{"gene":"ANKRD31","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2019,"finding":"ANKRD31 directly interacts with REC114 via a pleckstrin homology (PH) domain in REC114; a crystal structure defines the direct intermolecular contacts between the PH domain of REC114 and ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the pseudoautosomal region (PAR) and elsewhere on meiotic chromosomes, acting as a scaffold anchoring REC114 and other factors to specific genomic locations to regulate DSB formation.","method":"Crystal structure of REC114 PH domain bound to ANKRD31, in vivo localization by immunofluorescence, Ankrd31 knockout mouse model with phenotypic analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vivo functional validation in knockout mouse, multiple orthogonal methods in single study","pmids":["31003867"],"is_preprint":false},{"year":2019,"finding":"ANKRD31 is a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. ANKRD31 deficiency causes delayed recombination initiation genome-wide, alters DSB distribution (reduced selectivity for normal hotspot sites), and abolishes uniquely high DSB rates in pseudoautosomal regions (PARs) of X and Y chromosomes, leading to failure of sex chromosome crossover and meiotic arrest in spermatocytes.","method":"Ankrd31 knockout mouse, immunofluorescence of axis-associated DSB-promoting proteins, cytological analysis of recombination markers, chromosome spread analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple defined cellular and molecular phenotypes, replicated independently by two labs simultaneously","pmids":["31000436","31003867"],"is_preprint":false},{"year":2020,"finding":"ANKRD31, together with MEI4, is required for the hyperaccumulation of DSB-promoting factors in the PAR and for the PAR-specific chromosome axis elongation and sister chromatid separation that precede DSB formation. ANKRD31 is needed for the specialized PAR chromatin and higher-order structure that promotes recombination, but this requirement is independent of REC8 or HORMAD1.","method":"Mouse knockout models (Ankrd31, Mei4), super-resolution and structured illumination microscopy of PAR ultrastructure, immunofluorescence of DSB factors, genetic epistasis with Rec8 and Hormad1 mutants","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — multiple knockouts with genetic epistasis, direct ultrastructural imaging with functional consequence, replicated across labs","pmids":["32461690"],"is_preprint":false},{"year":2023,"finding":"The ANKRD31-REC114 interaction is essential for ANKRD31 function: mice with C-terminal truncation of ANKRD31 that eliminates the REC114 interaction (without disrupting other known partners) phenocopy Ankrd31 null mutants, with delayed DSB formation, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. A missense mutation that partially disrupts the interaction shows dosage-dependent intermediate phenotypes.","method":"Genome-edited mice with specific Ankrd31 missense and truncation alleles, immunofluorescence of DSB markers, cytological recombination analysis, genetic complementation/dosage experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — structure-informed mutagenesis in vivo with multiple alleles and dosage analysis, rigorous controls separating interaction-specific effects","pmids":["37976262"],"is_preprint":false},{"year":2023,"finding":"IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and ANKRD31, indicating that ANKRD31 competes with IHO1 and TOPOVIBL for binding to the REC114 PH domain, and that REC114 may act as a regulatory platform mediating mutually exclusive interactions with several partners.","method":"AlphaFold2 structural modeling combined with biochemical characterization (pulldowns, SEC-MALS), in vitro interaction assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — structural modeling validated by in vitro biochemistry, competitive binding established with multiple partners","pmids":["37431931"],"is_preprint":false},{"year":2024,"finding":"Without IHO1-HORMAD1 interaction (loss of primary seeding pathway), residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters on chromosome axes. ANKRD31 thus represents a complementary pathway to IHO1-HORMAD1 for targeting the DSB machinery to chromosome axes.","method":"Mouse genetic epistasis (IHO1 interaction mutants combined with Ankrd31 analysis), immunofluorescence of DSB-factor clusters on chromosome axes, quantitative cytology","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in mouse with defined molecular