{"gene":"ANKRD31","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2019,"finding":"ANKRD31 controls number, timing, and location of meiotic DNA double-strand breaks (DSBs). Spermatocytes lacking ANKRD31 have altered DSB locations, fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes, and show delayed and/or fewer recombination sites but paradoxically more DSBs overall, indicating DSB dysregulation.","method":"Knockout mouse model (Ankrd31-deficient spermatocytes), cytological and molecular analysis of recombination","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotypes, replicated independently in two concurrent papers (PMID:31003867 and PMID:31000436)","pmids":["31003867","31000436"],"is_preprint":false},{"year":2019,"finding":"A crystal structure reveals that REC114 contains a pleckstrin homology (PH) domain that directly contacts ANKRD31 through intermolecular interactions. ANKRD31 stabilizes REC114 association with the PAR and elsewhere in vivo, functioning as a scaffold that anchors REC114 and other DSB-promoting factors to specific genomic locations.","method":"Crystal structure determination, in vivo chromatin association assays in mouse spermatocytes","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vivo functional validation, replicated in subsequent structural/biochemical work (PMID:37431931)","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 genome-wide delayed recombination initiation, reduced selectivity for DSB hotspot sites, and loss of a specialized PAR-axis domain highly enriched for DSB-promoting proteins.","method":"Knockout mouse model, immunofluorescence/cytology, ChIP, genome-wide DSB mapping","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with multiple orthogonal molecular readouts, replicated independently","pmids":["31000436"],"is_preprint":false},{"year":2020,"finding":"MEI4 and ANKRD31 proteins are required for the hyperaccumulation of DSB-promoting factors in the PAR, elongation of PAR chromosome axes, and separation of sister chromatids prior to DSB formation — processes linked to heterochromatic mo-2 minisatellite arrays. These events make the PAR the hottest DSB segment in the male mouse genome.","method":"Knockout mouse models, super-resolution and conventional immunofluorescence, cytological axis measurements","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO in vivo with multiple orthogonal structural and molecular methods","pmids":["32461690"],"is_preprint":false},{"year":2021,"finding":"ANKRD31 physically interacts with epithelial cell-cell junction proteins in the epididymis. Loss of ANKRD31 in knockout male mice disrupts the blood-epididymal barrier (BEB) due to cell-to-cell junction anomalies, resulting in oligo-astheno-teratozoospermia and infertility.","method":"Ankrd31 knockout mouse model, co-immunoprecipitation of ANKRD31 with junction proteins in wild-type epididymis, histology","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus KO phenotype in single lab; junction partner identities and mechanism not fully defined","pmids":["34820371"],"is_preprint":false},{"year":2021,"finding":"Pathogenic heterozygous variants in ANKRD31 identified in premature ovarian insufficiency (POI) patients disrupt the interaction between ANKRD31 and the DSB-formation factor REC114, exerting their pathogenic effect via haploinsufficiency, indicating dosage-dependent control of ovarian function.","method":"Exome sequencing in POI patients, functional interaction assays (disruption of ANKRD31-REC114 interaction by variants)","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protein-interaction disruption assay plus clinical genetics, single lab","pmids":["34257419"],"is_preprint":false},{"year":2023,"finding":"The REC114 PH domain interacts with ANKRD31 and with IHO1 and TOPOVIBL at the same surface, indicating mutually exclusive interactions. REC114 acts as a regulatory platform where ANKRD31 competes with other partners for binding.","method":"AlphaFold2 structural modeling combined with in vitro biochemical characterization and size exclusion chromatography-multi-angle light scattering (SEC-MALS)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural modeling validated by multiple biochemical assays in single lab","pmids":["37431931"],"is_preprint":false},{"year":2023,"finding":"Complete disruption of the ANKRD31-REC114 interaction (by C-terminal truncation of ANKRD31) mimics the Ankrd31 null phenotype: delayed global DSB formation, defects in DSB repair, and failure to target DSBs to the PARs. Substantial but incomplete disruption (missense mutation) delays DSB formation but leaves recombination, repair, and DSB locations near normal. A dosage effect was observed when combining partial-loss and null alleles.","method":"Genome-edited mouse models carrying specific Ankrd31 missense and truncation mutations, cytological DSB assays, crossover analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple allelic series in mice with orthogonal molecular and cytological readouts, peer-reviewed","pmids":["37976262"],"is_preprint":false},{"year":2024,"finding":"When the IHO1-HORMAD1 axis-seeding pathway is disrupted, residual meiotic DSBs become dependent on ANKRD31, which enhances both the seeding and growth of DSB-machinery clusters on chromosome axes, demonstrating that ANKRD31 and the IHO1-HORMAD1 pathway act in complementary, partially redundant routes for DSB-machinery condensation.","method":"Compound mouse mutant analysis (IHO1-HORMAD1 interaction mutant combined with Ankrd31 mutant), immunofluorescence quantification of DSB-factor foci","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in vivo using clean compound mutants with quantitative cytological readouts, published in peer-reviewed journal","pmids":["38580643"],"is_preprint":false},{"year":2023,"finding":"MEI1 variants identified in non-obstructive azoospermia patients disrupt MEI1 interactions with ANKRD31 (as well as IHO1, REC114, and MEI4) as detected by co-immunoprecipitation, consistent with ANKRD31 being a component of