{"gene":"PF3D7_1333700","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2006,"finding":"PF3D7_1333700 (CenH3) was identified as a histone variant encoded in the P. falciparum genome; however, mass spectrometry of acid-extracted histones failed to detect CenH3 protein synthesis, while all other histones were confirmed, suggesting either low abundance or restricted expression.","method":"Genome analysis and mass spectrometry proteomics; real-time RT-PCR; immunoblotting","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry plus gene expression analysis from a single lab; negative detection result for the protein itself is explicitly stated","pmids":["16410041"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"CenH3 (PfCENH3) occupies a defined ~4–4.5 kb chromatin region on each P. falciparum chromosome as determined by ChIP-seq, co-localises with histone variant PfH2A.Z at centromeres, and is absent from pericentric heterochromatin. Immunofluorescence showed that centromeres marked by CenH3 cluster to a single nuclear location prior to and during schizogony (mitosis/cytokinesis) but dissociate soon after erythrocyte invasion.","method":"Chromatin immunoprecipitation followed by sequencing (ChIP-seq); immunofluorescence assay (IFA)","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide ChIP-seq combined with IFA across the cell cycle, two orthogonal methods, single lab with comprehensive coverage of all 14 chromosomes","pmids":["22507744"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"The C-terminal histone fold domain (HFD) of CenH3, specifically the CATD region containing LLAL residues, is essential for centromere targeting and functional chromosome segregation, as demonstrated by complementation of yeast Cse4p temperature-sensitive mutants. CenH3 was also shown to associate with PfCENP-C (an inner kinetochore protein) by co-immunoprecipitation. Canonical P. falciparum histone H3 does not complement Cse4p function.","method":"Functional complementation assay in Saccharomyces cerevisiae (Cse4p ts mutant); site-directed mutagenesis of CATD/LLAL residues; co-immunoprecipitation","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis-based structure-function dissection combined with heterologous complementation and co-IP, single lab but multiple orthogonal methods","pmids":["24316361"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"CenH3 (PfCENH3) associates with PfCENP-C and with mitotic spindles in P. falciparum, as shown by pull-down assays and co-immunoprecipitation. ChIP-PCR confirmed that PfCENP-C occupies CenH3-marked centromeric loci in vivo, establishing CenH3 as a platform for inner kinetochore assembly.","method":"Co-immunoprecipitation / pull-down assay; ChIP-PCR","journal":"Malaria journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal co-IP and ChIP-PCR from single lab; CenH3 role inferred from its interaction partner's characterisation","pmids":["25476240"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"In P. falciparum gametocytes, CenH3 (PfCENH3) localises to chromatin captured by a non-mitotic nuclear hemispindle and participates in chromatin organisation during cellular elongation. Depolymerisation of microtubules with trifluralin disrupts CenH3/centromere organisation, indicating CenH3-marked centromeres are connected to the nuclear microtubule apparatus in gametocytes.","method":"3D electron microscopy (ultrastructure expansion microscopy); immunofluorescence; pharmacological microtubule depolymerisation with trifluralin","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct imaging with functional perturbation (drug treatment), single lab, specific phenotypic readout for CenH3 localisation","pmids":["36030238"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"finding":"Conditional disruption of kinetochore components PfNDC80 and PfNuf2 causes aberrant mitotic spindle organisation and disruption of the CenH3 (CENH3) centromere marker, demonstrating that proper kinetochore assembly is required to maintain normal CenH3 localisation and centromere integrity during schizogony.","method":"Conditional disruption (knockdown) of PfNDC80/PfNuf2; ultrastructure expansion microscopy; immunofluorescence with CenH3 as centromere marker","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via conditional disruption with direct imaging readout of CenH3 localisation; single lab but rigorous microscopy","pmids":["38987258"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"CenH3 (PfCENH3) was used as a core chromatin component target to validate DiBioCUT&Tag (dimerization-induced biotin ligase proximity labelling combined with anti-biotin CUT&Tag) in P. falciparum, confirming its utility as a centromeric reference mark for genome-wide chromatin profiling from low-input samples.","method":"CUT&Tag; DiBioCUT&Tag (proximity biotinylation followed by CUT&Tag); comparison with ChIP-seq","journal":"Cell reports methods","confidence":"Low","confidence_rationale":"Tier 3 / Weak — CenH3 used as a validation target in a methods paper; single lab, no new mechanistic insight beyond confirming centromeric localisation","pmids":["40675147"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_1333700 (CenH3/PfCENH3) is a centromere-specific histone H3 variant in P. falciparum whose CATD region within the histone fold domain is required for centromere targeting and chromosome segregation; it occupies ~4–4.5 kb centromeric chromatin regions on all 14 chromosomes (co-localising with PfH2A.Z), clusters to a single nuclear focus during schizogony and gametocyte development, associates with inner kinetochore protein PfCENP-C and mitotic spindle components, and its centromeric localisation is disrupted when kinetochore assembly (PfNDC80/PfNuf2) or microtubule dynamics are perturbed."