phenotype, multiple orthogonal methods","pmids":["38580643"],"is_preprint":false},{"year":2021,"finding":"Two pathogenic heterozygous variants in ANKRD31 identified in POI patients disrupt its interaction with REC114 (demonstrated by functional interaction assays), with haploinsufficiency as the mechanism, indicating dosage-dependent pathogenic effects on ovarian function.","method":"Exome sequencing in POI patients, functional interaction assays (co-immunoprecipitation/Y2H-type assay) for ANKRD31-REC114 interaction with patient variants","journal":"Genetics in medicine : official journal of the American College of Medical Genetics","confidence":"Medium","confidence_rationale":"Tier 2-3 — interaction assay with patient variants, single lab study","pmids":["34257419"],"is_preprint":false},{"year":2021,"finding":"ANKRD31 is expressed in the epididymis and physically interacts with epithelial junction proteins. Ankrd31 knockout male mice show infertility with oligo-astheno-teratozoospermia and disruption of the blood-epididymal barrier due to cell-to-cell junction anomalies, suggesting a role in epididymal integrity beyond meiosis.","method":"Ankrd31 knockout mouse model, co-immunoprecipitation of ANKRD31 with epithelial junction proteins in wild-type epididymides, histological and morphological analysis","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP plus KO phenotype, single lab","pmids":["34820371"],"is_preprint":false},{"year":2026,"finding":"Biallelic pathogenic MEI1 variants disrupt MEI1 interactions with ANKRD31, IHO1, REC114, and MEI4 as demonstrated by co-immunoprecipitation assays, placing ANKRD31 in a complex with MEI1 and other DSB-promoting factors.","method":"Co-immunoprecipitation assays with patient-derived MEI1 variants","journal":"Journal of assisted reproduction and genetics","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP showing interaction, but ANKRD31 is not the primary subject; single method","pmids":["41706353"],"is_preprint":false}],"current_model":"ANKRD31 functions as a modular scaffold protein in meiotic DSB formation: it directly binds the PH domain of REC114 (via a structurally defined interface that competes with IHO1 and TOPOVIBL binding) and anchors REC114 and other DSB-promoting factors to meiotic chromosome axes, thereby controlling the timing, number, and genomic location of DSBs—with an especially critical role in targeting DSBs to the pseudoautosomal regions of sex chromosomes to ensure crossover formation and correct meiotic segregation."},"narrative":{"teleology":[{"year":2019,"claim":"The discovery that ANKRD31 directly binds REC114 via a PH-domain interface and that its loss delays DSBs genome-wide while abolishing PAR-targeted DSBs established ANKRD31 as the first identified scaffold specifically targeting DSB-promoting complexes to defined chromosomal locations during meiosis.","evidence":"Crystal structure of REC114 PH domain–ANKRD31 complex, Ankrd31 knockout mouse with cytological and immunofluorescence analysis of DSB markers","pmids":["31003867","31000436"],"confidence":"High","gaps":["How ANKRD31 recognizes PAR-specific genomic features was unknown","Whether the REC114 interaction is sufficient for all ANKRD31 functions was untested","Relationship to other DSB-promoting factors such as MEI4 and IHO1 was undefined"]},{"year":2020,"claim":"Demonstrating that ANKRD31 and MEI4 are jointly required for PAR axis elongation, sister chromatid separation, and hyperaccumulation of DSB factors at the PAR—independently of REC8 and HORMAD1—revealed that ANKRD31 shapes higher-order chromosome architecture to create a recombination-permissive environment.","evidence":"Super-resolution and structured illumination microscopy of PAR ultrastructure in Ankrd31, Mei4, Rec8, and Hormad1 knockout mice with genetic epistasis analysis","pmids":["32461690"],"confidence":"High","gaps":["Whether ANKRD31 directly mediates axis structural changes or acts indirectly through DSB-factor recruitment was unresolved","The molecular basis of the ANKRD31–MEI4 functional interaction was not defined"]},{"year":2021,"claim":"Identification of heterozygous ANKRD31 variants in POI patients that disrupt REC114 binding provided the first evidence that ANKRD31 haploinsufficiency causes human meiotic disease, extending its role from mouse genetics to human reproductive pathology.","evidence":"Exome sequencing in POI patients combined with functional interaction assays for ANKRD31-REC114 binding with patient-derived