{"gene":"CENPH","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1999,"finding":"CENP-H is a constitutive kinetochore protein containing a coiled-coil structure and a nuclear localization signal, specifically and constitutively localized at kinetochores throughout the cell cycle.","method":"Protein isolation, sequence analysis, immunofluorescence with anti-CENP-H antibody","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — initial characterization using immunofluorescence, single lab, consistent with subsequent studies","pmids":["10488063"],"is_preprint":false},{"year":2001,"finding":"CENP-H is required for targeting CENP-C, but not CENP-A, to the centromere in vertebrate cells; loss of CENP-H causes metaphase arrest consistent with centromere dysfunction, establishing a hierarchical centromere assembly pathway in which CENP-A localization is upstream of both CENP-C and CENP-H.","method":"Conditional loss-of-function knockout in chicken DT40 cells, immunocytochemistry","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with defined cellular phenotype, epistasis established, replicated by independent RNAi study","pmids":["11500386"],"is_preprint":false},{"year":2005,"finding":"The functional region of CENP-H interacts with Hec1, a member of the Nuf2 complex, both by yeast two-hybrid and coimmunoprecipitation; CENP-H and Hec1 form stable associations at centromeres during mitosis (by FRAP), suggesting the Nuf2 complex acts as a connector between inner and outer kinetochores.","method":"Yeast two-hybrid, coimmunoprecipitation, FRAP (photobleaching experiments) in chicken DT40 cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and yeast two-hybrid plus FRAP, multiple orthogonal methods in one study","pmids":["15713649"],"is_preprint":false},{"year":2006,"finding":"RNAi knockdown of CENP-H in human HEp-2 cells to <5% of normal levels causes misaligned chromosomes and multipolar spindles, slightly reduces CENP-C levels at kinetochores, reduces CENP-E at misaligned chromosomes, but does not cause mitotic arrest and leaves hBubR1 localization normal, indicating CENP-H is required for kinetochore architecture including CENP-E recruitment.","method":"RNAi knockdown, immunofluorescence, Western blot in human HEp-2 cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotypes and protein-level readouts, single lab","pmids":["16875666"],"is_preprint":false},{"year":2007,"finding":"CENP-H and CENP-C co-localize to discontinuous CENP-A chromatin subdomains at human neocentromeres, defining an inner kinetochore chromatin structure consistent with higher-order chromatin looping models.","method":"Chromatin immunoprecipitation on CHIP (ChIP-on-chip) using BAC and PCR-amplicon microarrays","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-on-chip with multiple resolution microarrays, single lab","pmids":["17651496"],"is_preprint":false},{"year":2009,"finding":"CENP-H and CENP-K form a stable ~1:1 stoichiometry subcomplex (resistant to high salt) purified by tandem affinity purification; bioinformatic analysis indicates both are enriched in coiled-coil regions, and their interacting functional regions map to their N- and C-terminals, suggesting heterodimeric coiled-coil formation within the inner kinetochore.","method":"Tandem affinity purification (TAP) from HEK293 cells expressing TAP-CENP-K, bioinformatic coiled-coil analysis","journal":"Science in China. Series C, Life sciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — TAP purification with domain-mapping analysis, single lab, functional validation lacking","pmids":["19381461"],"is_preprint":false},{"year":2009,"finding":"TRIM36 interacts with CENP-H (identified by yeast two-hybrid) and co-localizes with alpha-tubulin; TRIM36 has ubiquitin ligase activity and its overexpression decelerates the cell cycle, suggesting a functional link between TRIM36 and CENP-H in chromosome segregation.","method":"Yeast two-hybrid, immunofluorescence, cell cycle assay","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid interaction only, no direct co-IP confirmation, single lab","pmids":["19232519"],"is_preprint":false},{"year":2010,"finding":"Loss of cenph in zebrafish (stac mutant) causes mitotic chromosome missegregation, G2/M arrest, hyperactivation of the intrinsic apoptosis pathway (partially blocked by p53 knockdown), and embryonic lethality; heterozygosity for cenph reduces invasive tumor development, establishing CENPH as essential for mitosis and linking it to tumor suppression in vivo.","method":"Transposon insertional mutant, antisense morpholino knockdown, mRNA rescue, p53 co-knockdown epistasis, in vivo tumor incidence assay in zebrafish","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic approaches (mutant + morpholino + mRNA rescue + epistasis), defined cellular and