{"gene":"CENPA","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2006,"finding":"CENP-A nucleosomes directly recruit a proximal nucleosome-associated complex (NAC) comprising CENP-M, CENP-N, CENP-T, CENP-U(50), CENP-C, and CENP-H. Assembly of the NAC requires CENP-M, CENP-N, and CENP-T. FACT and nucleophosmin-1 are stably recruited to CENP-A nucleosomes independently of the NAC. Seven distal CAD components (CENP-K, -L, -O, -P, -Q, -R, -S) assemble on the NAC. Disruption of the NAC causes chromosome alignment and segregation errors.","method":"Mass spectrometry, co-immunoprecipitation, RNAi depletion with mitotic phenotype readout","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, MS interactome, RNAi with defined cellular phenotype; foundational study replicated extensively","pmids":["16622419"],"is_preprint":false},{"year":2000,"finding":"Cenpa null mouse embryos fail to survive beyond E6.5 with severe mitotic defects. Cenpa is required for kinetochore targeting of Cenpc; loss of Cenpa causes diffuse Cenpb foci and complete loss of discrete Cenpc centromere signal, indicating Cenpa organizes centromeric chromatin and is epistatic to Cenpc localization.","method":"Gene targeting (knockout mouse), immunofluorescence microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular and organismal phenotype, replicated in subsequent studies","pmids":["10655499"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of HJURP bound to a CENP-A–H4 heterodimer shows HJURP binds a pre-nucleosomal CENP-A–H4 heterodimer; the C-terminal β-sheet domain of HJURP caps the DNA-binding surface of CENP-A–H4, preventing spontaneous DNA association. A novel CENP-A-specific surface distinguishes it from H3 in HJURP binding.","method":"X-ray crystallography, in vitro binding assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical validation; independently corroborated by other structural studies","pmids":["21478274"],"is_preprint":false},{"year":2015,"finding":"CENP-C binds CENP-A nucleosomes and reshapes the octameric histone core: it rigidifies both surface and internal nucleosome structure and modulates terminal DNA to match the loose DNA wrap characteristic of native centromeric CENP-A nucleosomes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, demonstrating collaboration between CENP-C and CENP-A in maintaining centromere identity.","method":"Purified-component reconstitution, FRET, single-molecule PIFE, auxin-inducible degron depletion","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with multiple orthogonal biophysical methods plus in vivo depletion; single rigorous study","pmids":["25954010"],"is_preprint":false},{"year":2014,"finding":"Polo-like kinase 1 (Plk1) is required to initiate CENP-A deposition in human cells by promoting localization of the Mis18 complex to centromeres. CDK inhibits Mis18 complex assembly. A two-step Plk1/CDK regulatory paradigm controls cell-cycle-restricted CENP-A deposition; bypassing both steps uncouples deposition from cell-cycle progression and causes mitotic defects.","method":"RNAi/chemical inhibition, FRAP, auxin-inducible degron, epistasis with phosphomutants","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, genetic epistasis, functional rescue experiments in human cells","pmids":["25036634"],"is_preprint":false},{"year":2014,"finding":"Cdk1 phosphorylates CENP-A at Ser68 during early mitosis, which eliminates binding of CENP-A to its assembly chaperone HJURP, thereby preventing premature centromeric loading before mitotic exit. PP1α dephosphorylates Ser68 at mitotic exit, enabling HJURP-mediated centromeric deposition.","method":"In vitro kinase assay, phosphomutant rescue, co-immunoprecipitation, cell synchronization","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay plus phosphomutant cell biology with mechanistic follow-up; multiple methods","pmids":["25556658"],"is_preprint":false},{"year":2015,"finding":"CUL4A-RBX1-COPS8 E3 ligase monoubiquitylates CENP-A on K124, which is required for CENP-A interaction with chaperone HJURP and for centromere localization. The K124R mutation reduces HJURP binding and abolishes centromere loading; addition of monoubiquitin to K124R restores both HJURP interaction and centromere localization.","method":"In vitro ubiquitylation assay, co-immunoprecipitation, CRISPR knockin, immunofluorescence","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — biochemical reconstitution of ubiquitylation, rescue with monoubiquitin fusion, CRISPR knockin in vivo validation","pmids":["25727006"],"is_preprint":false},{"year":2018,"finding":"HJURP transiently associates with pre-existing CENP-A nucleosomes during S phase and is required for CENP-A nucleosome inheritance through DNA replication. HJURP co-purifies with the MCM2-7 helicase complex, and MCM2 and HJURP simultaneously bind CENP-A, defining a mechanism for retaining CENP-A during replication fork passage.","method":"BioID proximity labeling, co-immunoprecipitation, conditional depletion (auxin), quantitative imaging","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — BioID plus reciprocal Co-IP plus auxin-inducible depletion with defined S-phase phenotype; multiple orthogonal methods","pmids":["30293838"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of the human inner kinetochore CCAN complex bound to a CENP-A nucleosome reconstituted on α-satellite DNA show that CCAN forms edge-on contacts with the CENP-A nucleosome; a linker DNA segment threads through the central CENP-LN channel; CENP-TWSX partially wraps linker DNA. This topological entrapment of linker DNA by CCAN provides a mechanism for kinetochores to withstand spindle forces.","method":"Cryo-electron microscopy, reconstitution of CENP-A nucleosome on α-satellite DNA","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with fully reconstituted complex","pmids":["35420891"],"is_preprint":false},{"year":2016,"finding":"Cell-cycle control of CENP-A assembly requires two targets: a single Cdk phosphorylation site in licensing factor M18BP1 and a cyclin A binding site in HJURP. Simultaneous mutation of both sites completely uncouples CENP-A assembly from cell-cycle phase, allowing assembly under high Cdk activity indistinguishable from G1 assembly. Cdk-mediated inhibition works by sequestering active factors away from the centromere.","method":"Phosphomutant expression, quantitative live-cell imaging, CENP-A incorporation assays, cell-cycle staging","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with phosphomutants, multiple orthogonal methods, complete uncoupling from cell cycle demonstrated","pmids":["28017591"],"is_preprint":false},{"year":2005,"finding":"In CENP-A-depleted chicken DT40 cells, inner kinetochore proteins CENP-I, CENP-H, and CENP-C and outer components Nuf2/Hec1, Mad2, and CENP-E are mislocalised, whereas BubR1 and INCENP are efficiently recruited. CENP-A-depleted cells show chromosome congression defects and a specific defect in maintaining BubR1 at kinetochores under checkpoint activation, indicating CENP-A is required for a mitotic-checkpoint-competent kinetochore.","method":"Conditional gene disruption (DT40), immunofluorescence, epistasis of kinetochore component localization","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with systematic kinetochore protein localization analysis; multiple components tested","pmids":["15870271"],"is_preprint":false},{"year":2001,"finding":"CENP-A is phosphorylated at a serine residue (equivalent to H3 Ser10) in a cell-cycle-dependent manner distinct from histone H3. CENP-A phosphorylation begins in prophase, peaks in prometaphase, and disappears in telophase—after both pericentric and genome-wide H3 phosphorylation. Prekinetochore duplication (CENP-A foci doubling) occurs before complete H3 phosphorylation in G2.","method":"Phospho-specific antibody ELISA, western blot, quantitative immunocytochemistry","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-specific antibody with multiple readout methods in single lab; kinase not identified in this paper","pmids":["11171370"],"is_preprint":false},{"year":2009,"finding":"CENP-A is rapidly recruited to DNA double-strand breaks (DSBs) in human and mouse cells, along with centromere-associated proteins CENP-N, CENP-T, and CENP-U. The centromere-targeting domain (CATD) of CENP-A is necessary and sufficient for recruitment to DSBs. CENP-A accumulation at breaks is enhanced by active NHEJ but does not require DNA-PKcs, Ligase IV, or H2AX.","method":"Multiphoton laser microirradiation, I-SceI endonuclease-induced DSBs, CENP-A domain truncation/mutation, immunofluorescence","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with domain mapping; single lab, multiple complementary approaches","pmids":["19717431"],"is_preprint":false},{"year":2014,"finding":"CENP-A overexpression leads to ectopic enrichment at sites of active histone turnover in a heterotypic CENP-A/H3.3–H4 tetramer. Ectopic localization depends on H3.3 chaperone DAXX rather than the dedicated CENP-A chaperone HJURP. Ectopic CENP-A nucleosomes occlude CTCF binding and confer DNA damage tolerance, both dependent on DAXX.","method":"ChIP-seq, co-immunoprecipitation, RNAi knockdown, CTCF binding assay, cell survival assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-seq plus Co-IP plus RNAi epistasis plus functional assays; multiple orthogonal methods identifying DAXX as chaperone for ectopic CENP-A","pmids":["24530302"],"is_preprint":false},{"year":2012,"finding":"Human CENP-A nucleosomes undergo cell-cycle-dependent structural transitions in vivo: they exist as tetramers (half-nucleosomes) after replication and convert to octamers before the next replication, accompanied by reversible chaperone binding, chromatin fiber folding changes, and histone fold domain modifications on CENP-A and H4.","method":"AFM, mass spectrometry of PTMs, chromatin fractionation, cell-cycle synchronization","journal":"Cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biophysical and biochemical methods in single lab; octameric structure contested by other papers (see PMID:28235947)","pmids":["22817894"],"is_preprint":false},{"year":2017,"finding":"Human centromeric CENP-A chromatin is exclusively an octameric nucleosome at all cell-cycle phases (G1 and G2), containing equimolar CENP-A, H2A, H2B, and H4 with no H3. CENP-A nucleosomes protect DNA lengths centered on 133 bp, consistent with octamers with DNA unwrapping at entry and exit. Solid-state nanopore analyses confirm nucleosomal size.","method":"ChIP-seq with new reference models, MNase protection assay, nanopore analysis, quantitative mass spectrometry","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal structural and genomic methods; contradicts tetramer model with rigorous controls across cell-cycle phases","pmids":["28235947"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structures of centromeric tri-nucleosomes show that H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture with