{"gene":"IHO1","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2016,"finding":"IHO1 (CCDC36) directly interacts with HORMAD1 and is essential for meiotic DNA double-strand break (DSB) formation in mice. IHO1 and conserved SPO11-auxiliary proteins MEI4 and REC114 assemble chromatin-bound recombinosomes on meiotic chromosome axes that are predicted activators of DSB formation. HORMAD1 is required for robust recruitment of IHO1 to unsynapsed axes and efficient formation/stabilization of these recombinosomes.","method":"Co-immunoprecipitation, yeast two-hybrid, immunofluorescence localization, mouse knockout/loss-of-function with meiotic phenotype readout","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, direct interaction demonstrated, mouse KO with defined meiotic phenotype, replicated in multiple subsequent studies","pmids":["27723721"],"is_preprint":false},{"year":2018,"finding":"IHO1, MEI4, and REC114 form a ternary complex in mouse spermatocytes on the axes of meiotic chromosomes. MEI4 forms a stable complex with REC114, and the REC114 C-terminal domain interacts with the MEI4 N-terminal domain in vitro. The REC114 N-terminal domain has structural similarity to Pleckstrin homology (PH) domains.","method":"Co-immunoprecipitation in spermatocytes, in vitro complex reconstitution, X-ray crystallography of REC114 N-terminal domain","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, structural analysis, and Co-IP in native tissue; replicated by later studies","pmids":["30569039"],"is_preprint":false},{"year":2017,"finding":"CXXC1 interacts with IHO1 in mouse meiosis, providing a potential molecular link between PRDM9-marked recombination hotspots and the DSB machinery on the chromosome axis, analogous to the yeast Spp1-Mer2 interaction.","method":"Yeast two-hybrid assay, Co-immunoprecipitation","journal":"Chromosoma","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus Co-IP, single lab, functional consequence inferred by analogy rather than direct experiment","pmids":["28527011"],"is_preprint":false},{"year":2017,"finding":"IHO1 is the mammalian ortholog of yeast Mer2/Rec15 and fungal Asy2, establishing evolutionary conservation of the DSB initiation complex scaffold across fungi, plants, and mammals. In Sordaria, the Mer2 ortholog mediates assembly of recombination-initiation complexes and DSBs, as well as homolog pairing and chromosome compaction.","method":"Genetic and cytological analysis of Mer2 mutants in Sordaria, immunolocalization, sequence conservation analysis","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional analysis in a model fungus with multiple mutant alleles and orthogonal cytological methods; orthology to mammalian IHO1 established but mammalian experiments not performed in this paper","pmids":["29021238"],"is_preprint":false},{"year":2019,"finding":"ANKRD31 associates with the DSB-promoting protein complex (including IHO1) on meiotic chromosome axes and is required for normal spatiotemporal patterning of DSB formation, including high rates of DSBs at pseudoautosomal regions (PARs). Loss of ANKRD31 causes delayed and redistributed DSB formation and failure of X-Y chromosome recombination.","method":"Mouse knockout, immunofluorescence co-localization with IHO1 and other DSB factors, cytological analysis of DSB markers","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — mouse KO with defined molecular and cytological phenotypes, co-localization of IHO1 with ANKRD31 complex, replicated by later structural studies","pmids":["31000436"],"is_preprint":false},{"year":2019,"finding":"STAG3 (meiotic cohesin) and REC8 promote axis localization of IHO1 (along with HORMAD1 and MEI4) in spermatocytes. STAG3 deficiency reduces IHO1 recruitment to chromosome axes, demonstrating that meiotic cohesin complexes are upstream of IHO1 axis association.","method":"Mouse knockout (Stag3-deficient), immunofluorescence localization of IHO1 and associated proteins, genetic epistasis","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mouse KO with specific localization phenotype for IHO1, single lab","pmids":["30853435"],"is_preprint":false},{"year":2021,"finding":"DSBs restrict the DSB machinery via at least four distinct negative feedback pathways in mice: (1) ATM kinase activation by DSBs restricts pre-DSB recombinosome numbers without affecting IHO1 axis levels; (2) ATR kinase locally depletes IHO1 near DSB sites; (3) DSB-enabled homolog synapsis promotes depletion of IHO1 and pre-DSB recombinosomes from synapsed axes; (4) DSBs and three DDR kinases (ATM, ATR, PRKDC) enable stage-specific global depletion of IHO1 from all axes.","method":"Mouse genetic models (ATM, ATR, PRKDC inhibition/knockout), immunofluorescence quantification of IHO1 and recombinosome proteins on chromosome axes, epistasis analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic perturbations with specific IHO1 localization readouts, four mechanistically distinct pathways established with orthogonal experiments","pmids":["33619545"],"is_preprint":false},{"year":2021,"finding":"In zebrafish spermatocytes, IHO1 dissociation from chromosome axes occurs in a