organismal phenotypes","pmids":["20573960"],"is_preprint":false},{"year":2015,"finding":"CSPP1 binds CENP-H both in vitro and in vivo; CSPP1 depletion impairs chromosome oscillation and spindle assembly checkpoint satisfaction similarly to CENP-H depletion; disrupting the CENP-H/CSPP1 interaction with a membrane-permeable competing peptide causes mitotic arrest and chromosome segregation defects; CSPP1 overexpression decreases kinetochore movement speed, establishing CSPP1 as a CENP-H-interacting regulator of kinetochore–microtubule dynamics.","method":"In vitro binding assay, co-immunoprecipitation, RNAi depletion, competing peptide perturbation, live-cell imaging of chromosome movement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo binding confirmed, functional perturbation with peptide competitor, multiple orthogonal methods, single lab","pmids":["26378239"],"is_preprint":false},{"year":2016,"finding":"CenpH is required for meiotic G2/M transition in mouse oocytes: depletion of CenpH reduces cyclin B1 protein levels, attenuates MPF (maturation-promoting factor) activation, and severely impairs meiotic resumption; CenpH protects cyclin B1 from APC/C(Cdh1)-mediated destruction; exogenous cyclin B1 rescues the G2/M transition defect; however, CenpH depletion does not affect spindle organization or cell cycle progression after germinal vesicle breakdown.","method":"Morpholino injection in mouse oocytes, Western blot, rescue by exogenous cyclin B1 expression, epistasis with APC/CCdh1 pathway","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — morpholino KD with biochemical readouts, mRNA rescue, epistasis with APC/CCdh1, multiple orthogonal approaches in one study","pmids":["27993978"],"is_preprint":false},{"year":2017,"finding":"CENPH interacts physically with GOLPH3 (confirmed by co-immunoprecipitation, GST pull-down, His-tag pull-down, and confocal colocalization); through this interaction, CENPH attenuates both mTORC1 and mTORC2 signaling and reduces sensitivity to the mTOR inhibitor rapamycin in colorectal cancer cells.","method":"Co-immunoprecipitation, GST pull-down, His-tag pull-down, laser scanning confocal microscopy, Western blot for mTOR pathway components, MTT/colony formation assays","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal binding assays confirming CENPH–GOLPH3 interaction, functional pathway readout, single lab","pmids":["28819418"],"is_preprint":false},{"year":2017,"finding":"CENP-H knockdown in hepatocellular carcinoma Hep3B cells inhibits proliferation, induces apoptosis with activation of cleaved caspase-3, and increases Bax/Bcl-2 ratio at both mRNA and protein levels, placing CENP-H upstream of the mitochondrial (intrinsic) apoptotic pathway.","method":"siRNA knockdown, MTT assay, colony formation, transmission electron microscopy, Western blot, qRT-PCR, xenograft mouse model with IHC","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with multiple cellular and in vivo readouts, consistent mechanistic pathway placement, single lab","pmids":["28498417"],"is_preprint":false}],"current_model":"CENP-H is a constitutive inner kinetochore component (part of the CCAN) that is required for recruiting CENP-C (but not CENP-A) to centromeres, interacts with the Nuf2/Hec1 complex to connect inner and outer kinetochores, forms a stable heterodimeric coiled-coil subcomplex with CENP-K, cooperates with CSPP1 to regulate kinetochore–microtubule dynamics and chromosome oscillation, protects cyclin B1 from APC/C(Cdh1)-mediated degradation to enable meiotic G2/M transition, and modulates mTOR signaling through interaction with GOLPH3."},"narrative":{"mechanistic_narrative":"CENP-H is a constitutive inner kinetochore protein that localizes to centromeres throughout the cell cycle and is required for proper kinetochore assembly and chromosome segregation [PMID:10488063, PMID:11500386]. Within the centromere assembly hierarchy, CENP-A localization is upstream of CENP-H, and CENP-H is in turn required to recruit CENP-C—but not CENP-A—to the centromere, with its loss producing centromere dysfunction and metaphase arrest [PMID:11500386]; CENP-H and CENP-C co-occupy discontinuous CENP-A chromatin subdomains at neocentromeres, defining the inner kinetochore chromatin architecture [PMID:17651496]. CENP-H forms a stable, salt-resistant ~1:1 coiled-coil heterodimer with CENP-K and connects the inner kinetochore to the outer kinetochore through interaction with Hec1 of the Nuf2 complex [PMID:15713649, PMID:19381461]. Functionally, CENP-H cooperates with CSPP1 to regulate kinetochore–microtubule dynamics, chromosome oscillation, and spindle assembly checkpoint satisfaction [PMID:26378239]. CENP-H is essential for mitosis in vivo: its loss in zebrafish causes chromosome missegregation, G2/M arrest, and p53-dependent intrinsic