outward-facing linker DNA path between nucleosomes, distinct from H3-H3-H3 tri-nucleosomes with inward-facing DNA. This exposes the CENP-A nucleosome to solvent in condensed chromatin.","method":"Cryo-electron microscopy, tri-nucleosome reconstitution","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure of reconstituted complex; single study but high-resolution structural method","pmids":["31711756"],"is_preprint":false},{"year":2018,"finding":"Aurora A kinase phosphorylates CENP-A at serine 7 (S7) at inner centromeres in mitosis, protecting bioriented chromosomes against cohesion fatigue. Expression of non-phosphorylatable CENP-A S7A weakens sister chromatid cohesion only when centromeres are under spindle tension. Aurora A is recruited to centromeres in a Bub1-dependent manner.","method":"Phosphomutant CENP-A expression, chromosome cohesion assays, Aurora A inhibition, immunofluorescence","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphomutant phenotype with kinase inhibition and epistasis; single lab; note that PMID:30635586 disputes S7 phosphorylation controlling centromere function","pmids":["29760389"],"is_preprint":false},{"year":2019,"finding":"Phosphorylation of CENP-A on serine 7 is dispensable for correct CENP-C recruitment, faithful chromosome segregation, and long-term cell viability, as demonstrated by gene targeting at both endogenous CENP-A alleles and gene replacement with S7A in human cells.","method":"Gene targeting at endogenous loci (CRISPR), gene replacement, immunofluorescence, live-cell imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — allelic replacement at endogenous locus with rigorous controls; directly contradicts prior claim about S7 phosphorylation","pmids":["30635586"],"is_preprint":false},{"year":2019,"finding":"CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail. The CENP-C central region (CENP-C_CR) binds the CENP-A nucleosome with high affinity via an extended hydrophobic area involving CENP-A V532 and V533. CENP-C binding further loosens terminal DNA wrapping (through H2A C-tail destabilization) and rigidifies the H4 N-terminal tail in a conformation favoring H4K20 monomethylation.","method":"Cryo-EM, in vitro binding assays, nucleosome reconstitution, mutagenesis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus in vitro biochemistry plus mutagenesis identifying specific residues; multiple orthogonal methods","pmids":["31475439"],"is_preprint":false},{"year":2019,"finding":"CDK1 phosphorylates CENP-C in its C-terminal CENP-A binding motif, which facilitates CENP-C binding to the CENP-A nucleosome in vitro and in vivo. This phosphorylation promotes CENP-C kinetochore localization during mitosis. The CENP-A–CENP-C interaction is critical for long-term cell viability in human RPE-1 cells.","method":"In vitro kinase assay, co-immunoprecipitation, phosphomutant rescue, immunofluorescence, cell viability assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay plus in vivo phosphomutant analysis plus cell viability; multiple orthogonal methods","pmids":["31676716"],"is_preprint":false},{"year":2015,"finding":"CENP-B forms a stable complex with the CENP-A nucleosome in vitro, preferentially when the CENP-B box DNA is located at the proximal edge of the nucleosome. The CENP-B DNA-binding domain specifically interacts with the CENP-A–H4 complex (but not H3.1–H4). In vivo, CENP-B binding near the CENP-A nucleosome substantially stabilizes CENP-A on alphoid DNA.","method":"In vitro nucleosome reconstitution, pulldown assay, in vivo chromatin stability assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution with defined position-dependent binding plus in vivo validation","pmids":["25916850"],"is_preprint":false},{"year":2018,"finding":"Centromeric transcription by RNA Pol II temporally coincides with de novo dCENP-A deposition in Drosophila. Short inhibition of transcription impairs CENP-A incorporation into stable (salt-resistant) chromatin but does not prevent initial targeting to centromeres, revealing two stability states of newly loaded CENP-A: a salt-sensitive association and a salt-resistant incorporated form. Transcription-mediated chromatin remodeling drives the transition to fully incorporated nucleosomes.","method":"Drosophila tissue culture, transcription inhibitor treatment, SNAP-tag pulse-chase, chromatin fractionation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — novel dCENP-A loading system with pharmacological and genetic dissection; multiple orthogonal assays","pmids":["29626011"],"is_preprint":false},{"year":2020,"finding":"Spt6, a histone chaperone and transcription elongation factor, prevents loss of old CENP-A nucleosomes during centromeric transcription. Spt6 directly binds dCENP-A in vitro; phosphomimetic dCENP-A mutants reduce Spt6 association, and non-phosphorylatable dCENP-A accumulates at centromeres. Spt6 acts as a conserved CENP-A maintenance factor in both Drosophila and human cells.","method":"Co-immunoprecipitation/direct binding assay, SNAP-tag pulse-chase, phosphomutant analysis, immunofluorescence","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated, conserved across two species, phosphomutant epistasis, multiple orthogonal approaches","pmids":["32522980"],"is_preprint":false},{"year":2021,"finding":"CENP-A chromatin protects centromeric alpha-satellite DNA from replication stress during S phase. Rapid CENP-A removal specifically in S phase causes R-loop accumulation with increased centromeric transcripts, impaired replication fork progression, recombination at alpha-satellites, and anaphase bridges leading to chromosome breakage and translocations at centromeric regions.","method":"Auxin-inducible degron for cell-cycle-specific CENP-A removal, DNA-RNA immunoprecipitation, DNA fiber assay, cytogenetics","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-cycle-specific conditional depletion with multiple orthogonal readouts; mechanistically links CENP-A to R-loop suppression and replication fork protection","pmids":["33653953"],"is_preprint":false},{"year":2016,"finding":"CENP-A ubiquitylation (K124) is inherited between cell divisions through CENP-A dimerization. Pre-existing ubiquitylated CENP-A is necessary for recruitment of newly synthesized CENP-A to the centromere. In vivo and in vitro experiments with dimerization mutants show that inheritance of K124 ubiquitylation requires CENP-A dimerization. Overexpression of monoubiquitin-fused CENP-A induces neocentromeres at non-centromeric regions.","method":"In vivo and in vitro dimerization mutant analysis, co-immunoprecipitation, immunofluorescence, ectopic CENP-A expression","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dimerization mutants plus biochemical assays in single lab; builds on prior K124 ubiquitylation work","pmids":["27052173"],"is_preprint":false},{"year":2019,"finding":"CENP-A HJURP nucleosome clusters form rosette-like structures (~250–300 nm) around HJURP during G1 phase. 2D/3D super-resolution microscopy shows HJURP localizes to the center of these rosettes and acts as a nucleation point for CENP-A deposition, providing structural insight into centromeric chromatin organization during loading.","method":"Super-resolution microscopy (STORM/PALM), co-localization analysis, segmentation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by super-resolution with HJURP as nucleation point; single lab, imaging-based","pmids":["31570711"],"is_preprint":false},{"year":2014,"finding":"Cell-cycle-dependent recruitment of HJURP to centromeres depends on timely CDK-mediated phosphorylation of HJURP. A non-phosphorylatable HJURP mutant localizes prematurely to centromeres in S and G2, causing premature CENP-A loading and cell-cycle delays. Once at centromeres, HJURP promotes CENP-A deposition through a unique DNA-binding domain.","method":"Phosphomutant expression, cell-cycle synchronization, immunofluorescence, DNA binding assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphomutant epistasis, DNA-binding domain mapping, multiple cell-cycle readouts","pmids":["25001279"],"is_preprint":false},{"year":2015,"finding":"In a cell-free CENP-A assembly system, two distinct domains of CENP-A within existing nucleosomes are required for new CENP-A assembly. CENP-A nucleosomes recruit CENP-C and M18BP1 independently, and CENP-C recruitment depends on the density of underlying CENP-A nucleosomes.","method":"Cell-free CENP-A assembly system (Xenopus extract), domain truncation analysis, chromatin binding assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution with domain-mapping; identifies two independent CENP-A-to-assembly-factor pathways","pmids":["26076692"],"is_preprint":false},{"year":2017,"finding":"At yeast centromeres, Cse4/CENP-A histone-fold domain interacts with inner kinetochore protein Mif2/CENP-C. Mif2 contacts one side of the nucleosome dyad engaging both Cse4 residues and AT-rich centromeric DNA through a contiguous DNA- and histone-binding domain (DHBD) containing the CENP-C motif, an AT hook, and RK clusters. Human CENP-C has two related DHBDs with preference for AT-rich DNA.","method":"Biochemical binding assays, mutagenesis, ChIP, structural modeling","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical reconstitution plus mutagenesis plus ChIP; identifies dual nucleosome-binding mechanism","pmids":["29074736"],"is_preprint":false},{"year":2002,"finding":"PARP-2 co-immunoprecipitates with CENP-A (Cenpa) and CENP-B (Cenpb) at active centromeres in a cell-cycle-dependent manner (accumulating prometaphase/metaphase, disassociating by telophase). PARP-2 does not interact with CENP-C. PARP-2 centromere binding is sequence-independent and enhanced by microtubule-inhibiting drugs.","method":"Co-immunoprecipitation, immunofluorescence, pseudodicentric chromosome analysis","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus cell-cycle localization; interaction with CENP-A established but functional mechanism less defined","pmids":["12217960"],"is_preprint":false},{"year":2012,"finding":"In Saccharomyces cerevisiae, Cse4/CENP-A (centromeric H3 variant) is methylated on arginine 37 (R37). Absence of R37 methylation reduces levels of Mtw1/MIND and Ctf19 complex components at the centromere (but not Cse4 itself), causing growth defects, G2/M arrest, and chromosome segregation errors. This modification regulates recruitment of linker kinetochore components.","method":"Mass spectrometry, methylation mutants, genetic epistasis with kinetochore mutants, ChIP","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification plus genetic epistasis plus ChIP in yeast; single lab","pmids":["22615363"],"is_preprint":false},{"year":2017,"finding":"CENPT bridges adjacent CENPA nucleosomes on young human α-satellite dimers. Sequential ChIP-seq shows CENPT is centered between two well-positioned CENPA nucleosomes over the CENPB box, forming a CENPA/CENPB/CENPC/CENPT complex nuclease-protected over an α-satellite dimer. CENPT interacts