DSB-dependent manner; persistent IHO1 foci are observed in spo11 mutant spermatocytes where no DSBs are formed, while IHO1 signal kinetics are otherwise similar to wild type.","method":"Zebrafish spo11 mutant analysis, immunofluorescence localization of Iho1 on chromosome axes","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function (spo11 mutant) with specific IHO1 localization phenotype in zebrafish, single study","pmids":["33842489"],"is_preprint":false},{"year":2022,"finding":"REC114 directly interacts with TOPOVIBL (the SPO11 accessory subunit), and this interaction is required for efficient DSB formation genome-wide. IHO1 is part of the accessory factor network (IHO1-REC114-MEI4-MEI1) controlling TOPOVIL catalytic activity. Point mutations in TOPOVIBL that disrupt REC114 binding strongly reduce DSBs in oocytes and sub-telomeric regions in spermatocytes.","method":"Co-immunoprecipitation, structural analysis of conserved interacting domains, mouse point-mutation knockin models, DSB monitoring genome-wide","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural domain identification, in vivo point mutation knockin, genome-wide DSB quantification; IHO1 placed in this complex network","pmids":["36396648"],"is_preprint":false},{"year":2022,"finding":"SKP1 (subunit of SCF E3 ubiquitin ligase) restrains accumulation of the pre-DSB complex (IHO1-REC114-MEI4) on the meiotic chromosome axis. Loss of SKP1 leads to aberrant IHO1 localization. FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and proteasomal degradation, thereby modulating the pre-DSB complex.","method":"Mouse conditional knockout of Skp1, co-immunoprecipitation of FBXO47-SKP1-HORMAD1, ubiquitination assay in HEK293T cells, immunofluorescence of IHO1 axis localization","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mouse KO with IHO1-specific phenotype, co-IP and ubiquitination assay in cell line, single lab","pmids":["35489071"],"is_preprint":false},{"year":2023,"finding":"IHO1 forms coiled-coil-based tetramers. IHO1 directly interacts with the PH domain of REC114, recognizing the same surface used by TOPOVIBL and ANKRD31, suggesting REC114 acts as a regulatory platform for mutually exclusive interactions with multiple partners including IHO1. REC114 forms homodimers and assembles with MEI4 as a 2:1 heterotrimer that further dimerizes, constituting a ternary IHO1-REC114-MEI4 complex.","method":"AlphaFold2 structural modeling, biochemical characterization (analytical ultracentrifugation, SEC-SAXS, pulldown assays), mutagenesis of interaction interfaces","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural modeling validated by biochemical reconstitution and mutagenesis, detailed stoichiometry established for the IHO1-REC114-MEI4 complex","pmids":["37431931"],"is_preprint":false},{"year":2024,"finding":"IHO1 acts as a seed/platform on chromosome axes for biogenesis of DSB-machinery clusters. Axial IHO1 platforms are based on IHO1-HORMAD1 interaction. IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), placing DDK upstream of IHO1 axis association. ANKRD31 enhances both seeding and growth of DSB-machinery clusters independently, providing a complementary pathway when IHO1-HORMAD1 interaction is disrupted.","method":"Mouse genetic models, DDK chemical inhibition, phosphorylation analysis of IHO1, immunofluorescence quantification of IHO1 cluster formation on axes, epistasis with ANKRD31","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and pharmacological perturbations, specific IHO1 phosphorylation and localization readouts, epistasis analysis with ANKRD31","pmids":["38580643"],"is_preprint":false},{"year":2024,"finding":"IHO1 anchors DSB sites (marked by PRDM9 binding) to chromosome axis components HORMAD1 and SYCP3. PRDM9 promotes recruitment of MEI4 and IHO1 to chromatin, and IHO1 in turn tethers these sites to the axis. IHO1, HORMAD1, and SYCP3 remain associated at DSB ends during DSB repair.","method":"ChIP-seq identification of axis-associated genomic sites, co-localization immunofluorescence, mouse genetic models showing PRDM9-dependent IHO1 and MEI4 recruitment","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-seq combined with genetic perturbations and immunofluorescence, multiple orthogonal methods establishing PRDM9→IHO1→axis tethering hierarchy","pmids":["38657614"],"is_preprint":false},{"year":2026,"finding":"MEI1 variants that cause non-obstructive azoospermia disrupt interactions of MEI1 with IHO1 (as well as ANKRD31, REC114, MEI4), as demonstrated by co-immunoprecipitation assays, establishing that IHO1 is a direct binding partner of MEI1 in the meiotic DSB machinery.","method":"Co-immunoprecipitation of MEI1 variants with IHO1 and other meiotic DSB proteins","journal":"Journal of assisted reproduction and genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP assay, single study, interaction disruption used diagnostically rather than mechanistically characterized","pmids":["41706353"],"is_preprint":false}],"current_model":"IHO1 (CCDC36/Mer2 ortholog) is an essential scaffold protein of the meiotic DSB machinery that directly binds HORMAD1 on unsynapsed chromosome axes; this interaction, modulated by DDK-dependent phosphorylation of IHO1, seeds the assembly of pre-DSB recombinosome clusters composed of IHO1 tetramers, REC114 (with which it interacts via REC114's PH domain), and MEI4, which collectively activate the SPO11-TOPOVIBL (TOPOVIL) catalytic complex to generate programmed DSBs; IHO1 is in turn recruited to PRDM9-marked chromatin sites and anchors DSB sites to axis components HORMAD1 and SYCP3, while DSB formation itself triggers negative feedback through ATM- and ATR-dependent local and global depletion of IHO1 from chromosome axes, with additional regulation by the SCF-FBXO47 ubiquitin ligase acting on HORMAD1."