transfer (FRET) approach with recombinant human histones and centromere DNA, we found that the nucleosome shape change directed by CENP-A is dominated by lateral passing of two DNA gyres (gyre sliding). A nonhistone centromere protein, CENP-C, binds and reshapes the nucleosome, sliding the DNA gyres back to positions similar to those in canonical nucleosomes containing conventional histone H3. The model that we generated to explain the CENP-A-nucleosome transition provides an example of a shape change imposed by external binding proteins and has notable implications for understanding of the epigenetic basis of the faithful inheritance of centromere location on chromosomes.

Centromeres are specified epigenetically, and the histone H3 variant CENP-A is assembled into the chromatin of all active centromeres. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to mark physically centromere location. Here we report the crystal structure of a subnucleosomal heterotetramer, human (CENP-A-H4)(2), that reveals three distinguishing properties encoded by the residues that comprise the CENP-A targeting domain (CATD; ref. 2): (1) a CENP-A-CENP-A interface that is substantially rotated relative to the H3-H3 interface; (2) a protruding loop L1 of the opposite charge as that on H3; and (3) strong hydrophobic contacts that rigidify the CENP-A-H4 interface. Residues involved in the CENP-A-CENP-A rotation are required for efficient incorporation into centromeric chromatin, indicating specificity for an unconventional nucleosome shape. DNA topological analysis indicates that CENP-A-containing nucleosomes are octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals. Our results indicate that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis.

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

Mammalian genomes are replicated in a cell type-specific order and in coordination with transcription and chromatin organization. Currently, single-cell replication studies require individual processing of sorted cells, yielding a limited number (<100) of cells. Here, we develop Kronos scRT, a software for single-cell Replication Timing (scRT) analysis. Kronos scRT does not require a specific platform or cell sorting, which allows investigating large datasets obtained from asynchronous cells. By applying our tool to published data as well as droplet-based single-cell whole-genome sequencing data generated in this study, we exploit scRT from thousands of cells for different mouse and human cell lines. Our results demonstrate that although genomic regions are frequently replicated around their population average RT, replication can occur stochastically throughout S phase. Altogether, Kronos scRT allows fast and comprehensive investigations of the RT programme at the single-cell resolution for both homogeneous and heterogeneous cell populations.

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.

Cytosolic DNA activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity, and cancer. cGAS is considered to be a sequence-independent DNA sensor with limited access to nuclear DNA because of compartmentalization. However, the nuclear envelope is a dynamic barrier, and cGAS is present in the nucleus. Here, we identify determinants of nuclear cGAS localization and activation. We show that nuclear-localized cGAS synthesizes cGAMP and induces innate immune activation of dendritic cells, although cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knockin mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent on LINE elements. The non-enzymatic N-terminal domain of cGAS determines nucleo-cytoplasmic localization, enrichment on centromeres, and activation of nuclear-localized cGAS. These results reveal a preferential functional association of nuclear cGAS with centromeres.

Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that whereas surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Furthermore, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.