apoptosis, and CENPH heterozygosity reduces tumor invasion [PMID:20573960]. In meiosis, CENP-H is required for the oocyte G2/M transition by protecting cyclin B1 from APC/C(Cdh1)-mediated degradation, thereby enabling MPF activation [PMID:27993978]. Beyond its kinetochore role, CENP-H physically interacts with GOLPH3 to attenuate mTORC1/mTORC2 signaling [PMID:28819418].","teleology":[{"year":1999,"claim":"Established CENP-H as a bona fide constitutive kinetochore component, defining a structural protein present throughout the cell cycle rather than a transiently recruited factor.","evidence":"Protein isolation, sequence analysis, and immunofluorescence with anti-CENP-H antibody","pmids":["10488063"],"confidence":"Medium","gaps":["Did not define functional partners or the assembly hierarchy","Coiled-coil and NLS inferred from sequence, not functionally tested"]},{"year":2001,"claim":"Positioned CENP-H within the centromere assembly hierarchy by showing it is required to recruit CENP-C but not CENP-A, establishing CENP-A as the upstream determinant.","evidence":"Conditional loss-of-function knockout in chicken DT40 cells with immunocytochemistry","pmids":["11500386"],"confidence":"High","gaps":["Molecular basis of CENP-C recruitment not defined","Direct vs indirect dependence not resolved"]},{"year":2005,"claim":"Identified the physical bridge between inner and outer kinetochore by showing CENP-H stably associates with Hec1 of the Nuf2 complex.","evidence":"Yeast two-hybrid, reciprocal co-immunoprecipitation, and FRAP in chicken DT40 cells","pmids":["15713649"],"confidence":"High","gaps":["Stoichiometry and structural interface with Hec1 unresolved","Functional consequence of disrupting the interaction not tested"]},{"year":2006,"claim":"Extended CENP-H's architectural role to human cells, showing it supports CENP-E recruitment and chromosome alignment without being strictly required for mitotic arrest.","evidence":"RNAi knockdown with immunofluorescence and Western blot in human HEp-2 cells","pmids":["16875666"],"confidence":"Medium","gaps":["Mechanism of CENP-E recruitment not defined","Partial CENP-C reduction not mechanistically explained"]},{"year":2007,"claim":"Defined the inner kinetochore chromatin organization by mapping CENP-H and CENP-C to discontinuous CENP-A subdomains, supporting higher-order chromatin looping.","evidence":"ChIP-on-chip on neocentromeres using BAC and PCR-amplicon microarrays","pmids":["17651496"],"confidence":"Medium","gaps":["Looping model not directly demonstrated","Restricted to neocentromere context"]},{"year":2009,"claim":"Resolved a core subcomplex by demonstrating CENP-H and CENP-K form a stable ~1:1 coiled-coil heterodimer within the inner kinetochore.","evidence":"Tandem affinity purification from HEK293 cells with bioinformatic coiled-coil domain mapping","pmids":["19381461"],"confidence":"Medium","gaps":["No high-resolution structure of the heterodimer","Functional validation of mapped interaction regions lacking"]},{"year":2010,"claim":"Established CENP-H as essential for mitosis in vivo and linked it to tumor suppression, connecting kinetochore integrity to organismal viability and cancer.","evidence":"Zebrafish transposon mutant, morpholino, mRNA rescue, p53 epistasis, and in vivo tumor incidence assay","pmids":["20573960"],"confidence":"High","gaps":["Molecular trigger of intrinsic apoptosis downstream of missegregation not defined","Mechanism of tumor suppression beyond mitotic fidelity unclear"]},{"year":2015,"claim":"Identified CSPP1 as a CENP-H-interacting regulator of kinetochore–microtubule dynamics and showed the interaction is functionally required for chromosome oscillation and checkpoint satisfaction.","evidence":"In vitro binding, co-IP, RNAi, competing peptide perturbation, and live-cell imaging of chromosome movement","pmids":["26378239"],"confidence":"High","gaps":["Structural basis of CENP-H/CSPP1 interface not defined","How the interaction couples to microtubule attachment kinetics unresolved"]},{"year":2016,"claim":"Revealed a meiotic role distinct from kinetochore architecture, showing CENP-H protects cyclin B1 from APC/C(Cdh1) to drive the oocyte G2/M transition.","evidence":"Morpholino in mouse oocytes, Western blot, exogenous cyclin B1 rescue, and APC/C(Cdh1) epistasis","pmids":["27993978"],"confidence":"High","gaps":["Direct mechanism by which CENP-H shields cyclin B1 from APC/C unknown","Whether this function is kinetochore-dependent unresolved"]},{"year":2017,"claim":"Uncovered a non-kinetochore signaling function in which CENP-H binds GOLPH3 to attenuate mTORC1/mTORC2, and placed CENP-H upstream of the intrinsic apoptotic pathway in cancer cells.","evidence":"Co-IP, GST and His pull-downs, confocal colocalization, mTOR pathway Western blot in colorectal cancer cells; siRNA