with CENPB/CENPC complex rather than H3 nucleosomes in vivo.","method":"Comparative and sequential ChIP-seq, base-pair-resolution genomic readout of protein-protein interactions","journal":"Genome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — sequential ChIP-seq with base-pair resolution; single lab; contradicts prior model but uses rigorous method","pmids":["27384170"],"is_preprint":false},{"year":2024,"finding":"CENP-A functions as an m6A reader of centromeric RNA (cenRNA): m6A-modified cenRNA stabilizes centromeric localization of CENP-A during S phase. Mutations at CENP-A Leu61 and Arg63 abolish cenRNA binding and cause loss of centromere-bound CENP-A during S phase, compromised centromere integrity, and abnormal chromosome separation.","method":"m6A methylation mapping, CENP-A mutagenesis, RNA-protein binding assays, live-cell imaging, tumor xenograft","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct biochemical demonstration of RNA-binding by CENP-A with specific residues identified, functional rescue assays, in vivo and in vitro validation","pmids":["39305902"],"is_preprint":false},{"year":2021,"finding":"CENP-A overexpression causes CIN with lagging chromosomes and micronuclei due to reduced localization of kinetochore proteins, resulting in defective kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE also reduces cell adhesion gene expression and increases invasion.","method":"Inducible overexpression, kinetochore protein quantification by immunofluorescence, live-cell imaging, xenograft","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative kinetochore protein analysis plus live-cell imaging in defined overexpression system; single lab","pmids":["33620383"],"is_preprint":false},{"year":2021,"finding":"CENPA overexpression promotes prostate cancer cell growth through a non-centromeric function as a transcriptional regulator that modulates expression of proliferation, cell-cycle, and centromere/kinetochore genes, as revealed by integrated ChIP-seq and RNA-seq experiments.","method":"ChIP-seq, RNA-seq, gain/loss-of-function experiments, tissue microarray","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus RNA-seq identifies genomic binding and gene regulation; single lab; non-centromeric transcriptional function","pmids":["32371391"],"is_preprint":false},{"year":2021,"finding":"CENPA recruits histone acetyltransferase GCN-5 to the promoter of KPNA2 to induce transcription activation in colon cancer cells. This non-centromeric transcriptional activity of CENPA promotes glycolysis and growth. H3K27ac modification is detected at the KPNA2 promoter upon CENPA binding.","method":"ChIP assay, co-immunoprecipitation, RNAi knockdown, rescue experiments, metabolic assays","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus Co-IP plus functional rescue; single lab demonstrating GCN-5 recruitment mechanism","pmids":["34508688"],"is_preprint":false},{"year":2021,"finding":"Phosphorylation of CENP-A Ser68 primes polyubiquitin-mediated proteasomal degradation of CENP-A during mitotic phase. DCAF11 (WDR23) is the E3 ligase mediating this polyubiquitination via K49 and K124. Mutation of K49R/K124R or DCAF11 deletion abrogates proper degradation and causes CENP-A mislocalization.","method":"In vivo ubiquitination assay, proteasome inhibitor treatment, DCAF11 depletion/knockout, immunofluorescence","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identification of E3 ligase with site-specific mutations plus depletion; single lab","pmids":["34758320"],"is_preprint":false},{"year":2023,"finding":"HJURP phosphorylation by CDK prevents HJURP interaction with CENP-C in metaphase, blocking delivery of soluble CENP-A to centromeres. Non-phosphorylatable HJURP constitutively binds CENP-C in metaphase but is insufficient for new CENP-A assembly. M18BP1.S subunit of the Mis18 complex competitively inhibits HJURP access to CENP-C. Two inhibitory activities together repress CENP-A assembly in metaphase.","method":"X. laevis egg extract cell-free assembly system, phosphomutant analysis, co-immunoprecipitation, competitive binding assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution system with defined biochemical pathway, phosphomutant analysis, competitive binding; comprehensive mechanistic dissection","pmids":["37141119"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structure of chicken CENP-A nucleosome bound to ggKNL2 (Mis18 complex component with CENP-C-like motif) shows ggKNL2 simultaneously recognizes the CENP-A C-terminal tail and the RG-loop using its CENP-C-like motif to distinguish CENP-A from H3. ggKNL2 changes its centromere binding partner during cell cycle progression.","method":"Cryo-EM structure, biochemical binding assays, cell biology validation, cell-cycle analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure plus biochemical and cell-biology validation; identifies dual recognition mechanism","pmids":["36744604"],"is_preprint":false},{"year":2023,"finding":"CENP-A and CENP-B collaborate to establish an open centromeric chromatin state. CENP-A incorporation increases chromatin fiber dynamics. This increased dynamics allow CENP-B DNA access; bound CENP-B further opens chromatin fiber structure and induces nucleosomal DNA unwrapping. Removal of CENP-A increases CENP-B mobility in cells.","method":"Single-molecule fluorescence, cryo-EM, FRAP (in cells), chromatin fiber reconstitution","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule fluorescence plus cryo-EM plus in-cell FRAP; multiple orthogonal methods demonstrating cooperative chromatin remodeling","pmids":["38086807"],"is_preprint":false},{"year":2024,"finding":"DNAJC9 (a J-domain protein for H3-H4 folding) restricts CENP-A mislocalization. Its depletion promotes CENP-A interaction with MCM2, which drives CENP-A deposition at ectopic sites. H3.3 depletion also causes CENP-A mislocalization. This defines MCM2 as a driver of ectopic CENP-A deposition when H3-H4 supply chains are disrupted.","method":"Genome-wide RNAi screen, global interactome analysis, MCM2 depletion epistasis, immunofluorescence","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen plus interactome plus epistasis; single lab, multiple methods","pmids":["38600242"],"is_preprint":false},{"year":2016,"finding":"Diaphanous formin mDia2 is required for stable replenishment of new CENP-A at centromeres in G1. Depletion of mDia2 causes prolonged centromere association of HJURP. mDia2 acts downstream of the MgcRacGAP-dependent small GTPase pathway in regulating CENP-A nucleosome assembly. Nuclear localization of mDia2 is required for this function.","method":"Quantitative imaging, pulse-chase, constitutively active mutant rescue, RNAi epistasis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pulse-chase plus epistasis experiments; single lab; pathway position established","pmids":["27185834"],"is_preprint":false},{"year":2019,"finding":"Quiescent cells (G0-arrested and prophase I-arrested oocytes) actively and continuously incorporate new CENP-A at centromeres using the canonical CENP-A deposition machinery. Plk1 is required specifically for G1 (not quiescent) CENP-A deposition, while transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence reduces CENP-A levels and perturbs chromosome segregation upon re-entry into division.","method":"Pulse-chase labeling, chemical inhibitors (Plk1/transcription), conditional depletion, quantitative imaging","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pulse-chase plus genetic/chemical dissection in multiple quiescent cell types; single lab","pmids":["31422918"],"is_preprint":false},{"year":2017,"finding":"CENP-A acetylation at K124 causes tightening of the histone core and diminishes CENP-C binding (by computational modeling and CENP-A K124Q/K124A mutants). CENP-A K124 switches from acetylation at G1/S to monomethylation during early/mid-S phase. The HAT p300 is implicated in K124 acetylation. K124 mutations alter centromeric replication timing and cause modest increases in mitotic errors.","method":"Computational modeling, in vivo phosphomutant analysis, mass spectrometry of native CENP-A, HAT inhibitor experiments","journal":"Epigenetics & chromatin","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification of modification switching plus mutant cell biology; single lab, some computational predictions included","pmids":["28396698"],"is_preprint":false}],"current_model":"CENP-A is a centromere-specific histone H3 variant that epigenetically marks centromere identity by forming octameric nucleosomes with loosely wrapped DNA; these nucleosomes are assembled during late mitosis/early G1 in a cell-cycle-controlled process requiring the dedicated chaperone HJURP (which binds a pre-nucleosomal CENP-A–H4 heterodimer), the Mis18 licensing complex (recruited by Plk1), and CDK inhibition of HJURP and M18BP1 in other cell-cycle phases; Ser68 phosphorylation by Cdk1 prevents premature HJURP binding, while K124 monoubiquitylation by CUL4A-RBX1-COPS8 is required for HJURP interaction and centromere loading; once incorporated, CENP-A nucleosomes are stabilized by CENP-C (which reshapes and rigidifies the nucleosome), by Spt6-mediated protection during centromeric transcription, and by HJURP-MCM2-mediated retention during DNA replication; CENP-A nucleosomes directly recruit the CCAN (via CENP-C and CENP-N binding specific CENP-A residues) and the outer kinetochore, are recognized as m6A-modified centromeric RNA readers for S-phase centromere stability, and collaborate with CENP-B to create an open centromeric chromatin state accessible to kinetochore factors."