variants","pmids":["34257419"],"confidence":"Medium","gaps":["Number of patients is limited; independent replication in larger cohorts is lacking","Whether the variants cause meiotic DSB defects in human oocytes was not directly shown","Potential additional functions of ANKRD31 in oogenesis beyond DSB formation were not explored"]},{"year":2023,"claim":"Structure-informed in vivo mutagenesis showed that the REC114-binding interface is the essential functional output of ANKRD31: truncation alleles eliminating REC114 contact phenocopy nulls, and partial-disruption alleles produce dosage-dependent intermediate phenotypes, separating REC114 binding from other potential ANKRD31 activities.","evidence":"Genome-edited mice carrying Ankrd31 missense and truncation alleles targeting the REC114-interaction surface, with cytological recombination and DSB marker analysis","pmids":["37976262"],"confidence":"High","gaps":["Whether ANKRD31 has REC114-independent functions in specific genomic contexts remained untested","Structural basis of ANKRD31 interactions with partners other than REC114 was unknown"]},{"year":2023,"claim":"Showing that IHO1 and TOPOVIBL compete with ANKRD31 for the same REC114 PH-domain surface reframed ANKRD31 as one component of a mutually exclusive interaction network, positioning REC114 as a regulatory hub rather than a passive ANKRD31 partner.","evidence":"AlphaFold2 structural modeling validated by pulldowns and SEC-MALS competition assays in vitro","pmids":["37431931"],"confidence":"High","gaps":["The temporal order and regulatory logic of partner switching on REC114 in vivo was not determined","Whether competition is regulated by post-translational modifications was unknown"]},{"year":2024,"claim":"Genetic epistasis between IHO1 interaction mutants and ANKRD31 revealed that ANKRD31 provides a complementary, partially redundant pathway for seeding and growing DSB-machinery clusters on axes, explaining residual DSB activity when the primary IHO1-HORMAD1 seeding mechanism is lost.","evidence":"Mouse genetic epistasis combining IHO1 interaction mutants with Ankrd31 analysis, quantitative immunofluorescence of DSB-factor clusters","pmids":["38580643"],"confidence":"High","gaps":["Molecular mechanism by which ANKRD31 seeds clusters independently of IHO1-HORMAD1 is undefined","Whether other scaffold proteins contribute to a third pathway remains unknown"]},{"year":null,"claim":"The mechanism by which ANKRD31 specifically recognizes PAR sequences or PAR-associated chromatin features to create a uniquely recombination-permissive environment, and the in vivo dynamics of competitive partner switching on the REC114 PH domain, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No DNA-binding or chromatin-recognition domain in ANKRD31 has been identified","In vivo partner switching kinetics on REC114 have not been measured","Whether ANKRD31 has meiosis-independent functions in somatic tissues requires systematic investigation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,3,5]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,2]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,1,2,3,5]}],"complexes":[],"partners":["REC114","MEI4","IHO1","TOPOVIBL","MEI1"],"other_free_text":[]},"mechanistic_narrative":"ANKRD31 is a meiosis-specific scaffold protein that controls the timing, number, and genomic distribution of programmed DNA double-strand breaks (DSBs) by anchoring DSB-promoting factors to chromosome axes. It directly binds the pleckstrin homology domain of REC114 through a structurally defined interface that is competitively shared with IHO1 and TOPOVIBL, and this interaction is essential for ANKRD31 function, as structure-guided mutations that specifically disrupt REC114 binding phenocopy the null mutant [PMID:31003867, PMID:37976262, PMID:37431931]. ANKRD31 is indispensable for the hyperaccumulation of DSB machinery and the specialized axis elongation at pseudoautosomal regions (PARs), and its loss abolishes the uniquely high PAR DSB rates required for obligate sex-chromosome crossovers, causing meiotic arrest in spermatocytes [PMID:31000436, PMID:32461690]. Heterozygous variants in ANKRD31 that disrupt REC114 binding have been identified as a cause of primary ovarian insufficiency (POI) in humans, consistent with dosage-sensitive requirements for meiotic DSB formation [PMID:34257419]."