knockdown with apoptosis assays and xenografts in hepatocellular carcinoma","pmids":["28819418","28498417"],"confidence":"Medium","gaps":["Mechanism linking CENP-H/GOLPH3 to mTOR regulation not defined","Whether the signaling role is separable from the kinetochore role unclear"]},{"year":null,"claim":"The structural basis of CENP-H's integration into the CCAN and how its distinct kinetochore, meiotic cyclin B1, and mTOR-signaling functions are mechanistically coordinated remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of CENP-H within the inner kinetochore","Direct biochemical mechanism of cyclin B1 protection unknown","Connection between CENP-H/GOLPH3 binding and mTOR modulation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,2,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,8]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,3,7,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7,11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10]}],"complexes":["CCAN (inner kinetochore)","CENP-H/CENP-K heterodimer"],"partners":["CENPK","NDC80/HEC1","CENPC","CSPP1","GOLPH3","TRIM36"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H3R5","full_name":"Centromere protein H","aliases":["Interphase centromere complex protein 35"],"length_aa":247,"mass_kda":28.5,"function":"Component of the CENPA-NAC (nucleosome-associated) complex, a complex that plays a central role in assembly of kinetochore proteins, mitotic progression and chromosome segregation. The CENPA-NAC complex recruits the CENPA-CAD (nucleosome distal) complex and may be involved in incorporation of newly synthesized CENPA into centromeres. Required for chromosome congression and efficiently align the chromosomes on a metaphase plate","subcellular_location":"Nucleus; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q9H3R5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPH","classification":"Common Essential","n_dependent_lines":969,"n_total_lines":1208,"dependency_fraction":0.8021523178807947},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CENPH","total_profiled":1310},"omim":[{"mim_id":"611511","title":"MLF1-INTERACTING PROTEIN; MLF1IP","url":"https://www.omim.org/entry/611511"},{"mim_id":"611510","title":"CENTROMERIC PROTEIN T; CENPT","url":"https://www.omim.org/entry/611510"},{"mim_id":"611509","title":"CENTROMERIC PROTEIN N; CENPN","url":"https://www.omim.org/entry/611509"},{"mim_id":"611506","title":"CENTROMERIC PROTEIN Q; CENPQ","url":"https://www.omim.org/entry/611506"},{"mim_id":"611505","title":"CENTROMERIC PROTEIN P; CENPP","url":"https://www.omim.org/entry/611505"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli rim","reliability":"Supported"},{"location":"Mitotic chromosome","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":31.8},{"tissue":"testis","ntpm":27.8}],"url":"https://www.proteinatlas.org/search/CENPH"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q9H3R5","domains":[{"cath_id":"1.20.5","chopping":"77-116_127-189","consensus_level":"medium","plddt":93.7156,"start":77,"end":189},{"cath_id":"4.10.810","chopping":"193-240","consensus_level":"high","plddt":88.5969,"start":193,"end":240}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H3R5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H3R5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H3R5-F1-predicted_aligned_error_v6.png","plddt_mean":81.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPH","jax_strain_url":"https://www.jax.org/strain/search?query=CENPH"},"sequence":{"accession":"Q9H3R5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H3R5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H3R5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H3R5"}},"corpus_meta":[{"pmid":"11500386","id":"PMC_11500386","title":"CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11500386","citation_count":145,"is_preprint":false},{"pmid":"17651496","id":"PMC_17651496","title":"Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres.","date":"2007","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/17651496","citation_count":69,"is_preprint":false},{"pmid":"10488063","id":"PMC_10488063","title":"Characterization of a novel kinetochore protein, CENP-H.