},"narrative":{"mechanistic_narrative":"CENP-A is a centromere-specific histone H3 variant that epigenetically marks centromere identity and is essential for building a functional kinetochore; its loss in mouse embryos causes early lethality with mitotic catastrophe and abolishes recruitment of CENP-C and downstream kinetochore components [PMID:10655499, PMID:15870271]. Centromeric CENP-A chromatin forms octameric nucleosomes that protect ~133 bp of DNA with characteristic loose entry/exit wrapping [PMID:28235947], and these nucleosomes directly nucleate the inner kinetochore: they assemble a nucleosome-associated complex of CCAN proteins (CENP-M/N/T/U/C/H) whose integrity is required for accurate chromosome alignment and segregation [PMID:16622419], with cryo-EM showing CCAN making edge-on contacts and topologically entrapping linker DNA to resist spindle forces [PMID:35420891]. CENP-C reads the CENP-A nucleosome through an extended hydrophobic interface (CENP-A V532/V533), reshapes and rigidifies the histone core, and unwraps terminal DNA via the H2A C-terminal tail, a collaboration required to retain CENP-A at centromeres [PMID:25954010, PMID:31475439]. CENP-A also cooperates with CENP-B to generate an open, dynamic centromeric chromatin state accessible to kinetochore factors [PMID:25916850, PMID:38086807]. CENP-A deposition is strictly cell-cycle-restricted to mitotic exit/G1 by a dedicated machinery: the chaperone HJURP caps a pre-nucleosomal CENP-A–H4 heterodimer [PMID:21478274] and is licensed by the Plk1-recruited Mis18/M18BP1 complex, while CDK activity inhibits assembly by phosphorylating M18BP1 and sequestering HJURP [PMID:25036634, PMID:28017591]. Multiple CENP-A modifications gate this process — Cdk1 phosphorylation of Ser68 blocks premature HJURP binding [PMID:25556658], and CUL4A-RBX1-COPS8-mediated K124 monoubiquitylation is required for HJURP interaction and centromere loading [PMID:25727006]. Once incorporated, CENP-A nucleosomes are retained through DNA replication by an HJURP–MCM2 mechanism [PMID:30293838], protected during centromeric transcription by Spt6 [PMID:32522980], and stabilized in S phase through CENP-A recognition of m6A-modified centromeric RNA via Leu61/Arg63 [PMID:39305902]; CENP-A chromatin also suppresses R-loops and protects centromeric replication, preventing breakage and translocations [PMID:33653953]. Beyond the centromere, CENP-A overexpression drives ectopic, DAXX-dependent deposition that occludes CTCF and confers DNA-damage tolerance [PMID:24530302], and contributes to chromosomal instability and tumor phenotypes [PMID:33620383].","teleology":[{"year":2000,"claim":"Established that CENP-A is genetically upstream of centromere organization, answering whether it is required to build the kinetochore at all.","evidence":"Cenpa knockout mouse embryos with immunofluorescence of centromere proteins","pmids":["10655499"],"confidence":"High","gaps":["Did not resolve which kinetochore proteins bind CENP-A directly versus indirectly","No molecular mechanism for CENP-C recruitment"]},{"year":2005,"claim":"Defined the hierarchy of kinetochore assembly, showing CENP-A is required for both inner CCAN and outer kinetochore recruitment and for checkpoint competence.","evidence":"Conditional CENP-A disruption in chicken DT40 cells with systematic component localization","pmids":["15870271"],"confidence":"High","gaps":["Did not distinguish direct from indirect recruitment","Mechanism of BubR1 maintenance defect unresolved"]},{"year":2006,"claim":"Identified the CENP-A nucleosome-associated complex (NAC/CCAN), defining the molecular bridge from CENP-A chromatin to the kinetochore.","evidence":"Mass spectrometry interactome, reciprocal Co-IP, and RNAi with mitotic phenotype","pmids":["16622419"],"confidence":"High","gaps":["Did not determine atomic contacts between CENP-A and CCAN","Role of FACT and nucleophosmin-1 left undefined"]},{"year":2011,"claim":"Resolved how the dedicated chaperone HJURP selectively recognizes CENP-A, answering the basis of CENP-A-specific assembly.","evidence":"X-ray crystallography of HJURP–CENP-A–H4 with in vitro binding","pmids":["21478274"],"confidence":"High","gaps":["Did not address how HJURP is delivered to centromeres","Mechanism of DNA deposition not captured by structure"]},{"year":2014,"claim":"Built the cell-cycle control logic of CENP-A deposition, showing a two-step Plk1-activating/CDK-inhibiting paradigm and CENP-A Ser68 phosphorylation as a brake on premature HJURP binding.","evidence":"RNAi/inhibitor, FRAP, degron, phosphomutant kinase assays and rescue in human cells","pmids":["25036634","25556658","25001279"],"confidence":"High","gaps":["Did not enumerate all CDK substrates in the pathway","Phosphatase regulation of multiple sites incompletely mapped"]},{"year":2015,"claim":"Established post-translational gating and stabilization of CENP-A: K124 monoubiquitylation by CUL4A-RBX1-COPS8 is required for HJURP binding/loading, and CENP-C and CENP-B stabilize incorporated nucleosomes.","evidence":"In vitro ubiquitylation, CRISPR knockin, reconstitution/FRET, single-molecule PIFE, and in vivo stability assays","pmids":["25727006","25954010","25916850"],"confidence":"High","gaps":["Did not define the deubiquitylase or full PTM crosstalk","CENP-C-induced reshaping not yet linked to atomic interface (resolved later)"]},{"year":2017,"claim":"Resolved the long-standing octamer-versus-tetramer debate, establishing CENP-A chromatin as an octameric nucleosome at all cell-cycle phases with characteristic DNA unwrapping.","evidence":"ChIP-seq with new reference models, MNase protection, nanopore, and quantitative mass spectrometry","pmids":["28235947"],"confidence":"High","gaps":["Does not reconcile earlier in vivo transition observations (idx 14)","Dynamics of unwrapping in living cells not directly measured"]},{"year":2018,"claim":"Explained how CENP-A survives DNA replication, identifying an HJURP–MCM2 retention mechanism coupling CENP-A inheritance to the replication fork.","evidence":"BioID, reciprocal Co-IP, auxin-inducible depletion, and quantitative imaging","pmids":["30293838"],"confidence":"High","gaps":["Did not quantify fraction of CENP-A retained versus newly deposited","Coordination with leading/lagging strand not resolved"]},{"year":2019,"claim":"Provided atomic-level mechanism for CENP-C reading and unwrapping the CENP-A nucleosome and for licensing factor (KNL2) discrimination of CENP-A from H3.","evidence":"Cryo-EM, in vitro binding, and mutagenesis of specific residues","pmids":["31475439","36744604","31676716"],"confidence":"High","gaps":["Functional consequence of H4 tail rigidification on K20me1 not fully tested in vivo","Did not capture full CCAN engagement"]},{"year":2021,"claim":"Defined a genome-protective role for CENP-A in S phase, showing CENP-A chromatin suppresses R-loops and protects centromeric replication against breakage.","evidence":"Cell-cycle-specific auxin degron, DRIP, DNA fiber assays, and cytogenetics","pmids":["33653953"],"confidence":"High","gaps":["Mechanism linking CENP-A nucleosome to R-loop resolution unknown","Did not identify the transcription/replication conflict resolving factors"]},{"year":2022,"claim":"Revealed how the inner kinetochore mechanically couples to the CENP-A nucleosome, showing CCAN topologically entraps linker DNA to withstand spindle forces.","evidence":"Cryo-EM of reconstituted CCAN–CENP-A nucleosome on α-satellite DNA","pmids":["35420891"],"confidence":"High","gaps":["Static structure does not show force response dynamics","Outer kinetochore connection not in the reconstitution"]},{"year":2024,"claim":"Identified an RNA-reading function of CENP-A, showing it binds m6A-modified centromeric RNA via Leu61/Arg63 to stabilize centromere localization in S phase.","evidence":"m6A mapping, CENP-A mutagenesis, RNA-protein binding, live-cell imaging, and xenografts","pmids":["39305902"],"confidence":"High","gaps":["m6A writer/eraser controlling cenRNA at centromeres not defined here","Structural basis of CENP-A–RNA contact not resolved"]},{"year":null,"claim":"How the diverse CENP-A modifications (Ser68/Ser7/K124 ubiquitylation, acetylation, methylation), RNA-reading, and chaperone choices are integrated into a single quantitative inheritance program — and how ectopic/overexpressed CENP-A is normally restrained — remains incompletely defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling competing PTM functions (e.g. S7 phosphorylation dispensability)","Mechanism restricting ectopic DAXX/MCM2-driven deposition only partially mapped","Disease relevance of CENP-A overexpression in cancer lacks causal in vivo proof"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[15,8]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,15,8]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[33]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[35,36]}],"localization":[{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[1,0,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,13]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[10,8]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,10,0,4]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[3,15,40]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[24,12]}],"complexes":["CENP-A nucleosome","CCAN/NAC (inner kinetochore)"],"partners":["HJURP","CENP-C","CENP-B","CENP-N","CENP-T","MCM2","SPT6","M18BP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P49450","full_name":"Histone H3-like centromeric protein A","aliases":["Centromere autoantigen A","Centromere protein A","CENP-A"],"length_aa":140,"mass_kda":16.0,"function":"Histone H3-like nucleosomal protein that is specifically found in centromeric nucleosomes (PubMed:11756469, PubMed:14667408, PubMed:15282608, PubMed:15475964, PubMed:15702419, PubMed:17651496, PubMed:19114591, PubMed:20739937, PubMed:27499292, PubMed:7962047, PubMed:9024683). Replaces conventional H3 in the nucleosome core of centromeric chromatin that serves as an assembly site for the inner kinetochore (PubMed:18072184). The presence of CENPA subtly modifies the nucleosome structure and the way DNA is wrapped around the nucleosome and gives rise to protruding DNA ends that are less well-ordered and rigid compared to nucleosomes containing histone H3 (PubMed:26878239, PubMed:27499292). May serve as an epigenetic mark that propagates centromere identity through replication and cell division (PubMed:15282608, PubMed:15475964, PubMed:20739937, PubMed:21478274, PubMed:26878239). Required for recruitment and assembly of kinetochore proteins, and as a consequence required for progress through mitosis, chromosome segregation and cytokinesis (PubMed:11756469, PubMed:14667408, PubMed:18072184, PubMed:23818633, PubMed:25556658, PubMed:27499292)","subcellular_location":"Nucleus; Chromosome, centromere","url":"https://www.uniprot.org/uniprotkb/P49450/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPA","classification":"Common Essential","n_dependent_lines":1134,"n_total_lines":1208,"dependency_fraction":0.9387417218543046},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000115163","cell_line_id":"CID000925","localizations":[{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleolus_gc","grade":1}],"interactors":[{"gene":"HJURP","stoichiometry":0.2},{"gene":"ARGLU1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000925","total_profiled":1310},"omim":[{"mim_id":"618139","title":"MIS18-BINDING PROTEIN 1; MIS18BP1","url":"https://www.omim.org/entry/618139"},{"mim_id":"618137","title":"MIS18 KINETOCHORE PROTEIN A: MIS18A","url":"https://www.omim.org/entry/618137"},{"mim_id":"616401","title":"SPINDLE APPARATUS COILED-COIL PROTEIN 1; SPDL1","url":"https://www.omim.org/entry/616401"},{"mim_id":"612667","title":"HOLLIDAY JUNCTION RECOGNITION PROTEIN; HJURP","url":"https://www.omim.org/entry/612667"},{"mim_id":"611511","title":"MLF1-INTERACTING PROTEIN; MLF1IP","url":"https://www.omim.org/entry/611511"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":12.4}],"url":"https://www.proteinatlas.org/search/CENPA"},"hgnc":{"alias_symbol":["CENP-A","CenH3"],"prev_symbol":[]},"alphafold":{"accession":"P49450","domains":[{"cath_id":"1.10.20.10","chopping":"44-134","consensus_level":"medium","plddt":95.0744,"start":44,"end":134}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P49450","model_url":"https://alphafold.ebi.ac.uk/files/AF-P49450-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P49450-F1-predicted_aligned_error_v6.png","plddt_mean":81.