},"prefetch_data":{"uniprot":{"accession":"Q8N7Z5","full_name":"Ankyrin repeat domain-containing protein 31","aliases":[],"length_aa":1873,"mass_kda":210.8,"function":"Required for DNA double-strand breaks (DSBs) formation during meiotic recombination. Regulates the spatial and temporal patterns of pre-DSB recombinosome assembly and recombination activity by acting as a scaffold that anchors REC114 and other factors to specific genomic locations, thereby regulating DSB formation. Plays a key role in recombination in the pseudoautosomal regions of sex chromosomes","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q8N7Z5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANKRD31","classification":"Not Classified","n_dependent_lines":35,"n_total_lines":1208,"dependency_fraction":0.028973509933774833},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ANKRD31","total_profiled":1310},"omim":[{"mim_id":"618423","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 31; ANKRD31","url":"https://www.omim.org/entry/618423"},{"mim_id":"618421","title":"REC114 MEIOTIC RECOMBINATION PROTEIN; REC114","url":"https://www.omim.org/entry/618421"},{"mim_id":"618417","title":"MEIOTIC DOUBLE-STRANDED BREAK FORMATION PROTEIN 4; MEI4","url":"https://www.omim.org/entry/618417"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Centrosome","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":4.6}],"url":"https://www.proteinatlas.org/search/ANKRD31"},"hgnc":{"alias_symbol":["FLJ40191"],"prev_symbol":[]},"alphafold":{"accession":"Q8N7Z5","domains":[{"cath_id":"1.25.40.20","chopping":"476-610","consensus_level":"medium","plddt":89.2124,"start":476,"end":610},{"cath_id":"1.25.40.20","chopping":"1153-1276","consensus_level":"medium","plddt":91.7482,"start":1153,"end":1276},{"cath_id":"1.20.58.70","chopping":"1330-1434","consensus_level":"medium","plddt":77.1473,"start":1330,"end":1434},{"cath_id":"-","chopping":"1700-1780","consensus_level":"high","plddt":85.2994,"start":1700,"end":1780}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N7Z5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N7Z5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N7Z5-F1-predicted_aligned_error_v6.png","plddt_mean":43.09},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANKRD31","jax_strain_url":"https://www.jax.org/strain/search?query=ANKRD31"},"sequence":{"accession":"Q8N7Z5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N7Z5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N7Z5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N7Z5"}},"corpus_meta":[{"pmid":"31473137","id":"PMC_31473137","title":"Genome-wide association analysis of dementia and its clinical endophenotypes reveal novel loci associated with Alzheimer's disease and three causality networks: The GR@ACE project.","date":"2019","source":"Alzheimer's & dementia : the journal of the Alzheimer's Association","url":"https://pubmed.ncbi.nlm.nih.gov/31473137","citation_count":126,"is_preprint":false},{"pmid":"34794894","id":"PMC_34794894","title":"Genetics of ovarian insufficiency and defects of folliculogenesis.","date":"2021","source":"Best practice & research. Clinical endocrinology & metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/34794894","citation_count":92,"is_preprint":false},{"pmid":"31003867","id":"PMC_31003867","title":"REC114 Partner ANKRD31 Controls Number, Timing, and Location of Meiotic DNA Breaks.","date":"2019","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/31003867","citation_count":88,"is_preprint":false},{"pmid":"32461690","id":"PMC_32461690","title":"Ensuring meiotic DNA break formation in the mouse pseudoautosomal region.","date":"2020","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/32461690","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 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diagnostics.","date":"2026","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/41706353","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.02.11.25322045","title":"Genome-wide association study of susceptibility to acute respiratory distress syndrome","date":"2025-02-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.11.25322045","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10796,"output_tokens":2284,"usd":0.033324},"stage2":{"model":"claude-opus-4-6","input_tokens":5578,"output_tokens":2023,"usd":0.117697},"total_usd":0.151021,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"ANKRD31 directly interacts with REC114 via a pleckstrin homology (PH) domain in REC114; a crystal structure defines the direct intermolecular contacts between the PH domain of REC114 and ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the pseudoautosomal region (PAR) and elsewhere on meiotic chromosomes, acting as a scaffold anchoring REC114 and other factors to specific genomic locations to regulate DSB formation.