},"narrative":{"mechanistic_narrative":"IHO1 (CCDC36), the mammalian ortholog of yeast Mer2/Rec15 and fungal Asy2, is an essential scaffold protein that nucleates the assembly of meiotic DNA double-strand break (DSB) machinery on chromosome axes [PMID:27723721, PMID:29021238]. IHO1 directly binds the axis protein HORMAD1, an interaction required for its robust recruitment to unsynapsed axes, and together with the SPO11-auxiliary proteins MEI4 and REC114 it forms chromatin-bound recombinosomes that promote DSB formation [PMID:27723721, PMID:30569039]. Structurally, IHO1 forms coiled-coil-based tetramers and engages the pleckstrin-homology (PH) domain of REC114 using the same surface that REC114 employs for TOPOVIBL and ANKRD31, so REC114 acts as a regulatory platform for mutually exclusive interactions, while REC114 itself dimerizes and assembles with MEI4 as a higher-order ternary complex [PMID:37431931, PMID:30569039]. This IHO1-REC114-MEI4 accessory network controls the catalytic activity of the SPO11-TOPOVIBL (TOPOVIL) complex that generates programmed DSBs [PMID:36396648]. IHO1 seeds and grows DSB-machinery clusters on the axis through its HORMAD1 interaction, a process dependent on DDK-mediated IHO1 phosphorylation, and it anchors PRDM9-marked DSB sites to the axis components HORMAD1 and SYCP3 [PMID:38580643, PMID:38657614]. Upstream, meiotic cohesin (STAG3/REC8) promotes IHO1 axis localization, and the SCF-FBXO47 ubiquitin ligase restrains pre-DSB complex accumulation via HORMAD1 turnover [PMID:30853435, PMID:35489071]. Once DSBs form, multiple ATM-, ATR-, and PRKDC-dependent negative-feedback pathways locally and globally deplete IHO1 from chromosome axes, limiting further break formation [PMID:33619545, PMID:33842489].","teleology":[{"year":2016,"claim":"Established the founding link between an axis-bound scaffold and meiotic break formation by showing IHO1 binds HORMAD1 and is required for DSBs and recombinosome assembly with MEI4 and REC114.","evidence":"Co-IP, yeast two-hybrid, immunofluorescence, and mouse knockout with meiotic phenotype","pmids":["27723721"],"confidence":"High","gaps":["Stoichiometry and architecture of the IHO1-containing recombinosome not resolved","Mechanism by which recombinosomes activate SPO11 not defined"]},{"year":2017,"claim":"Placed IHO1 in an evolutionarily conserved DSB-initiation framework by identifying it as the Mer2/Rec15/Asy2 ortholog with conserved roles in recombination, pairing, and chromosome compaction.","evidence":"Genetic and cytological analysis of Mer2 orthologs in Sordaria with sequence conservation","pmids":["29021238"],"confidence":"Medium","gaps":["Mammalian functions inferred by orthology, not tested in this work","Whether pairing/compaction roles transfer to IHO1 unknown"]},{"year":2017,"claim":"Proposed a route from PRDM9-marked hotspots to the axis-bound machinery by identifying a CXXC1-IHO1 interaction analogous to yeast Spp1-Mer2.","evidence":"Yeast two-hybrid and Co-IP in mouse meiosis","pmids":["28527011"],"confidence":"Medium","gaps":["Functional consequence inferred by analogy rather than tested directly","Whether CXXC1 recruits IHO1 to hotspots in vivo unresolved"]},{"year":2018,"claim":"Defined the molecular organization of the accessory complex by reconstituting an IHO1-MEI4-REC114 ternary complex and resolving the REC114 PH-like domain that mediates MEI4 binding.","evidence":"Co-IP in spermatocytes, in vitro reconstitution, and crystallography of REC114 N-terminal domain","pmids":["30569039"],"confidence":"High","gaps":["Atomic-level IHO1-REC114 interface not resolved here","Higher-order assembly stoichiometry not determined"]},{"year":2019,"claim":"Identified upstream axis-recruitment and spatial-patterning inputs to IHO1 through cohesin (STAG3/REC8) dependence and ANKRD31 co-assembly controlling DSB distribution.","evidence":"Mouse knockouts of Stag3 and Ankrd31 with IHO1 co-localization and DSB cytology","pmids":["30853435","31000436"],"confidence":"Medium","gaps":["Mechanism by which cohesin promotes IHO1 loading not defined","How ANKRD31 directs PAR-localized DSBs at the molecular level unclear"]},{"year":2021,"claim":"Revealed DSB-triggered negative feedback that limits the machinery, showing distinct ATM, ATR, and PRKDC pathways locally and globally deplete IHO1 from axes after breaks form.","evidence":"Mouse genetic/pharmacological perturbation of DDR kinases with IHO1 axis quantification, plus zebrafish spo11 mutant