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10488063","citation_count":59,"is_preprint":false},{"pmid":"19232519","id":"PMC_19232519","title":"TRIM36 interacts with the kinetochore protein CENP-H and delays cell cycle progression.","date":"2009","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/19232519","citation_count":41,"is_preprint":false},{"pmid":"15713649","id":"PMC_15713649","title":"The functional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15713649","citation_count":39,"is_preprint":false},{"pmid":"17016595","id":"PMC_17016595","title":"Increased expression of CENP-H gene in human oral squamous cell carcinomas harboring high-proliferative activity.","date":"2006","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/17016595","citation_count":23,"is_preprint":false},{"pmid":"28819418","id":"PMC_28819418","title":"CENPH Inhibits Rapamycin Sensitivity by Regulating GOLPH3-dependent mTOR Signaling Pathway in Colorectal Cancer.","date":"2017","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/28819418","citation_count":21,"is_preprint":false},{"pmid":"20573960","id":"PMC_20573960","title":"Interruption of cenph causes mitotic failure and embryonic death, and its haploinsufficiency suppresses cancer in zebrafish.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20573960","citation_count":20,"is_preprint":false},{"pmid":"28498417","id":"PMC_28498417","title":"CENP-H regulates the cell growth of human hepatocellular carcinoma cells through the mitochondrial apoptotic pathway.","date":"2017","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/28498417","citation_count":19,"is_preprint":false},{"pmid":"16875666","id":"PMC_16875666","title":"RNAi knockdown of human kinetochore protein CENP-H.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16875666","citation_count":18,"is_preprint":false},{"pmid":"26378239","id":"PMC_26378239","title":"Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26378239","citation_count":17,"is_preprint":false},{"pmid":"27993978","id":"PMC_27993978","title":"CenpH regulates meiotic G2/M transition by modulating the APC/CCdh1-cyclin B1 pathway in oocytes.","date":"2016","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/27993978","citation_count":11,"is_preprint":false},{"pmid":"23691478","id":"PMC_23691478","title":"Clinical Significance of CENP-H Expression in Uterine Cervical Cancer.","date":"2012","source":"Cancer biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23691478","citation_count":9,"is_preprint":false},{"pmid":"21392133","id":"PMC_21392133","title":"The Aspergillus nidulans CENP-E kinesin motor KipA interacts with the fungal homologue of the centromere-associated protein CENP-H at the kinetochore.","date":"2011","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/21392133","citation_count":9,"is_preprint":false},{"pmid":"19381461","id":"PMC_19381461","title":"CENP-K and CENP-H may form coiled-coils in the kinetochores.","date":"2009","source":"Science in China. Series C, Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/19381461","citation_count":8,"is_preprint":false},{"pmid":"23083211","id":"PMC_23083211","title":"Low prevalence of autoantibodies to CENP-H, -I, -K, -L, -M, -N, -T and -U in a Japanese cohort of anti-centromere positive samples.","date":"2012","source":"Immunopharmacology and immunotoxicology","url":"https://pubmed.ncbi.nlm.nih.gov/23083211","citation_count":4,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9065,"output_tokens":2992,"usd":0.036038,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10366,"output_tokens":3427,"usd":0.068752,"stage2_stop_reason":"end_turn"},"total_usd":0.10479,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"CENP-H is a constitutive kinetochore protein containing a coiled-coil structure and a nuclear localization signal, specifically and constitutively localized at kinetochores throughout the cell cycle.\",\n      \"method\": \"Protein isolation, sequence analysis, immunofluorescence with anti-CENP-H antibody\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — initial characterization using immunofluorescence, single lab, consistent with subsequent studies\",\n      \"pmids\": [\"10488063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CENP-H is required for targeting CENP-C, but not CENP-A, to the centromere in vertebrate cells; loss of CENP-H causes metaphase arrest consistent with centromere dysfunction, establishing a hierarchical centromere assembly pathway in which CENP-A localization is upstream of both CENP-C and CENP-H.\",\n      \"method\": \"Conditional loss-of-function knockout in chicken DT40 cells, immunocytochemistry\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with defined cellular phenotype, epistasis established, replicated by independent RNAi study\",\n      \"pmids\": [\"11500386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The functional region of CENP-H interacts with Hec1, a member of the Nuf2 complex, both by yeast two-hybrid and coimmunoprecipitation; CENP-H and Hec1 form stable associations at centromeres during mitosis (by FRAP), suggesting the Nuf2 complex acts as a connector between inner and outer kinetochores.