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPA","jax_strain_url":"https://www.jax.org/strain/search?query=CENPA"},"sequence":{"accession":"P49450","fasta_url":"https://rest.uniprot.org/uniprotkb/P49450.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P49450/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P49450"}},"corpus_meta":[{"pmid":"16622419","id":"PMC_16622419","title":"The human CENP-A centromeric nucleosome-associated complex.","date":"2006","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16622419","citation_count":594,"is_preprint":false},{"pmid":"10655499","id":"PMC_10655499","title":"Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice.","date":"2000","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10655499","citation_count":328,"is_preprint":false},{"pmid":"12417704","id":"PMC_12417704","title":"Centromeric retroelements and satellites interact with maize kinetochore protein CENH3.","date":"2002","source":"The Plant cell","url":"https://pubmed.ncbi.nlm.nih.gov/12417704","citation_count":296,"is_preprint":false},{"pmid":"24530302","id":"PMC_24530302","title":"Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24530302","citation_count":211,"is_preprint":false},{"pmid":"25954010","id":"PMC_25954010","title":"Chromosomes. 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Assembly of the NAC requires CENP-M, CENP-N, and CENP-T. FACT and nucleophosmin-1 are stably recruited to CENP-A nucleosomes independently of the NAC. Seven distal CAD components (CENP-K, -L, -O, -P, -Q, -R, -S) assemble on the NAC. Disruption of the NAC causes chromosome alignment and segregation errors.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, RNAi depletion with mitotic phenotype readout\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, MS interactome, RNAi with defined cellular phenotype; foundational study replicated extensively\",\n      \"pmids\": [\"16622419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cenpa null mouse embryos fail to survive beyond E6.5 with severe mitotic defects. Cenpa is required for kinetochore targeting of Cenpc; loss of Cenpa causes diffuse Cenpb foci and complete loss of discrete Cenpc centromere signal, indicating Cenpa organizes centromeric chromatin and is epistatic to Cenpc localization.\",\n      \"method\": \"Gene targeting (knockout mouse), immunofluorescence microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular and organismal phenotype, replicated in subsequent studies\",\n      \"pmids\": [\"10655499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of HJURP bound to a CENP-A–H4 heterodimer shows HJURP binds a pre-nucleosomal CENP-A–H4 heterodimer; the C-terminal β-sheet domain of HJURP caps the DNA-binding surface of CENP-A–H4, preventing spontaneous DNA association. A novel CENP-A-specific surface distinguishes it from H3 in HJURP binding.\",\n      \"method\": \"X-ray crystallography, in vitro binding assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical validation; independently corroborated by other structural studies\",\n      \"pmids\": [\"21478274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-C binds CENP-A nucleosomes and reshapes the octameric histone core: it rigidifies both surface and internal nucleosome structure and modulates terminal DNA to match the loose DNA wrap characteristic of native centromeric CENP-A nucleosomes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, demonstrating collaboration between CENP-C and CENP-A in maintaining centromere identity.\",\n      \"method\": \"Purified-component reconstitution, FRET, single-molecule PIFE, auxin-inducible degron depletion\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with multiple orthogonal biophysical methods plus in vivo depletion; single rigorous study\",\n      \"pmids\": [\"25954010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Polo-like kinase 1 (Plk1) is required to initiate CENP-A deposition in human cells by promoting localization of the Mis18 complex to centromeres. CDK inhibits Mis18 complex assembly. A two-step Plk1/CDK regulatory paradigm controls cell-cycle-restricted CENP-A deposition; bypassing both steps uncouples deposition from cell-cycle progression and causes mitotic defects.\",\n      \"method\": \"RNAi/chemical inhibition, FRAP, auxin-inducible degron, epistasis with phosphomutants\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, genetic epistasis, functional rescue experiments in human cells\",\n      \"pmids\": [\"25036634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cdk1 phosphorylates CENP-A at Ser68 during early mitosis, which eliminates binding of CENP-A to its assembly chaperone HJURP, thereby preventing premature centromeric loading before mitotic exit. PP1α dephosphorylates Ser68 at mitotic exit, enabling HJURP-mediated centromeric deposition.\",\n      \"method\": \"In vitro kinase assay, phosphomutant rescue, co-immunoprecipitation, cell synchronization\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay plus phosphomutant cell biology with mechanistic follow-up; multiple methods\",\n      \"pmids\": [\"25556658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CUL4A-RBX1-COPS8 E3 ligase monoubiquitylates CENP-A on K124, which is required for CENP-A interaction with chaperone HJURP and for centromere localization. The K124R mutation reduces HJURP binding and abolishes centromere loading; addition of monoubiquitin to K124R restores both HJURP interaction and centromere localization.\",\n      \"method\": \"In vitro ubiquitylation assay, co-immunoprecipitation, CRISPR knockin, immunofluorescence\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — biochemical reconstitution of ubiquitylation, rescue with monoubiquitin fusion, CRISPR knockin in vivo validation\",\n      \"pmids\": [\"25727006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HJURP transiently associates with pre-existing CENP-A nucleosomes during S phase and is required for CENP-A nucleosome inheritance through DNA replication. HJURP co-purifies with the MCM2-7 helicase complex, and MCM2 and HJURP simultaneously bind CENP-A, defining a mechanism for retaining CENP-A during replication fork passage.\",\n      \"method\": \"BioID proximity labeling, co-immunoprecipitation, conditional depletion (auxin), quantitative imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BioID plus reciprocal Co-IP plus auxin-inducible depletion with defined S-phase phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"30293838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of the human inner kinetochore CCAN complex bound to a CENP-A nucleosome reconstituted on α-satellite DNA show that CCAN forms edge-on contacts with the CENP-A nucleosome; a linker DNA segment threads through the central CENP-LN channel; CENP-TWSX partially wraps linker DNA. This topological entrapment of linker DNA by CCAN provides a mechanism for kinetochores to withstand spindle forces.\",\n      \"method\": \"Cryo-electron microscopy, reconstitution of CENP-A nucleosome on α-satellite DNA\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with fully reconstituted complex\",\n      \"pmids\": [\"35420891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cell-cycle control of CENP-A assembly requires two targets: a single Cdk phosphorylation site in licensing factor M18BP1 and a cyclin A binding site in HJURP. Simultaneous mutation of both sites completely uncouples CENP-A assembly from cell-cycle phase, allowing assembly under high Cdk activity indistinguishable from G1 assembly. Cdk-mediated inhibition works by sequestering active factors away from the centromere.\",\n      \"method\": \"Phosphomutant expression, quantitative live-cell imaging, CENP-A incorporation assays, cell-cycle staging\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with phosphomutants, multiple orthogonal methods, complete uncoupling from cell cycle demonstrated\",\n      \"pmids\": [\"28017591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In CENP-A-depleted chicken DT40 cells, inner kinetochore proteins CENP-I, CENP-H, and CENP-C and outer components Nuf2/Hec1, Mad2, and CENP-E are mislocalised, whereas BubR1 and INCENP are efficiently recruited. CENP-A-depleted cells show chromosome congression defects and a specific defect in maintaining BubR1 at kinetochores under checkpoint activation, indicating CENP-A is required for a mitotic-checkpoint-competent kinetochore.\",\n      \"method\": \"Conditional gene disruption (DT40), immunofluorescence, epistasis of kinetochore component localization\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with systematic kinetochore protein localization analysis; multiple components tested\",\n      \"pmids\": [\"15870271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CENP-A is phosphorylated at a serine residue (equivalent to H3 Ser10) in a cell-cycle-dependent manner distinct from histone H3. CENP-A phosphorylation begins in prophase, peaks in prometaphase, and disappears in telophase—after both pericentric and genome-wide H3 phosphorylation. Prekinetochore duplication (CENP-A foci doubling) occurs before complete H3 phosphorylation in G2.\",\n      \"method\": \"Phospho-specific antibody ELISA, western blot, quantitative immunocytochemistry\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-specific antibody with multiple readout methods in single lab; kinase not identified in this paper\",\n      \"pmids\": [\"11171370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CENP-A is rapidly recruited to DNA double-strand breaks (DSBs) in human and mouse cells, along with centromere-associated proteins CENP-N, CENP-T, and CENP-U. The centromere-targeting domain (CATD) of CENP-A is necessary and sufficient for recruitment to DSBs. CENP-A accumulation at breaks is enhanced by active NHEJ but does not require DNA-PKcs, Ligase IV, or H2AX.\",\n      \"method\": \"Multiphoton laser microirradiation, I-SceI endonuclease-induced DSBs, CENP-A domain truncation/mutation, immunofluorescence\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with domain mapping; single lab, multiple complementary approaches\",\n      \"pmids\": [\"19717431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CENP-A overexpression leads to ectopic enrichment at sites of active histone turnover in a heterotypic CENP-A/H3.3–H4 tetramer. Ectopic localization depends on H3.3 chaperone DAXX rather than the dedicated CENP-A chaperone HJURP. Ectopic CENP-A nucleosomes occlude CTCF binding and confer DNA damage tolerance, both dependent on DAXX.