\",\n      \"method\": \"Crystal structure of REC114 PH domain bound to ANKRD31, in vivo localization by immunofluorescence, Ankrd31 knockout mouse model with phenotypic analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vivo functional validation in knockout mouse, multiple orthogonal methods in single study\",\n      \"pmids\": [\"31003867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ANKRD31 is a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. ANKRD31 deficiency causes delayed recombination initiation genome-wide, alters DSB distribution (reduced selectivity for normal hotspot sites), and abolishes uniquely high DSB rates in pseudoautosomal regions (PARs) of X and Y chromosomes, leading to failure of sex chromosome crossover and meiotic arrest in spermatocytes.\",\n      \"method\": \"Ankrd31 knockout mouse, immunofluorescence of axis-associated DSB-promoting proteins, cytological analysis of recombination markers, chromosome spread analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple defined cellular and molecular phenotypes, replicated independently by two labs simultaneously\",\n      \"pmids\": [\"31000436\", \"31003867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ANKRD31, together with MEI4, is required for the hyperaccumulation of DSB-promoting factors in the PAR and for the PAR-specific chromosome axis elongation and sister chromatid separation that precede DSB formation. ANKRD31 is needed for the specialized PAR chromatin and higher-order structure that promotes recombination, but this requirement is independent of REC8 or HORMAD1.\",\n      \"method\": \"Mouse knockout models (Ankrd31, Mei4), super-resolution and structured illumination microscopy of PAR ultrastructure, immunofluorescence of DSB factors, genetic epistasis with Rec8 and Hormad1 mutants\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple knockouts with genetic epistasis, direct ultrastructural imaging with functional consequence, replicated across labs\",\n      \"pmids\": [\"32461690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The ANKRD31-REC114 interaction is essential for ANKRD31 function: mice with C-terminal truncation of ANKRD31 that eliminates the REC114 interaction (without disrupting other known partners) phenocopy Ankrd31 null mutants, with delayed DSB formation, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. A missense mutation that partially disrupts the interaction shows dosage-dependent intermediate phenotypes.\",\n      \"method\": \"Genome-edited mice with specific Ankrd31 missense and truncation alleles, immunofluorescence of DSB markers, cytological recombination analysis, genetic complementation/dosage experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — structure-informed mutagenesis in vivo with multiple alleles and dosage analysis, rigorous controls separating interaction-specific effects\",\n      \"pmids\": [\"37976262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and ANKRD31, indicating that ANKRD31 competes with IHO1 and TOPOVIBL for binding to the REC114 PH domain, and that REC114 may act as a regulatory platform mediating mutually exclusive interactions with several partners.\",\n      \"method\": \"AlphaFold2 structural modeling combined with biochemical characterization (pulldowns, SEC-MALS), in vitro interaction assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural modeling validated by in vitro biochemistry, competitive binding established with multiple partners\",\n      \"pmids\": [\"37431931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Without IHO1-HORMAD1 interaction (loss of primary seeding pathway), residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters on chromosome axes. ANKRD31 thus represents a complementary pathway to IHO1-HORMAD1 for targeting the DSB machinery to chromosome axes.\",\n      \"method\": \"Mouse genetic epistasis (IHO1 interaction mutants combined with Ankrd31 analysis), immunofluorescence of DSB-factor clusters on chromosome axes, quantitative cytology\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in mouse with defined molecular phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"38580643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Two pathogenic heterozygous variants in ANKRD31 identified in POI patients disrupt its interaction with REC114 (demonstrated by functional interaction assays), with haploinsufficiency as the mechanism, indicating dosage-dependent pathogenic effects on ovarian function.