},"narrative":{"mechanistic_narrative":"PF3D7_1333700 (CenH3) is the centromere-specific histone H3 variant of Plasmodium falciparum that defines centromeric chromatin and nucleates kinetochore assembly for chromosome segregation [PMID:22507744, PMID:24316361]. First identified as a genome-encoded histone variant that escaped detection in acid-extracted histone proteomics, consistent with low abundance or restricted expression [PMID:16410041], CenH3 was subsequently shown by ChIP-seq to occupy a discrete ~4–4.5 kb chromatin region on every P. falciparum chromosome, co-localising with the histone variant PfH2A.Z and excluded from pericentric heterochromatin [PMID:22507744]. Its C-terminal histone fold domain—specifically the CATD region and its LLAL residues—is required for centromere targeting and functional chromosome segregation, as canonical H3 fails but CenH3 complements the segregation defect of a yeast Cse4p temperature-sensitive mutant [PMID:24316361]. CenH3 provides the platform for inner kinetochore assembly by associating with PfCENP-C, which occupies CenH3-marked loci in vivo, and it further associates with the mitotic spindle [PMID:24316361, PMID:25476240]. Across the cell cycle CenH3-marked centromeres cluster to a single nuclear focus during schizogony [PMID:22507744] and, in gametocytes, are captured by a non-mitotic hemispindle in a microtubule-dependent manner [PMID:36030238]; conversely, CenH3's own centromeric localisation depends on intact kinetochore assembly, since conditional disruption of PfNDC80/PfNuf2 disrupts the CenH3 mark [PMID:38987258]. Beyond these findings, the biophysics of CenH3 nucleosome deposition and its dedicated chaperone machinery have not been characterized in the available corpus.","teleology":[{"year":2006,"claim":"Establishing whether P. falciparum encodes a centromeric H3 variant at all was the first step; genome analysis identified CenH3 but its protein was not detected proteomically, flagging low abundance or restricted expression as a defining feature.","evidence":"genome analysis, mass spectrometry of acid-extracted histones, RT-PCR and immunoblotting in P. falciparum","pmids":["16410041"],"confidence":"Medium","gaps":["Negative MS detection does not distinguish low abundance from absent expression","No localisation or functional data at this stage","Cell-cycle expression profile not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"Where CenH3 sits in the genome and how it behaves across the cell cycle was unknown; genome-wide ChIP-seq defined discrete ~4–4.5 kb centromeric domains on all chromosomes co-marked with PfH2A.Z, and imaging showed centromere clustering to a single focus during schizogony.","evidence":"ChIP-seq across all 14 chromosomes plus cell-cycle immunofluorescence in P. falciparum","pmids":["22507744"],"confidence":"High","gaps":["Sequence/structural determinants of centromere targeting not defined","Mechanism of centromere clustering unknown","Deposition machinery not identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Which region confers CenH3 identity and function was resolved by mapping targeting to the CATD/LLAL residues of the histone fold domain, with heterologous yeast complementation proving functional conservation and co-IP linking CenH3 to PfCENP-C.","evidence":"site-directed mutagenesis, functional complementation of a S. cerevisiae Cse4p ts mutant, and co-immunoprecipitation","pmids":["24316361"],"confidence":"High","gaps":["Structural basis of CATD-mediated targeting in P. falciparum not solved","Direct vs indirect nature of the CenH3–PfCENP-C interaction not established","In vivo requirement of LLAL residues in the parasite untested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Whether CenH3 serves as the assembly platform for the kinetochore was addressed by showing reciprocal association with PfCENP-C and mitotic spindles, with ChIP-PCR placing PfCENP-C at CenH3-marked loci in vivo.","evidence":"co-immunoprecipitation/pull-down and ChIP-PCR in P. falciparum","pmids":["25476240"],"confidence":"Medium","gaps":["Full inner kinetochore composition anchored by CenH3 not defined","Spindle association may be indirect","CenH3 role largely inferred from partner characterisation"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"Whether CenH3 organisation extends beyond mitosis was tested in gametocytes, showing CenH3-marked centromeres are captured by a non-mitotic hemispindle and depend