\",\n      \"method\": \"ChIP-seq, co-immunoprecipitation, RNAi knockdown, CTCF binding assay, cell survival assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-seq plus Co-IP plus RNAi epistasis plus functional assays; multiple orthogonal methods identifying DAXX as chaperone for ectopic CENP-A\",\n      \"pmids\": [\"24530302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human CENP-A nucleosomes undergo cell-cycle-dependent structural transitions in vivo: they exist as tetramers (half-nucleosomes) after replication and convert to octamers before the next replication, accompanied by reversible chaperone binding, chromatin fiber folding changes, and histone fold domain modifications on CENP-A and H4.\",\n      \"method\": \"AFM, mass spectrometry of PTMs, chromatin fractionation, cell-cycle synchronization\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biophysical and biochemical methods in single lab; octameric structure contested by other papers (see PMID:28235947)\",\n      \"pmids\": [\"22817894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human centromeric CENP-A chromatin is exclusively an octameric nucleosome at all cell-cycle phases (G1 and G2), containing equimolar CENP-A, H2A, H2B, and H4 with no H3. CENP-A nucleosomes protect DNA lengths centered on 133 bp, consistent with octamers with DNA unwrapping at entry and exit. Solid-state nanopore analyses confirm nucleosomal size.\",\n      \"method\": \"ChIP-seq with new reference models, MNase protection assay, nanopore analysis, quantitative mass spectrometry\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal structural and genomic methods; contradicts tetramer model with rigorous controls across cell-cycle phases\",\n      \"pmids\": [\"28235947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structures of centromeric tri-nucleosomes show that H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture with outward-facing linker DNA path between nucleosomes, distinct from H3-H3-H3 tri-nucleosomes with inward-facing DNA. This exposes the CENP-A nucleosome to solvent in condensed chromatin.\",\n      \"method\": \"Cryo-electron microscopy, tri-nucleosome reconstitution\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure of reconstituted complex; single study but high-resolution structural method\",\n      \"pmids\": [\"31711756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Aurora A kinase phosphorylates CENP-A at serine 7 (S7) at inner centromeres in mitosis, protecting bioriented chromosomes against cohesion fatigue. Expression of non-phosphorylatable CENP-A S7A weakens sister chromatid cohesion only when centromeres are under spindle tension. Aurora A is recruited to centromeres in a Bub1-dependent manner.\",\n      \"method\": \"Phosphomutant CENP-A expression, chromosome cohesion assays, Aurora A inhibition, immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphomutant phenotype with kinase inhibition and epistasis; single lab; note that PMID:30635586 disputes S7 phosphorylation controlling centromere function\",\n      \"pmids\": [\"29760389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Phosphorylation of CENP-A on serine 7 is dispensable for correct CENP-C recruitment, faithful chromosome segregation, and long-term cell viability, as demonstrated by gene targeting at both endogenous CENP-A alleles and gene replacement with S7A in human cells.\",\n      \"method\": \"Gene targeting at endogenous loci (CRISPR), gene replacement, immunofluorescence, live-cell imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — allelic replacement at endogenous locus with rigorous controls; directly contradicts prior claim about S7 phosphorylation\",\n      \"pmids\": [\"30635586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail. The CENP-C central region (CENP-C_CR) binds the CENP-A nucleosome with high affinity via an extended hydrophobic area involving CENP-A V532 and V533. CENP-C binding further loosens terminal DNA wrapping (through H2A C-tail destabilization) and rigidifies the H4 N-terminal tail in a conformation favoring H4K20 monomethylation.\",\n      \"method\": \"Cryo-EM, in vitro binding assays, nucleosome reconstitution, mutagenesis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus in vitro biochemistry plus mutagenesis identifying specific residues; multiple orthogonal methods\",\n      \"pmids\": [\"31475439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CDK1 phosphorylates CENP-C in its C-terminal CENP-A binding motif, which facilitates CENP-C binding to the CENP-A nucleosome in vitro and in vivo. This phosphorylation promotes CENP-C kinetochore localization during mitosis. The CENP-A–CENP-C interaction is critical for long-term cell viability in human RPE-1 cells.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, phosphomutant rescue, immunofluorescence, cell viability assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay plus in vivo phosphomutant analysis plus cell viability; multiple orthogonal methods\",\n      \"pmids\": [\"31676716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-B forms a stable complex with the CENP-A nucleosome in vitro, preferentially when the CENP-B box DNA is located at the proximal edge of the nucleosome. The CENP-B DNA-binding domain specifically interacts with the CENP-A–H4 complex (but not H3.1–H4). In vivo, CENP-B binding near the CENP-A nucleosome substantially stabilizes CENP-A on alphoid DNA.\",\n      \"method\": \"In vitro nucleosome reconstitution, pulldown assay, in vivo chromatin stability assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution with defined position-dependent binding plus in vivo validation\",\n      \"pmids\": [\"25916850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Centromeric transcription by RNA Pol II temporally coincides with de novo dCENP-A deposition in Drosophila. Short inhibition of transcription impairs CENP-A incorporation into stable (salt-resistant) chromatin but does not prevent initial targeting to centromeres, revealing two stability states of newly loaded CENP-A: a salt-sensitive association and a salt-resistant incorporated form. Transcription-mediated chromatin remodeling drives the transition to fully incorporated nucleosomes.\",\n      \"method\": \"Drosophila tissue culture, transcription inhibitor treatment, SNAP-tag pulse-chase, chromatin fractionation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — novel dCENP-A loading system with pharmacological and genetic dissection; multiple orthogonal assays\",\n      \"pmids\": [\"29626011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Spt6, a histone chaperone and transcription elongation factor, prevents loss of old CENP-A nucleosomes during centromeric transcription. Spt6 directly binds dCENP-A in vitro; phosphomimetic dCENP-A mutants reduce Spt6 association, and non-phosphorylatable dCENP-A accumulates at centromeres. Spt6 acts as a conserved CENP-A maintenance factor in both Drosophila and human cells.\",\n      \"method\": \"Co-immunoprecipitation/direct binding assay, SNAP-tag pulse-chase, phosphomutant analysis, immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated, conserved across two species, phosphomutant epistasis, multiple orthogonal approaches\",\n      \"pmids\": [\"32522980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CENP-A chromatin protects centromeric alpha-satellite DNA from replication stress during S phase. Rapid CENP-A removal specifically in S phase causes R-loop accumulation with increased centromeric transcripts, impaired replication fork progression, recombination at alpha-satellites, and anaphase bridges leading to chromosome breakage and translocations at centromeric regions.\",\n      \"method\": \"Auxin-inducible degron for cell-cycle-specific CENP-A removal, DNA-RNA immunoprecipitation, DNA fiber assay, cytogenetics\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-cycle-specific conditional depletion with multiple orthogonal readouts; mechanistically links CENP-A to R-loop suppression and replication fork protection\",\n      \"pmids\": [\"33653953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CENP-A ubiquitylation (K124) is inherited between cell divisions through CENP-A dimerization. Pre-existing ubiquitylated CENP-A is necessary for recruitment of newly synthesized CENP-A to the centromere. In vivo and in vitro experiments with dimerization mutants show that inheritance of K124 ubiquitylation requires CENP-A dimerization. Overexpression of monoubiquitin-fused CENP-A induces neocentromeres at non-centromeric regions.\",\n      \"method\": \"In vivo and in vitro dimerization mutant analysis, co-immunoprecipitation, immunofluorescence, ectopic CENP-A expression\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dimerization mutants plus biochemical assays in single lab; builds on prior K124 ubiquitylation work\",\n      \"pmids\": [\"27052173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CENP-A HJURP nucleosome clusters form rosette-like structures (~250–300 nm) around HJURP during G1 phase. 2D/3D super-resolution microscopy shows HJURP localizes to the center of these rosettes and acts as a nucleation point for CENP-A deposition, providing structural insight into centromeric chromatin organization during loading.\",\n      \"method\": \"Super-resolution microscopy (STORM/PALM), co-localization analysis, segmentation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by super-resolution with HJURP as nucleation point; single lab, imaging-based\",\n      \"pmids\": [\"31570711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cell-cycle-dependent recruitment of HJURP to centromeres depends on timely CDK-mediated phosphorylation of HJURP. A non-phosphorylatable HJURP mutant localizes prematurely to centromeres in S and G2, causing premature CENP-A loading and cell-cycle delays. Once at centromeres, HJURP promotes CENP-A deposition through a unique DNA-binding domain.\",\n      \"method\": \"Phosphomutant expression, cell-cycle synchronization, immunofluorescence, DNA binding assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphomutant epistasis, DNA-binding domain mapping, multiple cell-cycle readouts\",\n      \"pmids\": [\"25001279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In a cell-free CENP-A assembly system, two distinct domains of CENP-A within existing nucleosomes are required for new CENP-A assembly. CENP-A nucleosomes recruit CENP-C and M18BP1 independently, and CENP-C recruitment depends on the density of underlying CENP-A nucleosomes.\",\n      \"method\": \"Cell-free CENP-A assembly system (Xenopus extract), domain truncation analysis, chromatin binding assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution with domain-mapping; identifies two independent CENP-A-to-assembly-factor pathways\",\n      \"pmids\": [\"26076692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"At yeast centromeres, Cse4/CENP-A histone-fold domain interacts with inner kinetochore protein Mif2/CENP-C. Mif2 contacts one side of the nucleosome dyad engaging both Cse4 residues and AT-rich centromeric DNA through a contiguous DNA- and histone-binding domain (DHBD) containing the CENP-C motif, an AT hook, and RK clusters. Human CENP-C has two related DHBDs with preference for AT-rich DNA.