analysis","pmids":["33619545","33842489"],"confidence":"High","gaps":["Direct substrates and phospho-sites driving IHO1 depletion not identified","How global vs local depletion is partitioned mechanistically unclear"]},{"year":2022,"claim":"Connected the IHO1 accessory network to the catalytic core and to its ubiquitin-mediated restraint, mapping REC114-TOPOVIBL coupling and SCF-FBXO47 control of HORMAD1/pre-DSB complex levels.","evidence":"Structural and Co-IP analysis with TOPOVIBL point-mutant knockins and genome-wide DSB mapping; Skp1 cKO with FBXO47-HORMAD1 ubiquitination assays","pmids":["36396648","35489071"],"confidence":"Medium","gaps":["Direct IHO1 contribution to TOPOVIL activation not isolated from REC114-TOPOVIBL contact","Whether IHO1 itself is a ubiquitination target unknown"]},{"year":2023,"claim":"Defined IHO1 oligomeric architecture and the competitive logic of the assembly, showing IHO1 tetramerizes and binds the REC114 PH domain at a surface shared with TOPOVIBL and ANKRD31.","evidence":"AlphaFold2 modeling validated by AUC, SEC-SAXS, pulldowns, and interface mutagenesis","pmids":["37431931"],"confidence":"High","gaps":["In vivo functional consequence of mutually exclusive REC114 binding not tested","Dynamics of partner exchange during DSB formation unknown"]},{"year":2024,"claim":"Established IHO1 as the seed for cluster biogenesis and the bridge anchoring hotspots to the axis, showing DDK-dependent IHO1 phosphorylation drives axial platform formation and IHO1 tethers PRDM9-marked sites to HORMAD1 and SYCP3.","evidence":"Mouse genetics, DDK chemical inhibition, IHO1 phospho-analysis, ChIP-seq, and co-localization imaging","pmids":["38580643","38657614"],"confidence":"High","gaps":["DDK phospho-acceptor residues on IHO1 not mapped","How IHO1-axis tethering is released during repair not defined"]},{"year":2026,"claim":"Reinforced IHO1 as a direct MEI1 partner with clinical relevance, since azoospermia-associated MEI1 variants disrupt MEI1-IHO1 binding.","evidence":"Co-IP of MEI1 disease variants with IHO1 and other DSB proteins","pmids":["41706353"],"confidence":"Low","gaps":["Single Co-IP assay without reciprocal or in vivo validation","Functional role of the IHO1-MEI1 interaction in DSB formation not characterized"]},{"year":null,"claim":"How IHO1 platform assembly is mechanistically coupled to triggering SPO11-TOPOVIBL catalysis, and the precise phospho-regulation governing its loading and feedback removal, remain open.","evidence":"","pmids":[],"confidence":"High","gaps":["No direct demonstration that IHO1 stimulates SPO11 catalytic activity","DDK and DDR-kinase phospho-site maps on IHO1 incomplete","Structure of the full axis-bound IHO1 cluster not determined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,10,12]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[11,12]}],"localization":[{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0,6,12]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,11]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,11]}],"complexes":["IHO1-REC114-MEI4 pre-DSB recombinosome"],"partners":["HORMAD1","REC114","MEI4","CXXC1","ANKRD31","SYCP3","MEI1","SKP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IYA8","full_name":"Interactor of HORMAD1 protein 1","aliases":["Cancer/testis antigen 74","CT74","Coiled-coil domain-containing protein 36"],"length_aa":594,"mass_kda":66.3,"function":"Required for DNA double-strand breaks (DSBs) formation in unsynapsed regions during meiotic recombination. Probably acts by forming a complex with MEI4 and REC114, which activates DSBs formation in unsynapsed regions, an essential step to ensure completion of synapsis. Not required for HORMAD1 functions in pairing-independent synaptonemal complex formation, ATR recruitment to unsynapsed axes, meiotic silencing of unsynapsed chromatin (MSUC) or meiotic surveillance","subcellular_location":"Chromosome","url":"https://www.uniprot.org/uniprotkb/Q8IYA8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IHO1","classification":"Not Classified","n_dependent_lines":28,"n_total_lines":1208,"dependency_fraction":0.023178807947019868},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/IHO1","total_profiled":1310},"omim":[{"mim_id":"619190","title":"INTERACTOR OF HORMAD1 1; IHO1","url":"https://www.omim.org/entry/619190"},{"mim_id":"618421","title":"REC114 MEIOTIC RECOMBINATION PROTEIN; REC114","url":"https://www.omim.org/entry/618421"},{"mim_id":"609824","title":"HORMA DOMAIN-CONTAINING PROTEIN 1; HORMAD1","url":"https://www.omim.org/entry/609824"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":24.1}],"url":"https://www.proteinatlas.org/search/IHO1"},"hgnc":{"alias_symbol":["FLJ25320","CT74","LELA1"],"prev_symbol":["CCDC36"]},"alphafold":{"accession":"Q8IYA8","domains":[{"cath_id":"1.20.5","chopping":"116-216","consensus_level":"medium","plddt":96.7772,"start":116,"end":216}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IYA8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IYA8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IYA8-F1-predicted_aligned_error_v6.png","plddt_mean":54.66},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IHO1","jax_strain_url":"https://www.jax.org/strain/search?query=IHO1"},"sequence":{"accession":"Q8IYA8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IYA8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IYA8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IYA8"}},"corpus_meta":[{"pmid":"27723721","id":"PMC_27723721","title":"Meiotic