on intact microtubules.","evidence":"ultrastructure expansion 3D-EM, immunofluorescence, and trifluralin microtubule depolymerisation in P. falciparum gametocytes","pmids":["36030238"],"confidence":"Medium","gaps":["Molecular linker between CenH3 centromeres and the hemispindle unknown","Functional consequence for gametocyte development not defined","Pharmacological perturbation may have off-target effects"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"claim":"The directionality of the CenH3–kinetochore relationship was probed by showing that CenH3 localisation itself requires intact kinetochore assembly, since conditional loss of PfNDC80/PfNuf2 disrupts the CenH3 centromere mark.","evidence":"conditional disruption of PfNDC80/PfNuf2 with ultrastructure expansion microscopy using CenH3 as centromere marker in P. falciparum","pmids":["38987258"],"confidence":"Medium","gaps":["Whether CenH3 is lost or mislocalised is not distinguished","Direct vs indirect dependence unresolved","Effect on CenH3 deposition kinetics unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"CenH3 was leveraged as a validated centromeric reference mark to benchmark a low-input chromatin profiling method, reinforcing its status as the definitive centromere marker.","evidence":"CUT&Tag and DiBioCUT&Tag proximity labelling compared to ChIP-seq in P. falciparum","pmids":["40675147"],"confidence":"Low","gaps":["Methods validation only; no new CenH3 mechanism","Does not address deposition or dynamics","CenH3 used as a tool rather than the object of study"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The CenH3-specific deposition/chaperone machinery, the structural basis of CATD-directed centromere targeting in the parasite, and the epigenetic mechanism specifying centromere position remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No CenH3 chaperone or loading factor identified","No structure of a P. falciparum CenH3 nucleosome","Mechanism of centromere establishment/maintenance unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Inner kinetochore (CenH3–PfCENP-C association)"],"partners":["PFCENP-C","PFH2A.Z (CO-LOCALISATION AT CENTROMERES)"],"other_free_text":["Centromere-specific histone H3 variant occupying discrete ~4–4.5 kb domains on all 14 P. falciparum chromosomes [#1]","CATD/LLAL residues of the histone fold domain are required for centromere targeting; functionally complements yeast Cse4p [#2]","Centromeres cluster to a single nuclear focus during schizogony [#1] and are captured by a non-mitotic hemispindle in gametocytes [#4]","CenH3 localisation depends on intact kinetochore assembly (PfNDC80/PfNuf2) [#5]","Protein not detected by acid-extracted histone mass spectrometry, indicating low abundance or restricted expression [#0]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"16410041","id":"PMC_16410041","title":"The malaria parasite Plasmodium falciparum histones: organization, expression, and acetylation.","date":"2006","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/16410041","citation_count":154,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3","PfH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19351122","id":"PMC_19351122","title":"Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum.","date":"2009","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/19351122","citation_count":119,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22260922","id":"PMC_22260922","title":"Plasmodium falciparum histones induce endothelial proinflammatory response and barrier dysfunction.","date":"2012","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/22260922","citation_count":93,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22507744","id":"PMC_22507744","title":"Plasmodium falciparum centromeres display a unique epigenetic makeup and cluster prior to and during schizogony.","date":"2012","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/22507744","citation_count":59,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3","PfCenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36030238","id":"PMC_36030238","title":"Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36030238","citation_count":31,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfCenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38987258","id":"PMC_38987258","title":"Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38987258","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24316361","id":"PMC_24316361","title":"Plasmodium falciparum CENH3 is able to functionally complement Cse4p and its, C-terminus is essential for centromere function.","date":"2013","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/24316361","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3","PfCenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25476240","id":"PMC_25476240","title":"The dimerization domain of PfCENP-C is required for its functions as a centromere protein in human malaria parasite Plasmodium falciparum.","date":"2014","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/25476240","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3","PfCenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29935010","id":"PMC_29935010","title":"Centromere