\",\n      \"method\": \"Biochemical binding assays, mutagenesis, ChIP, structural modeling\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical reconstitution plus mutagenesis plus ChIP; identifies dual nucleosome-binding mechanism\",\n      \"pmids\": [\"29074736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PARP-2 co-immunoprecipitates with CENP-A (Cenpa) and CENP-B (Cenpb) at active centromeres in a cell-cycle-dependent manner (accumulating prometaphase/metaphase, disassociating by telophase). PARP-2 does not interact with CENP-C. PARP-2 centromere binding is sequence-independent and enhanced by microtubule-inhibiting drugs.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, pseudodicentric chromosome analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus cell-cycle localization; interaction with CENP-A established but functional mechanism less defined\",\n      \"pmids\": [\"12217960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In Saccharomyces cerevisiae, Cse4/CENP-A (centromeric H3 variant) is methylated on arginine 37 (R37). Absence of R37 methylation reduces levels of Mtw1/MIND and Ctf19 complex components at the centromere (but not Cse4 itself), causing growth defects, G2/M arrest, and chromosome segregation errors. This modification regulates recruitment of linker kinetochore components.\",\n      \"method\": \"Mass spectrometry, methylation mutants, genetic epistasis with kinetochore mutants, ChIP\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification plus genetic epistasis plus ChIP in yeast; single lab\",\n      \"pmids\": [\"22615363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CENPT bridges adjacent CENPA nucleosomes on young human α-satellite dimers. Sequential ChIP-seq shows CENPT is centered between two well-positioned CENPA nucleosomes over the CENPB box, forming a CENPA/CENPB/CENPC/CENPT complex nuclease-protected over an α-satellite dimer. CENPT interacts with CENPB/CENPC complex rather than H3 nucleosomes in vivo.\",\n      \"method\": \"Comparative and sequential ChIP-seq, base-pair-resolution genomic readout of protein-protein interactions\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — sequential ChIP-seq with base-pair resolution; single lab; contradicts prior model but uses rigorous method\",\n      \"pmids\": [\"27384170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CENP-A functions as an m6A reader of centromeric RNA (cenRNA): m6A-modified cenRNA stabilizes centromeric localization of CENP-A during S phase. Mutations at CENP-A Leu61 and Arg63 abolish cenRNA binding and cause loss of centromere-bound CENP-A during S phase, compromised centromere integrity, and abnormal chromosome separation.\",\n      \"method\": \"m6A methylation mapping, CENP-A mutagenesis, RNA-protein binding assays, live-cell imaging, tumor xenograft\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct biochemical demonstration of RNA-binding by CENP-A with specific residues identified, functional rescue assays, in vivo and in vitro validation\",\n      \"pmids\": [\"39305902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CENP-A overexpression causes CIN with lagging chromosomes and micronuclei due to reduced localization of kinetochore proteins, resulting in defective kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE also reduces cell adhesion gene expression and increases invasion.\",\n      \"method\": \"Inducible overexpression, kinetochore protein quantification by immunofluorescence, live-cell imaging, xenograft\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative kinetochore protein analysis plus live-cell imaging in defined overexpression system; single lab\",\n      \"pmids\": [\"33620383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CENPA overexpression promotes prostate cancer cell growth through a non-centromeric function as a transcriptional regulator that modulates expression of proliferation, cell-cycle, and centromere/kinetochore genes, as revealed by integrated ChIP-seq and RNA-seq experiments.\",\n      \"method\": \"ChIP-seq, RNA-seq, gain/loss-of-function experiments, tissue microarray\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus RNA-seq identifies genomic binding and gene regulation; single lab; non-centromeric transcriptional function\",\n      \"pmids\": [\"32371391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CENPA recruits histone acetyltransferase GCN-5 to the promoter of KPNA2 to induce transcription activation in colon cancer cells. This non-centromeric transcriptional activity of CENPA promotes glycolysis and growth. H3K27ac modification is detected at the KPNA2 promoter upon CENPA binding.\",\n      \"method\": \"ChIP assay, co-immunoprecipitation, RNAi knockdown, rescue experiments, metabolic assays\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus Co-IP plus functional rescue; single lab demonstrating GCN-5 recruitment mechanism\",\n      \"pmids\": [\"34508688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Phosphorylation of CENP-A Ser68 primes polyubiquitin-mediated proteasomal degradation of CENP-A during mitotic phase. DCAF11 (WDR23) is the E3 ligase mediating this polyubiquitination via K49 and K124. Mutation of K49R/K124R or DCAF11 deletion abrogates proper degradation and causes CENP-A mislocalization.\",\n      \"method\": \"In vivo ubiquitination assay, proteasome inhibitor treatment, DCAF11 depletion/knockout, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identification of E3 ligase with site-specific mutations plus depletion; single lab\",\n      \"pmids\": [\"34758320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HJURP phosphorylation by CDK prevents HJURP interaction with CENP-C in metaphase, blocking delivery of soluble CENP-A to centromeres. Non-phosphorylatable HJURP constitutively binds CENP-C in metaphase but is insufficient for new CENP-A assembly. M18BP1.S subunit of the Mis18 complex competitively inhibits HJURP access to CENP-C. Two inhibitory activities together repress CENP-A assembly in metaphase.\",\n      \"method\": \"X. laevis egg extract cell-free assembly system, phosphomutant analysis, co-immunoprecipitation, competitive binding assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution system with defined biochemical pathway, phosphomutant analysis, competitive binding; comprehensive mechanistic dissection\",\n      \"pmids\": [\"37141119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structure of chicken CENP-A nucleosome bound to ggKNL2 (Mis18 complex component with CENP-C-like motif) shows ggKNL2 simultaneously recognizes the CENP-A C-terminal tail and the RG-loop using its CENP-C-like motif to distinguish CENP-A from H3. ggKNL2 changes its centromere binding partner during cell cycle progression.\",\n      \"method\": \"Cryo-EM structure, biochemical binding assays, cell biology validation, cell-cycle analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure plus biochemical and cell-biology validation; identifies dual recognition mechanism\",\n      \"pmids\": [\"36744604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CENP-A and CENP-B collaborate to establish an open centromeric chromatin state. CENP-A incorporation increases chromatin fiber dynamics. This increased dynamics allow CENP-B DNA access; bound CENP-B further opens chromatin fiber structure and induces nucleosomal DNA unwrapping. Removal of CENP-A increases CENP-B mobility in cells.\",\n      \"method\": \"Single-molecule fluorescence, cryo-EM, FRAP (in cells), chromatin fiber reconstitution\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule fluorescence plus cryo-EM plus in-cell FRAP; multiple orthogonal methods demonstrating cooperative chromatin remodeling\",\n      \"pmids\": [\"38086807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DNAJC9 (a J-domain protein for H3-H4 folding) restricts CENP-A mislocalization. Its depletion promotes CENP-A interaction with MCM2, which drives CENP-A deposition at ectopic sites. H3.3 depletion also causes CENP-A mislocalization. This defines MCM2 as a driver of ectopic CENP-A deposition when H3-H4 supply chains are disrupted.\",\n      \"method\": \"Genome-wide RNAi screen, global interactome analysis, MCM2 depletion epistasis, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen plus interactome plus epistasis; single lab, multiple methods\",\n      \"pmids\": [\"38600242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Diaphanous formin mDia2 is required for stable replenishment of new CENP-A at centromeres in G1. Depletion of mDia2 causes prolonged centromere association of HJURP. mDia2 acts downstream of the MgcRacGAP-dependent small GTPase pathway in regulating CENP-A nucleosome assembly. Nuclear localization of mDia2 is required for this function.\",\n      \"method\": \"Quantitative imaging, pulse-chase, constitutively active mutant rescue, RNAi epistasis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pulse-chase plus epistasis experiments; single lab; pathway position established\",\n      \"pmids\": [\"27185834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Quiescent cells (G0-arrested and prophase I-arrested oocytes) actively and continuously incorporate new CENP-A at centromeres using the canonical CENP-A deposition machinery. Plk1 is required specifically for G1 (not quiescent) CENP-A deposition, while transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence reduces CENP-A levels and perturbs chromosome segregation upon re-entry into division.\",\n      \"method\": \"Pulse-chase labeling, chemical inhibitors (Plk1/transcription), conditional depletion, quantitative imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pulse-chase plus genetic/chemical dissection in multiple quiescent cell types; single lab\",\n      \"pmids\": [\"31422918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CENP-A acetylation at K124 causes tightening of the histone core and diminishes CENP-C binding (by computational modeling and CENP-A K124Q/K124A mutants). CENP-A K124 switches from acetylation at G1/S to monomethylation during early/mid-S phase. The HAT p300 is implicated in K124 acetylation. K124 mutations alter centromeric replication timing and cause modest increases in mitotic errors.