DNA break formation requires the unsynapsed chromosome axis-binding protein IHO1 (CCDC36) in mice.","date":"2016","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27723721","citation_count":141,"is_preprint":false},{"pmid":"30569039","id":"PMC_30569039","title":"Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with MEI4.","date":"2018","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/30569039","citation_count":80,"is_preprint":false},{"pmid":"31000436","id":"PMC_31000436","title":"Mouse ANKRD31 Regulates Spatiotemporal Patterning of Meiotic Recombination Initiation and Ensures Recombination between X and Y Sex Chromosomes.","date":"2019","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/31000436","citation_count":74,"is_preprint":false},{"pmid":"28527011","id":"PMC_28527011","title":"The PRDM9 KRAB domain is required for meiosis and involved in protein 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and is essential for meiotic DNA double-strand break (DSB) formation in mice. IHO1 and conserved SPO11-auxiliary proteins MEI4 and REC114 assemble chromatin-bound recombinosomes on meiotic chromosome axes that are predicted activators of DSB formation. HORMAD1 is required for robust recruitment of IHO1 to unsynapsed axes and efficient formation/stabilization of these recombinosomes.\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid, immunofluorescence localization, mouse knockout/loss-of-function with meiotic phenotype readout\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, direct interaction demonstrated, mouse KO with defined meiotic phenotype, replicated in multiple subsequent studies\",\n      \"pmids\": [\"27723721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IHO1, MEI4, and REC114 form a ternary complex in mouse spermatocytes on the axes of meiotic chromosomes. MEI4 forms a stable complex with REC114, and the REC114 C-terminal domain interacts with the MEI4 N-terminal domain in vitro. The REC114 N-terminal domain has structural similarity to Pleckstrin homology (PH) domains.\",\n      \"method\": \"Co-immunoprecipitation in spermatocytes, in vitro complex reconstitution, X-ray crystallography of REC114 N-terminal domain\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, structural analysis, and Co-IP in native tissue; replicated by later studies\",\n      \"pmids\": [\"30569039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CXXC1 interacts with IHO1 in mouse meiosis, providing a potential molecular link between PRDM9-marked recombination hotspots and the DSB machinery on the chromosome axis, analogous to the yeast Spp1-Mer2 interaction.\",\n      \"method\": \"Yeast two-hybrid assay, Co-immunoprecipitation\",\n      \"journal\": \"Chromosoma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus Co-IP, single lab, functional consequence inferred by analogy rather than direct experiment\",\n      \"pmids\": [\"28527011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"IHO1 is the mammalian ortholog of yeast Mer2/Rec15 and fungal Asy2, establishing evolutionary conservation of the DSB initiation complex scaffold across fungi, plants, and mammals. In Sordaria, the Mer2 ortholog mediates assembly of recombination-initiation complexes and DSBs, as well as homolog pairing and chromosome compaction.\",\n      \"method\": \"Genetic and cytological analysis of Mer2 mutants in Sordaria, immunolocalization, sequence conservation analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional analysis in a model fungus with multiple mutant alleles and orthogonal cytological methods; orthology to mammalian IHO1 established but mammalian experiments not performed in this paper\",\n      \"pmids\": [\"29021238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ANKRD31 associates with the DSB-promoting protein complex (including IHO1) on meiotic chromosome axes and is required for normal spatiotemporal patterning of DSB formation, including high rates of DSBs at pseudoautosomal regions (PARs). Loss of ANKRD31 causes delayed and redistributed DSB formation and failure of X-Y chromosome recombination.\",\n      \"method\": \"Mouse knockout, immunofluorescence co-localization with IHO1 and other DSB factors, cytological analysis of DSB markers\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mouse KO with defined molecular and cytological phenotypes, co-localization of IHO1 with ANKRD31 complex, replicated by later structural studies\",\n      \"pmids\": [\"31000436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"STAG3 (meiotic cohesin) and REC8 promote axis localization of IHO1 (along with HORMAD1 and MEI4) in spermatocytes. STAG3 deficiency reduces IHO1 recruitment to chromosome axes, demonstrating that meiotic cohesin complexes are upstream of IHO1 axis association.