and its associated proteins-what we know about them in Plasmodium falciparum.","date":"2018","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/29935010","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40675147","id":"PMC_40675147","title":"CUT&Tag and DiBioCUT&Tag enable investigation of the AT-rich epigenome of Plasmodium falciparum from low-input samples.","date":"2025","source":"Cell reports methods","url":"https://pubmed.ncbi.nlm.nih.gov/40675147","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CenH3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7531,"output_tokens":2142,"usd":0.027362,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7375,"output_tokens":3598,"usd":0.063412,"stage2_stop_reason":"end_turn"},"total_usd":0.090774,"stage1_batch_id":"msgbatch_011BPjN2BL33F8cJXD1r7V7T","stage2_batch_id":"msgbatch_01Qwg6vP7qxkpL6Gsc7tajsb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"PF3D7_1333700 (CenH3) was identified as a histone variant encoded in the P. falciparum genome; however, mass spectrometry of acid-extracted histones failed to detect CenH3 protein synthesis, while all other histones were confirmed, suggesting either low abundance or restricted expression.\",\n      \"method\": \"Genome analysis and mass spectrometry proteomics; real-time RT-PCR; immunoblotting\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry plus gene expression analysis from a single lab; negative detection result for the protein itself is explicitly stated\",\n      \"pmids\": [\"16410041\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CenH3 (PfCENH3) occupies a defined ~4–4.5 kb chromatin region on each P. falciparum chromosome as determined by ChIP-seq, co-localises with histone variant PfH2A.Z at centromeres, and is absent from pericentric heterochromatin. Immunofluorescence showed that centromeres marked by CenH3 cluster to a single nuclear location prior to and during schizogony (mitosis/cytokinesis) but dissociate soon after erythrocyte invasion.\",\n      \"method\": \"Chromatin immunoprecipitation followed by sequencing (ChIP-seq); immunofluorescence assay (IFA)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide ChIP-seq combined with IFA across the cell cycle, two orthogonal methods, single lab with comprehensive coverage of all 14 chromosomes\",\n      \"pmids\": [\"22507744\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The C-terminal histone fold domain (HFD) of CenH3, specifically the CATD region containing LLAL residues, is essential for centromere targeting and functional chromosome segregation, as demonstrated by complementation of yeast Cse4p temperature-sensitive mutants. CenH3 was also shown to associate with PfCENP-C (an inner kinetochore protein) by co-immunoprecipitation. Canonical P. falciparum histone H3 does not complement Cse4p function.\",\n      \"method\": \"Functional complementation assay in Saccharomyces cerevisiae (Cse4p ts mutant); site-directed mutagenesis of CATD/LLAL residues; co-immunoprecipitation\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis-based structure-function dissection combined with heterologous complementation and co-IP, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24316361\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CenH3 (PfCENH3) associates with PfCENP-C and with mitotic spindles in P. falciparum, as shown by pull-down assays and co-immunoprecipitation. ChIP-PCR confirmed that PfCENP-C occupies CenH3-marked centromeric loci in vivo, establishing CenH3 as a platform for inner kinetochore assembly.\",\n      \"method\": \"Co-immunoprecipitation / pull-down assay; ChIP-PCR\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal co-IP and ChIP-PCR from single lab; CenH3 role inferred from its interaction partner's characterisation\",\n      \"pmids\": [\"25476240\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In P. falciparum gametocytes, CenH3 (PfCENH3) localises to chromatin captured by a non-mitotic nuclear hemispindle and participates in chromatin organisation during cellular elongation. Depolymerisation of microtubules with trifluralin disrupts CenH3/centromere organisation, indicating CenH3-marked centromeres are connected to the nuclear microtubule apparatus in gametocytes.\",\n      \"method\": \"3D electron microscopy (ultrastructure expansion microscopy); immunofluorescence; pharmacological microtubule depolymerisation with trifluralin\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct imaging with functional perturbation (drug treatment), single lab, specific phenotypic readout for CenH3 localisation\",\n      \"pmids\": [\"36030238\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Conditional disruption of kinetochore components PfNDC80 and PfNuf2 causes aberrant mitotic spindle organisation and disruption of the CenH3 (CENH3) centromere marker, demonstrating that proper kinetochore assembly is required to maintain normal CenH3 localisation and centromere integrity during schizogony.