\",\n      \"method\": \"Yeast two-hybrid, coimmunoprecipitation, FRAP (photobleaching experiments) in chicken DT40 cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and yeast two-hybrid plus FRAP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"15713649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RNAi knockdown of CENP-H in human HEp-2 cells to <5% of normal levels causes misaligned chromosomes and multipolar spindles, slightly reduces CENP-C levels at kinetochores, reduces CENP-E at misaligned chromosomes, but does not cause mitotic arrest and leaves hBubR1 localization normal, indicating CENP-H is required for kinetochore architecture including CENP-E recruitment.\",\n      \"method\": \"RNAi knockdown, immunofluorescence, Western blot in human HEp-2 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotypes and protein-level readouts, single lab\",\n      \"pmids\": [\"16875666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CENP-H and CENP-C co-localize to discontinuous CENP-A chromatin subdomains at human neocentromeres, defining an inner kinetochore chromatin structure consistent with higher-order chromatin looping models.\",\n      \"method\": \"Chromatin immunoprecipitation on CHIP (ChIP-on-chip) using BAC and PCR-amplicon microarrays\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-on-chip with multiple resolution microarrays, single lab\",\n      \"pmids\": [\"17651496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CENP-H and CENP-K form a stable ~1:1 stoichiometry subcomplex (resistant to high salt) purified by tandem affinity purification; bioinformatic analysis indicates both are enriched in coiled-coil regions, and their interacting functional regions map to their N- and C-terminals, suggesting heterodimeric coiled-coil formation within the inner kinetochore.\",\n      \"method\": \"Tandem affinity purification (TAP) from HEK293 cells expressing TAP-CENP-K, bioinformatic coiled-coil analysis\",\n      \"journal\": \"Science in China. Series C, Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — TAP purification with domain-mapping analysis, single lab, functional validation lacking\",\n      \"pmids\": [\"19381461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TRIM36 interacts with CENP-H (identified by yeast two-hybrid) and co-localizes with alpha-tubulin; TRIM36 has ubiquitin ligase activity and its overexpression decelerates the cell cycle, suggesting a functional link between TRIM36 and CENP-H in chromosome segregation.\",\n      \"method\": \"Yeast two-hybrid, immunofluorescence, cell cycle assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid interaction only, no direct co-IP confirmation, single lab\",\n      \"pmids\": [\"19232519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Loss of cenph in zebrafish (stac mutant) causes mitotic chromosome missegregation, G2/M arrest, hyperactivation of the intrinsic apoptosis pathway (partially blocked by p53 knockdown), and embryonic lethality; heterozygosity for cenph reduces invasive tumor development, establishing CENPH as essential for mitosis and linking it to tumor suppression in vivo.\",\n      \"method\": \"Transposon insertional mutant, antisense morpholino knockdown, mRNA rescue, p53 co-knockdown epistasis, in vivo tumor incidence assay in zebrafish\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic approaches (mutant + morpholino + mRNA rescue + epistasis), defined cellular and organismal phenotypes\",\n      \"pmids\": [\"20573960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CSPP1 binds CENP-H both in vitro and in vivo; CSPP1 depletion impairs chromosome oscillation and spindle assembly checkpoint satisfaction similarly to CENP-H depletion; disrupting the CENP-H/CSPP1 interaction with a membrane-permeable competing peptide causes mitotic arrest and chromosome segregation defects; CSPP1 overexpression decreases kinetochore movement speed, establishing CSPP1 as a CENP-H-interacting regulator of kinetochore–microtubule dynamics.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation, RNAi depletion, competing peptide perturbation, live-cell imaging of chromosome movement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo binding confirmed, functional perturbation with peptide competitor, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"26378239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CenpH is required for meiotic G2/M transition in mouse oocytes: depletion of CenpH reduces cyclin B1 protein levels, attenuates MPF (maturation-promoting factor) activation, and severely impairs meiotic resumption; CenpH protects cyclin B1 from APC/C(Cdh1)-mediated destruction; exogenous cyclin B1 rescues the G2/M transition defect; however, CenpH depletion does not affect spindle organization or cell cycle progression after germinal vesicle breakdown.