\",\n      \"method\": \"Mouse knockout (Stag3-deficient), immunofluorescence localization of IHO1 and associated proteins, genetic epistasis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mouse KO with specific localization phenotype for IHO1, single lab\",\n      \"pmids\": [\"30853435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DSBs restrict the DSB machinery via at least four distinct negative feedback pathways in mice: (1) ATM kinase activation by DSBs restricts pre-DSB recombinosome numbers without affecting IHO1 axis levels; (2) ATR kinase locally depletes IHO1 near DSB sites; (3) DSB-enabled homolog synapsis promotes depletion of IHO1 and pre-DSB recombinosomes from synapsed axes; (4) DSBs and three DDR kinases (ATM, ATR, PRKDC) enable stage-specific global depletion of IHO1 from all axes.\",\n      \"method\": \"Mouse genetic models (ATM, ATR, PRKDC inhibition/knockout), immunofluorescence quantification of IHO1 and recombinosome proteins on chromosome axes, epistasis analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic perturbations with specific IHO1 localization readouts, four mechanistically distinct pathways established with orthogonal experiments\",\n      \"pmids\": [\"33619545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In zebrafish spermatocytes, IHO1 dissociation from chromosome axes occurs in a DSB-dependent manner; persistent IHO1 foci are observed in spo11 mutant spermatocytes where no DSBs are formed, while IHO1 signal kinetics are otherwise similar to wild type.\",\n      \"method\": \"Zebrafish spo11 mutant analysis, immunofluorescence localization of Iho1 on chromosome axes\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function (spo11 mutant) with specific IHO1 localization phenotype in zebrafish, single study\",\n      \"pmids\": [\"33842489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"REC114 directly interacts with TOPOVIBL (the SPO11 accessory subunit), and this interaction is required for efficient DSB formation genome-wide. IHO1 is part of the accessory factor network (IHO1-REC114-MEI4-MEI1) controlling TOPOVIL catalytic activity. Point mutations in TOPOVIBL that disrupt REC114 binding strongly reduce DSBs in oocytes and sub-telomeric regions in spermatocytes.\",\n      \"method\": \"Co-immunoprecipitation, structural analysis of conserved interacting domains, mouse point-mutation knockin models, DSB monitoring genome-wide\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural domain identification, in vivo point mutation knockin, genome-wide DSB quantification; IHO1 placed in this complex network\",\n      \"pmids\": [\"36396648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SKP1 (subunit of SCF E3 ubiquitin ligase) restrains accumulation of the pre-DSB complex (IHO1-REC114-MEI4) on the meiotic chromosome axis. Loss of SKP1 leads to aberrant IHO1 localization. FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and proteasomal degradation, thereby modulating the pre-DSB complex.\",\n      \"method\": \"Mouse conditional knockout of Skp1, co-immunoprecipitation of FBXO47-SKP1-HORMAD1, ubiquitination assay in HEK293T cells, immunofluorescence of IHO1 axis localization\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mouse KO with IHO1-specific phenotype, co-IP and ubiquitination assay in cell line, single lab\",\n      \"pmids\": [\"35489071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"IHO1 forms coiled-coil-based tetramers. IHO1 directly interacts with the PH domain of REC114, recognizing the same surface used by TOPOVIBL and ANKRD31, suggesting REC114 acts as a regulatory platform for mutually exclusive interactions with multiple partners including IHO1. REC114 forms homodimers and assembles with MEI4 as a 2:1 heterotrimer that further dimerizes, constituting a ternary IHO1-REC114-MEI4 complex.\",\n      \"method\": \"AlphaFold2 structural modeling, biochemical characterization (analytical ultracentrifugation, SEC-SAXS, pulldown assays), mutagenesis of interaction interfaces\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural modeling validated by biochemical reconstitution and mutagenesis, detailed stoichiometry established for the IHO1-REC114-MEI4 complex\",\n      \"pmids\": [\"37431931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IHO1 acts as a seed/platform on chromosome axes for biogenesis of DSB-machinery clusters. Axial IHO1 platforms are based on IHO1-HORMAD1 interaction. IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), placing DDK upstream of IHO1 axis association. ANKRD31 enhances both seeding and growth of DSB-machinery clusters independently, providing a complementary pathway when IHO1-HORMAD1 interaction is disrupted.\",\n      \"method\": \"Mouse genetic models, DDK chemical inhibition, phosphorylation analysis of IHO1, immunofluorescence quantification of IHO1 cluster formation on axes, epistasis with ANKRD31\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and pharmacological perturbations, specific IHO1 phosphorylation and localization readouts, epistasis analysis with ANKRD31\",\n      \"pmids\": [\"38580643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IHO1 anchors DSB sites (marked by PRDM9 binding) to chromosome axis components HORMAD1 and SYCP3. PRDM9 promotes recruitment of MEI4 and IHO1 to chromatin, and IHO1 in turn tethers these sites to the axis. IHO1, HORMAD1, and SYCP3 remain associated at DSB ends during DSB repair.