\",\n      \"method\": \"Conditional disruption (knockdown) of PfNDC80/PfNuf2; ultrastructure expansion microscopy; immunofluorescence with CenH3 as centromere marker\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via conditional disruption with direct imaging readout of CenH3 localisation; single lab but rigorous microscopy\",\n      \"pmids\": [\"38987258\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CenH3 (PfCENH3) was used as a core chromatin component target to validate DiBioCUT&Tag (dimerization-induced biotin ligase proximity labelling combined with anti-biotin CUT&Tag) in P. falciparum, confirming its utility as a centromeric reference mark for genome-wide chromatin profiling from low-input samples.\",\n      \"method\": \"CUT&Tag; DiBioCUT&Tag (proximity biotinylation followed by CUT&Tag); comparison with ChIP-seq\",\n      \"journal\": \"Cell reports methods\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — CenH3 used as a validation target in a methods paper; single lab, no new mechanistic insight beyond confirming centromeric localisation\",\n      \"pmids\": [\"40675147\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_1333700 (CenH3/PfCENH3) is a centromere-specific histone H3 variant in P. falciparum whose CATD region within the histone fold domain is required for centromere targeting and chromosome segregation; it occupies ~4–4.5 kb centromeric chromatin regions on all 14 chromosomes (co-localising with PfH2A.Z), clusters to a single nuclear focus during schizogony and gametocyte development, associates with inner kinetochore protein PfCENP-C and mitotic spindle components, and its centromeric localisation is disrupted when kinetochore assembly (PfNDC80/PfNuf2) or microtubule dynamics are perturbed.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_1333700 (CenH3) is the centromere-specific histone H3 variant of Plasmodium falciparum that defines centromeric chromatin and nucleates kinetochore assembly for chromosome segregation [#1, #2]. First identified as a genome-encoded histone variant that escaped detection in acid-extracted histone proteomics, consistent with low abundance or restricted expression [#0], CenH3 was subsequently shown by ChIP-seq to occupy a discrete ~4\\u20134.5 kb chromatin region on every P. falciparum chromosome, co-localising with the histone variant PfH2A.Z and excluded from pericentric heterochromatin [#1]. Its C-terminal histone fold domain\\u2014specifically the CATD region and its LLAL residues\\u2014is required for centromere targeting and functional chromosome segregation, as canonical H3 fails but CenH3 complements the segregation defect of a yeast Cse4p temperature-sensitive mutant [#2]. CenH3 provides the platform for inner kinetochore assembly by associating with PfCENP-C, which occupies CenH3-marked loci in vivo, and it further associates with the mitotic spindle [#2, #3]. Across the cell cycle CenH3-marked centromeres cluster to a single nuclear focus during schizogony [#1] and, in gametocytes, are captured by a non-mitotic hemispindle in a microtubule-dependent manner [#4]; conversely, CenH3's own centromeric localisation depends on intact kinetochore assembly, since conditional disruption of PfNDC80/PfNuf2 disrupts the CenH3 mark [#5]. Beyond these findings, the biophysics of CenH3 nucleosome deposition and its dedicated chaperone machinery have not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing whether P. falciparum encodes a centromeric H3 variant at all was the first step; genome analysis identified CenH3 but its protein was not detected proteomically, flagging low abundance or restricted expression as a defining feature.\",\n      \"evidence\": \"genome analysis, mass spectrometry of acid-extracted histones, RT-PCR and immunoblotting in P. falciparum\",\n      \"pmids\": [\"16410041\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative MS detection does not distinguish low abundance from absent expression\", \"No localisation or functional data at this stage\", \"Cell-cycle expression profile not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Where CenH3 sits in the genome and how it behaves across the cell cycle was unknown; genome-wide ChIP-seq defined discrete ~4\\u20134.5 kb centromeric domains on all chromosomes co-marked with PfH2A.Z, and imaging showed centromere clustering to a single focus during schizogony.\",\n      \"evidence\": \"ChIP-seq across all 14 chromosomes plus cell-cycle immunofluorescence in P. falciparum\",\n      \"pmids\": [\"22507744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sequence/structural determinants of centromere targeting not defined\", \"Mechanism of centromere clustering unknown\", \"Deposition machinery not identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Which region confers CenH3 identity and function was resolved by mapping targeting to the CATD/LLAL residues of the histone fold domain, with heterologous yeast complementation proving functional conservation and co-IP linking CenH3 to PfCENP-C.