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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]

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]

The CENP‐A nucleosome is a key structure for kinetochore assembly. Once the CENP‐A nucleosome is established in the centromere, additional proteins recognize the CENP‐A nucleosome to form a kinetochore. CENP‐C and CENP‐N are CENP‐A binding proteins. We previously demonstrated that vertebrate CENP‐C binding to the CENP‐A nucleosome is regulated by CDK1‐mediated CENP‐C phosphorylation. However, it is still unknown how the phosphorylation of CENP‐C regulates its binding to CENP‐A. It is also not completely understood how and whether CENP‐C and CENP‐N act together on the CENP‐A nucleosome. Here, using cryo‐electron microscopy (cryo‐EM) in combination with biochemical approaches, we reveal a stable CENP‐A nucleosome‐binding mode of CENP‐C through unique regions. The chicken CENP‐C structure bound to the CENP‐A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP‐C residue. The stable CENP‐A‐CENP‐C complex excludes CENP‐N from the CENP‐A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1‐mediated CENP‐C phosphorylation. Synopsis: Phosphorylation of kinetochore protein CENP‐C regulates its binding to the CENP‐A nucleosome. Cryo‐EM reveals how CENP‐C phosphorylation regulates CENP‐A binding and provides insights into a dynamic kinetochore assembly mechanism during mitosis. The C‐terminal region of CENP‐C adopts a stable fold upon CENP‐A nucleosome binding.CDK1 phosphorylation of CENP‐C stabilizes the complex structure with the CENP‐A nucleosome via an intramolecular interaction.CENP‐C binds the RG loop of the CENP‐A nucleosome, which is recognized by another CENP‐A‐binding protein, CENP‐N.The stable CENP‐A‐ CENP‐C complex excludes CENP‐N from the CENP‐A nucleosome. [ABSTRACT FROM AUTHOR]

Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP‐A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP‐A (CENP‐AOFF/ON). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP‐B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP‐A deposition. Indeed, lack of CENP‐B favors neocentromere formation under selective pressure. Occasionally, CENP‐B triggers centromere re‐activation initiated by CENP‐C, but not CENP‐A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP‐A‐based epigenetic loop. Finally, we identify a population of CENP‐A‐negative, CENP‐B/C‐positive resting CD4+ T cells capable to re‐express and reassembles CENP‐A upon cell cycle entry, demonstrating the physiological importance of the genetic memory. Synopsis: Despite centromere position being epigenetically defined, all human centromeres are built on long stretches of repetitive DNA. This work defines a genetic feature in human cells that provides memory for maintenance of native human centromeres, but is also capable of occasionally initiating epigenetic definition of centromeres. Preexisting CENP‐A nucleosomes are not required to avert sliding of centromere position, or to define a specific number of CENP‐A molecules.CENP‐B bound to centromeric DNA provides memory for maintenance of human centromeres, to prevent movement of centromere position.CENP‐C, but not CENP‐A, is the initiation factor for de novo centromere formation via its recruitment to CENP‐B-bound centromeric DNA.A subpopulation of resting T lymphocytes lacks CENP‐A, but is capable of re‐assembling it upon activation and cell cycle entry. [ABSTRACT FROM AUTHOR]

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP‐A‐containing nucleosomes. To preserve centromere identity, CENP‐A must be escorted to centromeres by a CENP‐A‐specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP‐A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X‐ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP‐A histone chaperone, binds both CENP‐A and the centromere receptor CENP‐C without the requirement for the Mis18 complex. While an N‐terminal CAL1 fragment wraps around CENP‐A/H4 through multiple physical contacts, a C‐terminal CAL1 fragment directly binds a CENP‐C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP‐A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP‐C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP‐C dimer. Overall, by demonstrating how CAL1 binds CENP‐A/H4 and CENP‐C, we provide key insights into the minimalistic principles underlying centromere maintenance. Synopsis: Epigenetic maintenance of centromeres requires specific deposition of histone H3 variant CENP‐A. X‐ray crystallography explains how the evolutionary distinct histone chaperone CAL1 combines both CENP‐A recognition and centromere targeting activities of the respective mammalian counterparts, HJURP and Mis18 complex. Crystal structures of Drosophila CAL1 bound to CENP‐A/H4 dimers show that CAL1 employs evolutionarily conserved and adaptive interactions to recognise CENP‐A/H4.CAL1 binding shields key CENP‐A/H4 surfaces required for nucleosome assembly, a key requirement for preventing untimely CENP‐A loading.CAL1 interactions with its centromeric receptor CENP‐C involve a short helical CAL1 segment spanning residues 890–913, and the CENP‐C cupin domain.CAL1 binds CENP‐C at a site stabilised by cupin domain dimerization, thereby restricting binding to one CAL1 molecule per CENP‐C dimer. [ABSTRACT FROM AUTHOR]

Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP‐A. New CENP‐A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP‐A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP‐A assembly is mediated in part by the three‐protein Mis18 complex (Mis18α, Mis18β, M18BP1). Here, we show that Xenopus M18BP1 localizes to centromeres during metaphase—prior to CENP‐A assembly—by binding to CENP‐C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP‐C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP‐C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP‐A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP‐A nucleosome assembly in interphase. Synopsis: Epigenetic specification of vertebrate centromeres depends on assembly of CENP‐A nucleosomes during interphase. In frogs, phosphorylation‐dependent targeting of the CENP‐A recruitment factor M18BP1 already during mitosis is crucial for its later nuclear localization and assembly role. CDK phosphorylation on Xenopus laevis M18BP1 is required for its interaction with CENP‐C in metaphase.Mutations that disrupt metaphase M18BP1 localization result in defective interphase localization and CENP‐A assembly.M18BP1 exchange across the nuclear envelope is limited.Metaphase M18BP1 localization may promote its retention on chromatin during nuclear envelope assembly. Phosphorylation‐dependent targeting to CENP‐C already during mitosis is crucial for correct M18BP1 nuclear localization and M18BP1‐mediated CENP‐A nucleosome assembly in interphase. [ABSTRACT FROM AUTHOR]

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein-A (CENP-A). The deposition of new centromeric nucleosomes requires the CENP-A-specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3-like domain of HJURP binds a single CENP-A-histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP-A nucleosome. How an octameric CENP-A nucleosome forms from individual CENP-A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C-terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP-A is deposited. Dimerization of HJURP is essential for the deposition of new CENP-A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP-A binding. These data provide a mechanism whereby the CENP-A pre-nucleosomal complex achieves assembly of the octameric CENP-A nucleosome through the dimerization of the CENP-A chaperone HJURP. [ABSTRACT FROM AUTHOR]

The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere-specific histone H3 variant CenH3, originally called CENP-A, which replaces canonical histone H3 at centromeres. CenH3-chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3-chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly. [ABSTRACT FROM AUTHOR]

We show that Trichostatin A (TSA)-induced partial histone hyperacetylation causes a unidirectional shift in the position of a previously defined binding domain for the centromere-specific histone H3 homologue CENP-A at a human neocentromere. The shif [ABSTRACT FROM AUTHOR]

Centromere protein A (CENP-A) is an essential centromere-specific histone H3 homologue. Using combined chromatin immunoprecipitation and DNA array analysis, we have defined a 330 kb CENP-A binding domain of a 10q25.3 neocentromere found on the human marker chromosome mardel(10). This domain is situated adjacent to the 80 kb region identified previously as the neocentromere site through tower-resolution immunofluorescence/FISH analysis of metaphase chromosomes. The 330 kb CENP-A binding domain shows a depletion of histone H3, providing evidence for the replacement of histone H3 by CENP-A within centromere-specific nucleosomes. The DNA within this domain has a high AT-content comparable to that of a-satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surrounding region. FISH analysis indicates that the normal 10q25.3 genomic region replicates around mid-S phase. Neocentromere formation is accompanied by a replication time tag around but not within the CENP-A binding region, with this tag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful model for dissecting the functional domains of complex higher eukaryotic centromeres. [ABSTRACT FROM AUTHOR]