\",\n      \"method\": \"Exome sequencing in POI patients, functional interaction assays (co-immunoprecipitation/Y2H-type assay) for ANKRD31-REC114 interaction with patient variants\",\n      \"journal\": \"Genetics in medicine : official journal of the American College of Medical Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interaction assay with patient variants, single lab study\",\n      \"pmids\": [\"34257419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ANKRD31 is expressed in the epididymis and physically interacts with epithelial junction proteins. Ankrd31 knockout male mice show infertility with oligo-astheno-teratozoospermia and disruption of the blood-epididymal barrier due to cell-to-cell junction anomalies, suggesting a role in epididymal integrity beyond meiosis.\",\n      \"method\": \"Ankrd31 knockout mouse model, co-immunoprecipitation of ANKRD31 with epithelial junction proteins in wild-type epididymides, histological and morphological analysis\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP plus KO phenotype, single lab\",\n      \"pmids\": [\"34820371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Biallelic pathogenic MEI1 variants disrupt MEI1 interactions with ANKRD31, IHO1, REC114, and MEI4 as demonstrated by co-immunoprecipitation assays, placing ANKRD31 in a complex with MEI1 and other DSB-promoting factors.\",\n      \"method\": \"Co-immunoprecipitation assays with patient-derived MEI1 variants\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP showing interaction, but ANKRD31 is not the primary subject; single method\",\n      \"pmids\": [\"41706353\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANKRD31 functions as a modular scaffold protein in meiotic DSB formation: it directly binds the PH domain of REC114 (via a structurally defined interface that competes with IHO1 and TOPOVIBL binding) and anchors REC114 and other DSB-promoting factors to meiotic chromosome axes, thereby controlling the timing, number, and genomic location of DSBs—with an especially critical role in targeting DSBs to the pseudoautosomal regions of sex chromosomes to ensure crossover formation and correct meiotic segregation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ANKRD31 is a meiosis-specific scaffold protein that controls the timing, number, and genomic distribution of programmed DNA double-strand breaks (DSBs) by anchoring DSB-promoting factors to chromosome axes. It directly binds the pleckstrin homology domain of REC114 through a structurally defined interface that is competitively shared with IHO1 and TOPOVIBL, and this interaction is essential for ANKRD31 function, as structure-guided mutations that specifically disrupt REC114 binding phenocopy the null mutant [PMID:31003867, PMID:37976262, PMID:37431931]. ANKRD31 is indispensable for the hyperaccumulation of DSB machinery and the specialized axis elongation at pseudoautosomal regions (PARs), and its loss abolishes the uniquely high PAR DSB rates required for obligate sex-chromosome crossovers, causing meiotic arrest in spermatocytes [PMID:31000436, PMID:32461690]. Heterozygous variants in ANKRD31 that disrupt REC114 binding have been identified as a cause of primary ovarian insufficiency (POI) in humans, consistent with dosage-sensitive requirements for meiotic DSB formation [PMID:34257419].\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"The discovery that ANKRD31 directly binds REC114 via a PH-domain interface and that its loss delays DSBs genome-wide while abolishing PAR-targeted DSBs established ANKRD31 as the first identified scaffold specifically targeting DSB-promoting complexes to defined chromosomal locations during meiosis.\",\n      \"evidence\": \"Crystal structure of REC114 PH domain–ANKRD31 complex, Ankrd31 knockout mouse with cytological and immunofluorescence analysis of DSB markers\",\n      \"pmids\": [\"31003867\", \"31000436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How ANKRD31 recognizes PAR-specific genomic features was unknown\",\n        \"Whether the REC114 interaction is sufficient for all ANKRD31 functions was untested\",\n        \"Relationship to other DSB-promoting factors such as MEI4 and IHO1 was undefined\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating that ANKRD31 and MEI4 are jointly required for PAR axis elongation, sister chromatid separation, and hyperaccumulation of DSB factors at the PAR—independently of REC8 and HORMAD1—revealed that ANKRD31 shapes higher-order chromosome architecture to create a recombination-permissive environment.