\",\n      \"evidence\": \"site-directed mutagenesis, functional complementation of a S. cerevisiae Cse4p ts mutant, and co-immunoprecipitation\",\n      \"pmids\": [\"24316361\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CATD-mediated targeting in P. falciparum not solved\", \"Direct vs indirect nature of the CenH3\\u2013PfCENP-C interaction not established\", \"In vivo requirement of LLAL residues in the parasite untested\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether CenH3 serves as the assembly platform for the kinetochore was addressed by showing reciprocal association with PfCENP-C and mitotic spindles, with ChIP-PCR placing PfCENP-C at CenH3-marked loci in vivo.\",\n      \"evidence\": \"co-immunoprecipitation/pull-down and ChIP-PCR in P. falciparum\",\n      \"pmids\": [\"25476240\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full inner kinetochore composition anchored by CenH3 not defined\", \"Spindle association may be indirect\", \"CenH3 role largely inferred from partner characterisation\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Whether CenH3 organisation extends beyond mitosis was tested in gametocytes, showing CenH3-marked centromeres are captured by a non-mitotic hemispindle and depend on intact microtubules.\",\n      \"evidence\": \"ultrastructure expansion 3D-EM, immunofluorescence, and trifluralin microtubule depolymerisation in P. falciparum gametocytes\",\n      \"pmids\": [\"36030238\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular linker between CenH3 centromeres and the hemispindle unknown\", \"Functional consequence for gametocyte development not defined\", \"Pharmacological perturbation may have off-target effects\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The directionality of the CenH3\\u2013kinetochore relationship was probed by showing that CenH3 localisation itself requires intact kinetochore assembly, since conditional loss of PfNDC80/PfNuf2 disrupts the CenH3 centromere mark.\",\n      \"evidence\": \"conditional disruption of PfNDC80/PfNuf2 with ultrastructure expansion microscopy using CenH3 as centromere marker in P. falciparum\",\n      \"pmids\": [\"38987258\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CenH3 is lost or mislocalised is not distinguished\", \"Direct vs indirect dependence unresolved\", \"Effect on CenH3 deposition kinetics unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"CenH3 was leveraged as a validated centromeric reference mark to benchmark a low-input chromatin profiling method, reinforcing its status as the definitive centromere marker.\",\n      \"evidence\": \"CUT&Tag and DiBioCUT&Tag proximity labelling compared to ChIP-seq in P. falciparum\",\n      \"pmids\": [\"40675147\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Methods validation only; no new CenH3 mechanism\", \"Does not address deposition or dynamics\", \"CenH3 used as a tool rather than the object of study\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The CenH3-specific deposition/chaperone machinery, the structural basis of CATD-directed centromere targeting in the parasite, and the epigenetic mechanism specifying centromere position remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No CenH3 chaperone or loading factor identified\", \"No structure of a P. falciparum CenH3 nucleosome\", \"Mechanism of centromere establishment/maintenance unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0030527\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000775\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0000776\", \"supporting_discovery_ids\": [2, 3, 5]},\n      {\"term_id\": \"GO:0000778\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007059\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0034508\", \"supporting_discovery_ids\": [2, 3, 5]},\n      {\"term_id\": \"GO:0007049\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"complexes\": [\n      \"Inner kinetochore (CenH3\\u2013PfCENP-C association)\"\n    ],\n    \"partners\": [\n      \"PfCENP-C\",\n      \"PfH2A.Z (co-localisation at centromeres)\"\n    ],\n    \"other_free_text\": [\n      \"Centromere-specific histone H3 variant occupying discrete ~4\\u20134.5 kb domains on all 14 P. falciparum chromosomes [#1]\",\n      \"CATD/LLAL residues of the histone fold domain are required for centromere targeting; functionally complements yeast Cse4p [#2]\",\n      \"Centromeres cluster to a single nuclear focus during schizogony [#1] and are captured by a non-mitotic hemispindle in gametocytes [#4]\",\n      \"CenH3 localisation depends on intact kinetochore assembly (PfNDC80/PfNuf2) [#5]\",\n      \"Protein not detected by acid-extracted histone mass spectrometry, indicating low abundance or restricted expression [#0]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1333700","name":null,"product":"histone H3-like centromeric protein CSE4","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1348400","name":null,"product":"histone H3-like centromeric protein CSE4, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1266900","name":null,"product":"histone H3-like centromeric protein CSE4, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"CenH3","evaluation":null}