\",\n      \"method\": \"ChIP-seq identification of axis-associated genomic sites, co-localization immunofluorescence, mouse genetic models showing PRDM9-dependent IHO1 and MEI4 recruitment\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-seq combined with genetic perturbations and immunofluorescence, multiple orthogonal methods establishing PRDM9→IHO1→axis tethering hierarchy\",\n      \"pmids\": [\"38657614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MEI1 variants that cause non-obstructive azoospermia disrupt interactions of MEI1 with IHO1 (as well as ANKRD31, REC114, MEI4), as demonstrated by co-immunoprecipitation assays, establishing that IHO1 is a direct binding partner of MEI1 in the meiotic DSB machinery.\",\n      \"method\": \"Co-immunoprecipitation of MEI1 variants with IHO1 and other meiotic DSB proteins\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP assay, single study, interaction disruption used diagnostically rather than mechanistically characterized\",\n      \"pmids\": [\"41706353\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IHO1 (CCDC36/Mer2 ortholog) is an essential scaffold protein of the meiotic DSB machinery that directly binds HORMAD1 on unsynapsed chromosome axes; this interaction, modulated by DDK-dependent phosphorylation of IHO1, seeds the assembly of pre-DSB recombinosome clusters composed of IHO1 tetramers, REC114 (with which it interacts via REC114's PH domain), and MEI4, which collectively activate the SPO11-TOPOVIBL (TOPOVIL) catalytic complex to generate programmed DSBs; IHO1 is in turn recruited to PRDM9-marked chromatin sites and anchors DSB sites to axis components HORMAD1 and SYCP3, while DSB formation itself triggers negative feedback through ATM- and ATR-dependent local and global depletion of IHO1 from chromosome axes, with additional regulation by the SCF-FBXO47 ubiquitin ligase acting on HORMAD1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IHO1 (CCDC36), the mammalian ortholog of yeast Mer2/Rec15 and fungal Asy2, is an essential scaffold protein that nucleates the assembly of meiotic DNA double-strand break (DSB) machinery on chromosome axes [#0, #3]. IHO1 directly binds the axis protein HORMAD1, an interaction required for its robust recruitment to unsynapsed axes, and together with the SPO11-auxiliary proteins MEI4 and REC114 it forms chromatin-bound recombinosomes that promote DSB formation [#0, #1]. Structurally, IHO1 forms coiled-coil-based tetramers and engages the pleckstrin-homology (PH) domain of REC114 using the same surface that REC114 employs for TOPOVIBL and ANKRD31, so REC114 acts as a regulatory platform for mutually exclusive interactions, while REC114 itself dimerizes and assembles with MEI4 as a higher-order ternary complex [#10, #1]. This IHO1-REC114-MEI4 accessory network controls the catalytic activity of the SPO11-TOPOVIBL (TOPOVIL) complex that generates programmed DSBs [#8]. IHO1 seeds and grows DSB-machinery clusters on the axis through its HORMAD1 interaction, a process dependent on DDK-mediated IHO1 phosphorylation, and it anchors PRDM9-marked DSB sites to the axis components HORMAD1 and SYCP3 [#11, #12]. Upstream, meiotic cohesin (STAG3/REC8) promotes IHO1 axis localization, and the SCF-FBXO47 ubiquitin ligase restrains pre-DSB complex accumulation via HORMAD1 turnover [#5, #9]. Once DSBs form, multiple ATM-, ATR-, and PRKDC-dependent negative-feedback pathways locally and globally deplete IHO1 from chromosome axes, limiting further break formation [#6, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established the founding link between an axis-bound scaffold and meiotic break formation by showing IHO1 binds HORMAD1 and is required for DSBs and recombinosome assembly with MEI4 and REC114.\",\n      \"evidence\": \"Co-IP, yeast two-hybrid, immunofluorescence, and mouse knockout with meiotic phenotype\",\n      \"pmids\": [\"27723721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the IHO1-containing recombinosome not resolved\", \"Mechanism by which recombinosomes activate SPO11 not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed IHO1 in an evolutionarily conserved DSB-initiation framework by identifying it as the Mer2/Rec15/Asy2 ortholog with conserved roles in recombination, pairing, and chromosome compaction.\",\n      \"evidence\": \"Genetic and cytological analysis of Mer2 orthologs in Sordaria with sequence conservation\",\n      \"pmids\": [\"29021238\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian functions inferred by orthology, not tested in this work\", \"Whether pairing/compaction roles transfer to IHO1 unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Proposed a route from PRDM9-marked hotspots to the axis-bound machinery by identifying a CXXC1-IHO1 interaction analogous to yeast Spp1-Mer2.