\",\n      \"method\": \"Morpholino injection in mouse oocytes, Western blot, rescue by exogenous cyclin B1 expression, epistasis with APC/CCdh1 pathway\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — morpholino KD with biochemical readouts, mRNA rescue, epistasis with APC/CCdh1, multiple orthogonal approaches in one study\",\n      \"pmids\": [\"27993978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CENPH interacts physically with GOLPH3 (confirmed by co-immunoprecipitation, GST pull-down, His-tag pull-down, and confocal colocalization); through this interaction, CENPH attenuates both mTORC1 and mTORC2 signaling and reduces sensitivity to the mTOR inhibitor rapamycin in colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, His-tag pull-down, laser scanning confocal microscopy, Western blot for mTOR pathway components, MTT/colony formation assays\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal binding assays confirming CENPH–GOLPH3 interaction, functional pathway readout, single lab\",\n      \"pmids\": [\"28819418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CENP-H knockdown in hepatocellular carcinoma Hep3B cells inhibits proliferation, induces apoptosis with activation of cleaved caspase-3, and increases Bax/Bcl-2 ratio at both mRNA and protein levels, placing CENP-H upstream of the mitochondrial (intrinsic) apoptotic pathway.\",\n      \"method\": \"siRNA knockdown, MTT assay, colony formation, transmission electron microscopy, Western blot, qRT-PCR, xenograft mouse model with IHC\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with multiple cellular and in vivo readouts, consistent mechanistic pathway placement, single lab\",\n      \"pmids\": [\"28498417\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENP-H is a constitutive inner kinetochore component (part of the CCAN) that is required for recruiting CENP-C (but not CENP-A) to centromeres, interacts with the Nuf2/Hec1 complex to connect inner and outer kinetochores, forms a stable heterodimeric coiled-coil subcomplex with CENP-K, cooperates with CSPP1 to regulate kinetochore–microtubule dynamics and chromosome oscillation, protects cyclin B1 from APC/C(Cdh1)-mediated degradation to enable meiotic G2/M transition, and modulates mTOR signaling through interaction with GOLPH3.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CENP-H is a constitutive inner kinetochore protein that localizes to centromeres throughout the cell cycle and is required for proper kinetochore assembly and chromosome segregation [#0, #1]. Within the centromere assembly hierarchy, CENP-A localization is upstream of CENP-H, and CENP-H is in turn required to recruit CENP-C—but not CENP-A—to the centromere, with its loss producing centromere dysfunction and metaphase arrest [#1]; CENP-H and CENP-C co-occupy discontinuous CENP-A chromatin subdomains at neocentromeres, defining the inner kinetochore chromatin architecture [#4]. CENP-H forms a stable, salt-resistant ~1:1 coiled-coil heterodimer with CENP-K and connects the inner kinetochore to the outer kinetochore through interaction with Hec1 of the Nuf2 complex [#2, #5]. Functionally, CENP-H cooperates with CSPP1 to regulate kinetochore–microtubule dynamics, chromosome oscillation, and spindle assembly checkpoint satisfaction [#8]. CENP-H is essential for mitosis in vivo: its loss in zebrafish causes chromosome missegregation, G2/M arrest, and p53-dependent intrinsic apoptosis, and CENPH heterozygosity reduces tumor invasion [#7]. In meiosis, CENP-H is required for the oocyte G2/M transition by protecting cyclin B1 from APC/C(Cdh1)-mediated degradation, thereby enabling MPF activation [#9]. Beyond its kinetochore role, CENP-H physically interacts with GOLPH3 to attenuate mTORC1/mTORC2 signaling [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established CENP-H as a bona fide constitutive kinetochore component, defining a structural protein present throughout the cell cycle rather than a transiently recruited factor.\",\n      \"evidence\": \"Protein isolation, sequence analysis, and immunofluorescence with anti-CENP-H antibody\",\n      \"pmids\": [\"10488063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define functional partners or the assembly hierarchy\", \"Coiled-coil and NLS inferred from sequence, not functionally tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Positioned CENP-H within the centromere assembly hierarchy by showing it is required to recruit CENP-C but not CENP-A, establishing CENP-A as the upstream determinant.\",\n      \"evidence\": \"Conditional loss-of-function knockout in chicken DT40 cells with immunocytochemistry\",\n      \"pmids\": [\"11500386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of CENP-C recruitment not defined\", \"Direct vs indirect dependence not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified the physical bridge between inner and outer kinetochore by showing CENP-H stably associates with Hec1 of the Nuf2 complex.