\",\n      \"evidence\": \"Super-resolution and structured illumination microscopy of PAR ultrastructure in Ankrd31, Mei4, Rec8, and Hormad1 knockout mice with genetic epistasis analysis\",\n      \"pmids\": [\"32461690\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ANKRD31 directly mediates axis structural changes or acts indirectly through DSB-factor recruitment was unresolved\",\n        \"The molecular basis of the ANKRD31–MEI4 functional interaction was not defined\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of heterozygous ANKRD31 variants in POI patients that disrupt REC114 binding provided the first evidence that ANKRD31 haploinsufficiency causes human meiotic disease, extending its role from mouse genetics to human reproductive pathology.\",\n      \"evidence\": \"Exome sequencing in POI patients combined with functional interaction assays for ANKRD31-REC114 binding with patient-derived variants\",\n      \"pmids\": [\"34257419\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Number of patients is limited; independent replication in larger cohorts is lacking\",\n        \"Whether the variants cause meiotic DSB defects in human oocytes was not directly shown\",\n        \"Potential additional functions of ANKRD31 in oogenesis beyond DSB formation were not explored\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Structure-informed in vivo mutagenesis showed that the REC114-binding interface is the essential functional output of ANKRD31: truncation alleles eliminating REC114 contact phenocopy nulls, and partial-disruption alleles produce dosage-dependent intermediate phenotypes, separating REC114 binding from other potential ANKRD31 activities.\",\n      \"evidence\": \"Genome-edited mice carrying Ankrd31 missense and truncation alleles targeting the REC114-interaction surface, with cytological recombination and DSB marker analysis\",\n      \"pmids\": [\"37976262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ANKRD31 has REC114-independent functions in specific genomic contexts remained untested\",\n        \"Structural basis of ANKRD31 interactions with partners other than REC114 was unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showing that IHO1 and TOPOVIBL compete with ANKRD31 for the same REC114 PH-domain surface reframed ANKRD31 as one component of a mutually exclusive interaction network, positioning REC114 as a regulatory hub rather than a passive ANKRD31 partner.\",\n      \"evidence\": \"AlphaFold2 structural modeling validated by pulldowns and SEC-MALS competition assays in vitro\",\n      \"pmids\": [\"37431931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The temporal order and regulatory logic of partner switching on REC114 in vivo was not determined\",\n        \"Whether competition is regulated by post-translational modifications was unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Genetic epistasis between IHO1 interaction mutants and ANKRD31 revealed that ANKRD31 provides a complementary, partially redundant pathway for seeding and growing DSB-machinery clusters on axes, explaining residual DSB activity when the primary IHO1-HORMAD1 seeding mechanism is lost.\",\n      \"evidence\": \"Mouse genetic epistasis combining IHO1 interaction mutants with Ankrd31 analysis, quantitative immunofluorescence of DSB-factor clusters\",\n      \"pmids\": [\"38580643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular mechanism by which ANKRD31 seeds clusters independently of IHO1-HORMAD1 is undefined\",\n        \"Whether other scaffold proteins contribute to a third pathway remains unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The mechanism by which ANKRD31 specifically recognizes PAR sequences or PAR-associated chromatin features to create a uniquely recombination-permissive environment, and the in vivo dynamics of competitive partner switching on the REC114 PH domain, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No DNA-binding or chromatin-recognition domain in ANKRD31 has been identified\",\n        \"In vivo partner switching kinetics on REC114 have not been measured\",\n        \"Whether ANKRD31 has meiosis-independent functions in somatic tissues requires systematic investigation\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"REC114\", \"MEI4\", \"IHO1\", \"TOPOVIBL\", \"MEI1\"],\n    \"other_free_text\": []\n  }\n}\n```"}