\",\n      \"method\": \"Computational modeling, in vivo phosphomutant analysis, mass spectrometry of native CENP-A, HAT inhibitor experiments\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification of modification switching plus mutant cell biology; single lab, some computational predictions included\",\n      \"pmids\": [\"28396698\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENP-A is a centromere-specific histone H3 variant that epigenetically marks centromere identity by forming octameric nucleosomes with loosely wrapped DNA; these nucleosomes are assembled during late mitosis/early G1 in a cell-cycle-controlled process requiring the dedicated chaperone HJURP (which binds a pre-nucleosomal CENP-A–H4 heterodimer), the Mis18 licensing complex (recruited by Plk1), and CDK inhibition of HJURP and M18BP1 in other cell-cycle phases; Ser68 phosphorylation by Cdk1 prevents premature HJURP binding, while K124 monoubiquitylation by CUL4A-RBX1-COPS8 is required for HJURP interaction and centromere loading; once incorporated, CENP-A nucleosomes are stabilized by CENP-C (which reshapes and rigidifies the nucleosome), by Spt6-mediated protection during centromeric transcription, and by HJURP-MCM2-mediated retention during DNA replication; CENP-A nucleosomes directly recruit the CCAN (via CENP-C and CENP-N binding specific CENP-A residues) and the outer kinetochore, are recognized as m6A-modified centromeric RNA readers for S-phase centromere stability, and collaborate with CENP-B to create an open centromeric chromatin state accessible to kinetochore factors.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CENP-A is a centromere-specific histone H3 variant that epigenetically marks centromere identity and is essential for building a functional kinetochore; its loss in mouse embryos causes early lethality with mitotic catastrophe and abolishes recruitment of CENP-C and downstream kinetochore components [#1, #10]. Centromeric CENP-A chromatin forms octameric nucleosomes that protect ~133 bp of DNA with characteristic loose entry/exit wrapping [#15], and these nucleosomes directly nucleate the inner kinetochore: they assemble a nucleosome-associated complex of CCAN proteins (CENP-M/N/T/U/C/H) whose integrity is required for accurate chromosome alignment and segregation [#0], with cryo-EM showing CCAN making edge-on contacts and topologically entrapping linker DNA to resist spindle forces [#8]. CENP-C reads the CENP-A nucleosome through an extended hydrophobic interface (CENP-A V532/V533), reshapes and rigidifies the histone core, and unwraps terminal DNA via the H2A C-terminal tail, a collaboration required to retain CENP-A at centromeres [#3, #19]. CENP-A also cooperates with CENP-B to generate an open, dynamic centromeric chromatin state accessible to kinetochore factors [#21, #40]. CENP-A deposition is strictly cell-cycle-restricted to mitotic exit/G1 by a dedicated machinery: the chaperone HJURP caps a pre-nucleosomal CENP-A–H4 heterodimer [#2] and is licensed by the Plk1-recruited Mis18/M18BP1 complex, while CDK activity inhibits assembly by phosphorylating M18BP1 and sequestering HJURP [#4, #9]. Multiple CENP-A modifications gate this process — Cdk1 phosphorylation of Ser68 blocks premature HJURP binding [#5], and CUL4A-RBX1-COPS8-mediated K124 monoubiquitylation is required for HJURP interaction and centromere loading [#6]. Once incorporated, CENP-A nucleosomes are retained through DNA replication by an HJURP–MCM2 mechanism [#7], protected during centromeric transcription by Spt6 [#23], and stabilized in S phase through CENP-A recognition of m6A-modified centromeric RNA via Leu61/Arg63 [#33]; CENP-A chromatin also suppresses R-loops and protects centromeric replication, preventing breakage and translocations [#24]. Beyond the centromere, CENP-A overexpression drives ectopic, DAXX-dependent deposition that occludes CTCF and confers DNA-damage tolerance [#13], and contributes to chromosomal instability and tumor phenotypes [#34].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that CENP-A is genetically upstream of centromere organization, answering whether it is required to build the kinetochore at all.\",\n      \"evidence\": \"Cenpa knockout mouse embryos with immunofluorescence of centromere proteins\",\n      \"pmids\": [\"10655499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which kinetochore proteins bind CENP-A directly versus indirectly\", \"No molecular mechanism for CENP-C recruitment\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the hierarchy of kinetochore assembly, showing CENP-A is required for both inner CCAN and outer kinetochore recruitment and for checkpoint competence.\",\n      \"evidence\": \"Conditional CENP-A disruption in chicken DT40 cells with systematic component localization\",\n      \"pmids\": [\"15870271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish direct from indirect recruitment\", \"Mechanism of BubR1 maintenance defect unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified the CENP-A nucleosome-associated complex (NAC/CCAN), defining the molecular bridge from CENP-A chromatin to the kinetochore.\",\n      \"evidence\": \"Mass spectrometry interactome, reciprocal Co-IP, and RNAi with mitotic phenotype\",\n      \"pmids\": [\"16622419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not determine atomic contacts between CENP-A and CCAN\", \"Role of FACT and nucleophosmin-1 left undefined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved how the dedicated chaperone HJURP selectively recognizes CENP-A, answering the basis of CENP-A-specific assembly.\",\n      \"evidence\": \"X-ray crystallography of HJURP–CENP-A–H4 with in vitro binding\",\n      \"pmids\": [\"21478274\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how HJURP is delivered to centromeres\", \"Mechanism of DNA deposition not captured by structure\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Built the cell-cycle control logic of CENP-A deposition, showing a two-step Plk1-activating/CDK-inhibiting paradigm and CENP-A Ser68 phosphorylation as a brake on premature HJURP binding.\",\n      \"evidence\": \"RNAi/inhibitor, FRAP, degron, phosphomutant kinase assays and rescue in human cells\",\n      \"pmids\": [\"25036634\", \"25556658\", \"25001279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not enumerate all CDK substrates in the pathway\", \"Phosphatase regulation of multiple sites incompletely mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established post-translational gating and stabilization of CENP-A: K124 monoubiquitylation by CUL4A-RBX1-COPS8 is required for HJURP binding/loading, and CENP-C and CENP-B stabilize incorporated nucleosomes.\",\n      \"evidence\": \"In vitro ubiquitylation, CRISPR knockin, reconstitution/FRET, single-molecule PIFE, and in vivo stability assays\",\n      \"pmids\": [\"25727006\", \"25954010\", \"25916850\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the deubiquitylase or full PTM crosstalk\", \"CENP-C-induced reshaping not yet linked to atomic interface (resolved later)\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the long-standing octamer-versus-tetramer debate, establishing CENP-A chromatin as an octameric nucleosome at all cell-cycle phases with characteristic DNA unwrapping.\",\n      \"evidence\": \"ChIP-seq with new reference models, MNase protection, nanopore, and quantitative mass spectrometry\",\n      \"pmids\": [\"28235947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not reconcile earlier in vivo transition observations (idx 14)\", \"Dynamics of unwrapping in living cells not directly measured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Explained how CENP-A survives DNA replication, identifying an HJURP–MCM2 retention mechanism coupling CENP-A inheritance to the replication fork.\",\n      \"evidence\": \"BioID, reciprocal Co-IP, auxin-inducible depletion, and quantitative imaging\",\n      \"pmids\": [\"30293838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not quantify fraction of CENP-A retained versus newly deposited\", \"Coordination with leading/lagging strand not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided atomic-level mechanism for CENP-C reading and unwrapping the CENP-A nucleosome and for licensing factor (KNL2) discrimination of CENP-A from H3.\",\n      \"evidence\": \"Cryo-EM, in vitro binding, and mutagenesis of specific residues\",\n      \"pmids\": [\"31475439\", \"36744604\", \"31676716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of H4 tail rigidification on K20me1 not fully tested in vivo\", \"Did not capture full CCAN engagement\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a genome-protective role for CENP-A in S phase, showing CENP-A chromatin suppresses R-loops and protects centromeric replication against breakage.\",\n      \"evidence\": \"Cell-cycle-specific auxin degron, DRIP, DNA fiber assays, and cytogenetics\",\n      \"pmids\": [\"33653953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking CENP-A nucleosome to R-loop resolution unknown\", \"Did not identify the transcription/replication conflict resolving factors\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed how the inner kinetochore mechanically couples to the CENP-A nucleosome, showing CCAN topologically entraps linker DNA to withstand spindle forces.\",\n      \"evidence\": \"Cryo-EM of reconstituted CCAN–CENP-A nucleosome on α-satellite DNA\",\n      \"pmids\": [\"35420891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Static structure does not show force response dynamics\", \"Outer kinetochore connection not in the reconstitution\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified an RNA-reading function of CENP-A, showing it binds m6A-modified centromeric RNA via Leu61/Arg63 to stabilize centromere localization in S phase.\",\n      \"evidence\": \"m6A mapping, CENP-A mutagenesis, RNA-protein binding, live-cell imaging, and xenografts\",\n      \"pmids\": [\"39305902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"m6A writer/eraser controlling cenRNA at centromeres not defined here\", \"Structural basis of CENP-A–RNA contact not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse CENP-A modifications (Ser68/Ser7/K124 ubiquitylation, acetylation, methylation), RNA-reading, and chaperone choices are integrated into a single quantitative inheritance program — and how ectopic/overexpressed CENP-A is normally restrained — remains incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling competing PTM functions (e.g. S7 phosphorylation dispensability)\", \"Mechanism restricting ectopic DAXX/MCM2-driven deposition only partially mapped\", \"Disease relevance of CENP-A overexpression in cancer lacks causal in vivo proof\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [15, 8]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 15, 8]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [33]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [35, 36]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [1, 0, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 13]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [10, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 10, 0, 4]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [3, 15, 40]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [24, 12]}\n    ],\n    \"complexes\": [\"CENP-A nucleosome\", \"CCAN/NAC (inner kinetochore)\"],\n    \"partners\": [\"HJURP\", \"CENP-C\", \"CENP-B\", \"CENP-N\", \"CENP-T\", \"MCM2\", \"Spt6\", \"M18BP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}