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-immunoprecipitation, and FRAP in chicken DT40 cells\",\n      \"pmids\": [\"15713649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural interface with Hec1 unresolved\", \"Functional consequence of disrupting the interaction not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended CENP-H's architectural role to human cells, showing it supports CENP-E recruitment and chromosome alignment without being strictly required for mitotic arrest.\",\n      \"evidence\": \"RNAi knockdown with immunofluorescence and Western blot in human HEp-2 cells\",\n      \"pmids\": [\"16875666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of CENP-E recruitment not defined\", \"Partial CENP-C reduction not mechanistically explained\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the inner kinetochore chromatin organization by mapping CENP-H and CENP-C to discontinuous CENP-A subdomains, supporting higher-order chromatin looping.\",\n      \"evidence\": \"ChIP-on-chip on neocentromeres using BAC and PCR-amplicon microarrays\",\n      \"pmids\": [\"17651496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Looping model not directly demonstrated\", \"Restricted to neocentromere context\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved a core subcomplex by demonstrating CENP-H and CENP-K form a stable ~1:1 coiled-coil heterodimer within the inner kinetochore.\",\n      \"evidence\": \"Tandem affinity purification from HEK293 cells with bioinformatic coiled-coil domain mapping\",\n      \"pmids\": [\"19381461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of the heterodimer\", \"Functional validation of mapped interaction regions lacking\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established CENP-H as essential for mitosis in vivo and linked it to tumor suppression, connecting kinetochore integrity to organismal viability and cancer.\",\n      \"evidence\": \"Zebrafish transposon mutant, morpholino, mRNA rescue, p53 epistasis, and in vivo tumor incidence assay\",\n      \"pmids\": [\"20573960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger of intrinsic apoptosis downstream of missegregation not defined\", \"Mechanism of tumor suppression beyond mitotic fidelity unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified CSPP1 as a CENP-H-interacting regulator of kinetochore–microtubule dynamics and showed the interaction is functionally required for chromosome oscillation and checkpoint satisfaction.\",\n      \"evidence\": \"In vitro binding, co-IP, RNAi, competing peptide perturbation, and live-cell imaging of chromosome movement\",\n      \"pmids\": [\"26378239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CENP-H/CSPP1 interface not defined\", \"How the interaction couples to microtubule attachment kinetics unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed a meiotic role distinct from kinetochore architecture, showing CENP-H protects cyclin B1 from APC/C(Cdh1) to drive the oocyte G2/M transition.\",\n      \"evidence\": \"Morpholino in mouse oocytes, Western blot, exogenous cyclin B1 rescue, and APC/C(Cdh1) epistasis\",\n      \"pmids\": [\"27993978\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism by which CENP-H shields cyclin B1 from APC/C unknown\", \"Whether this function is kinetochore-dependent unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Uncovered a non-kinetochore signaling function in which CENP-H binds GOLPH3 to attenuate mTORC1/mTORC2, and placed CENP-H upstream of the intrinsic apoptotic pathway in cancer cells.\",\n      \"evidence\": \"Co-IP, GST and His pull-downs, confocal colocalization, mTOR pathway Western blot in colorectal cancer cells; siRNA knockdown with apoptosis assays and xenografts in hepatocellular carcinoma\",\n      \"pmids\": [\"28819418\", \"28498417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking CENP-H/GOLPH3 to mTOR regulation not defined\", \"Whether the signaling role is separable from the kinetochore role unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of CENP-H's integration into the CCAN and how its distinct kinetochore, meiotic cyclin B1, and mTOR-signaling functions are mechanistically coordinated remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of CENP-H within the inner kinetochore\", \"Direct biochemical mechanism of cyclin B1 protection unknown\", \"Connection between CENP-H/GOLPH3 binding and mTOR modulation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 2, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 3, 7, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [\"CCAN (inner kinetochore)\", \"CENP-H/CENP-K heterodimer\"],\n    \"partners\": [\"CENPK\", \"NDC80/HEC1\", \"CENPC\", \"CSPP1\", \"GOLPH3\", \"TRIM36\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}