\",\n      \"evidence\": \"Yeast two-hybrid and Co-IP in mouse meiosis\",\n      \"pmids\": [\"28527011\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence inferred by analogy rather than tested directly\", \"Whether CXXC1 recruits IHO1 to hotspots in vivo unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the molecular organization of the accessory complex by reconstituting an IHO1-MEI4-REC114 ternary complex and resolving the REC114 PH-like domain that mediates MEI4 binding.\",\n      \"evidence\": \"Co-IP in spermatocytes, in vitro reconstitution, and crystallography of REC114 N-terminal domain\",\n      \"pmids\": [\"30569039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-level IHO1-REC114 interface not resolved here\", \"Higher-order assembly stoichiometry not determined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified upstream axis-recruitment and spatial-patterning inputs to IHO1 through cohesin (STAG3/REC8) dependence and ANKRD31 co-assembly controlling DSB distribution.\",\n      \"evidence\": \"Mouse knockouts of Stag3 and Ankrd31 with IHO1 co-localization and DSB cytology\",\n      \"pmids\": [\"30853435\", \"31000436\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which cohesin promotes IHO1 loading not defined\", \"How ANKRD31 directs PAR-localized DSBs at the molecular level unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed DSB-triggered negative feedback that limits the machinery, showing distinct ATM, ATR, and PRKDC pathways locally and globally deplete IHO1 from axes after breaks form.\",\n      \"evidence\": \"Mouse genetic/pharmacological perturbation of DDR kinases with IHO1 axis quantification, plus zebrafish spo11 mutant analysis\",\n      \"pmids\": [\"33619545\", \"33842489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates and phospho-sites driving IHO1 depletion not identified\", \"How global vs local depletion is partitioned mechanistically unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected the IHO1 accessory network to the catalytic core and to its ubiquitin-mediated restraint, mapping REC114-TOPOVIBL coupling and SCF-FBXO47 control of HORMAD1/pre-DSB complex levels.\",\n      \"evidence\": \"Structural and Co-IP analysis with TOPOVIBL point-mutant knockins and genome-wide DSB mapping; Skp1 cKO with FBXO47-HORMAD1 ubiquitination assays\",\n      \"pmids\": [\"36396648\", \"35489071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct IHO1 contribution to TOPOVIL activation not isolated from REC114-TOPOVIBL contact\", \"Whether IHO1 itself is a ubiquitination target unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined IHO1 oligomeric architecture and the competitive logic of the assembly, showing IHO1 tetramerizes and binds the REC114 PH domain at a surface shared with TOPOVIBL and ANKRD31.\",\n      \"evidence\": \"AlphaFold2 modeling validated by AUC, SEC-SAXS, pulldowns, and interface mutagenesis\",\n      \"pmids\": [\"37431931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo functional consequence of mutually exclusive REC114 binding not tested\", \"Dynamics of partner exchange during DSB formation unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established IHO1 as the seed for cluster biogenesis and the bridge anchoring hotspots to the axis, showing DDK-dependent IHO1 phosphorylation drives axial platform formation and IHO1 tethers PRDM9-marked sites to HORMAD1 and SYCP3.\",\n      \"evidence\": \"Mouse genetics, DDK chemical inhibition, IHO1 phospho-analysis, ChIP-seq, and co-localization imaging\",\n      \"pmids\": [\"38580643\", \"38657614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DDK phospho-acceptor residues on IHO1 not mapped\", \"How IHO1-axis tethering is released during repair not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Reinforced IHO1 as a direct MEI1 partner with clinical relevance, since azoospermia-associated MEI1 variants disrupt MEI1-IHO1 binding.\",\n      \"evidence\": \"Co-IP of MEI1 disease variants with IHO1 and other DSB proteins\",\n      \"pmids\": [\"41706353\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP assay without reciprocal or in vivo validation\", \"Functional role of the IHO1-MEI1 interaction in DSB formation not characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How IHO1 platform assembly is mechanistically coupled to triggering SPO11-TOPOVIBL catalysis, and the precise phospho-regulation governing its loading and feedback removal, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct demonstration that IHO1 stimulates SPO11 catalytic activity\", \"DDK and DDR-kinase phospho-site maps on IHO1 incomplete\", \"Structure of the full axis-bound IHO1 cluster not determined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 10, 12]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [11, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0, 6, 12]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"complexes\": [\"IHO1-REC114-MEI4 pre-DSB recombinosome\"],\n    \"partners\": [\"HORMAD1\", \"REC114\", \"MEI4\", \"CXXC1\", \"ANKRD31\", \"SYCP3\", \"MEI1\", \"SKP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}