the MEI1-containing DSB-promoting complex.","method":"Co-immunoprecipitation assays of mutant MEI1 with ANKRD31 and other meiotic factors","journal":"Journal of assisted reproduction and genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single co-IP result for the ANKRD31 interaction specifically; primarily a MEI1 study","pmids":["41706353"],"is_preprint":false}],"current_model":"ANKRD31 is a scaffold protein that localizes to meiotic chromosome axes and directly binds REC114 (via REC114's PH domain) to regulate the number, timing, and genomic distribution of meiotic DNA double-strand breaks, with a critical role in hyperactivating DSB formation at the pseudoautosomal regions of sex chromosomes; it also participates in the epididymal blood-epididymal barrier by interacting with cell-junction proteins, and its haploinsufficiency can cause premature ovarian insufficiency by impairing the ANKRD31-REC114 interaction in a dosage-dependent manner."},"narrative":{"mechanistic_narrative":"ANKRD31 is a meiotic scaffold protein that assembles with DSB-promoting factors on chromosome axes to control the number, timing, and genomic distribution of programmed meiotic DNA double-strand breaks (DSBs) [PMID:31003867, PMID:31000436]. Loss of ANKRD31 dysregulates DSBs genome-wide — delaying recombination initiation, reducing selectivity for hotspots, and paradoxically increasing total DSBs — while abolishing the specialized PAR-axis domain that normally makes the pseudoautosomal regions of the sex chromosomes the hottest DSB segment in the male genome [PMID:31003867, PMID:31000436, PMID:32461690]. Mechanistically, ANKRD31 binds directly to the pleckstrin homology (PH) domain of REC114, anchoring REC114 and associated DSB machinery to the PAR and to axis sites in vivo; this same REC114 PH surface is engaged competitively by IHO1 and TOPOVIBL, casting REC114 as a regulatory platform for mutually exclusive partner binding [PMID:31003867, PMID:37431931]. The ANKRD31–REC114 interaction is functionally central: complete disruption phenocopies the null (delayed DSBs, repair defects, loss of PAR targeting), whereas partial disruption only delays DSB timing, defining a dosage-dependent requirement [PMID:37976262]. ANKRD31 acts in a complementary, partially redundant route to the IHO1–HORMAD1 axis-seeding pathway, enhancing the seeding and growth of DSB-machinery clusters on axes [PMID:38580643]. Heterozygous ANKRD31 variants cause premature ovarian insufficiency through haploinsufficiency by disrupting the ANKRD31–REC114 interaction [PMID:34257419]. Beyond meiosis, ANKRD31 also interacts with epithelial cell-cell junction proteins in the epididymis, where its loss disrupts the blood-epididymal barrier and causes infertility [PMID:34820371].","teleology":[{"year":2019,"claim":"Establishing whether a dedicated factor governs where and when meiotic DSBs form, knockouts showed ANKRD31 is required to direct DSBs to the sex-chromosome PARs and to set their number and timing.","evidence":"Ankrd31-knockout mouse spermatocytes with cytological and molecular recombination analysis, replicated in two concurrent papers","pmids":["31003867","31000436"],"confidence":"High","gaps":["Did not resolve the molecular basis of PAR-specific targeting","Mechanism behind the paradoxical increase in total DSBs unexplained"]},{"year":2019,"claim":"To define how ANKRD31 acts at the molecular level, a crystal structure and in vivo assays showed it binds the REC114 PH domain directly and anchors REC114 to specific genomic sites, defining its scaffold function.","evidence":"Crystal structure determination plus in vivo chromatin association assays in mouse spermatocytes","pmids":["31003867"],"confidence":"High","gaps":["Did not establish which other DSB factors are co-recruited","Structural basis of PAR enrichment versus generic axis sites not distinguished"]},{"year":2020,"claim":"Addressing why the PAR is the hottest DSB region, ANKRD31 (with MEI4) was shown to drive hyperaccumulation of DSB factors, PAR axis elongation, and sister separation linked to mo-2 minisatellite arrays.","evidence":"Knockout mouse models with super-resolution immunofluorescence and cytological axis measurements","pmids":["32461690"],"confidence":"High","gaps":["How mo-2 minisatellite arrays recruit the machinery not defined","Causal link between axis elongation and DSB hyperactivation not fully resolved"]},{"year":2021,"claim":"Testing dosage-dependent human relevance, heterozygous ANKRD31 variants in POI patients were found to act by disrupting the ANKRD31–REC114 interaction via haploinsufficiency.","evidence":"Exome sequencing in POI patients with functional interaction-disruption assays","pmids":["34257419"],"confidence":"Medium","gaps":["Single-lab clinical cohort; broader genetic confirmation absent","Oocyte-level mechanistic consequences not directly examined"]},{"year":2021,"claim":"Probing a meiosis-independent role, ANKRD31 was found to interact with epididymal cell-junction proteins and to be required for blood-epididymal barrier integrity.","evidence":"Ankrd31-knockout mice with co-immunoprecipitation of junction proteins and histology","pmids":["34820371"],"confidence":"Medium","gaps":["Specific junction partner identities not defined","Single co-IP without reciprocal validation; molecular mechanism unresolved"]},{"year":2023,"claim":"Clarifying how REC114 integrates multiple inputs, structural and biochemical work showed ANKRD31, IHO1, and TOPOVIBL bind the same REC114 PH surface, establishing mutually exclusive, competitive interactions.","evidence":"AlphaFold2 modeling with in vitro biochemistry and SEC-MALS","pmids":["37431931"],"confidence":"High","gaps":["Did not define how competition is regulated in vivo","Temporal ordering of partner exchange unresolved"]},{"year":2023,"claim":"Dissecting the functional importance of the binding interface, an allelic series showed full ANKRD31–REC114 disruption phenocopies the null while partial disruption only delays DSB timing, defining a dosage-graded requirement.","evidence":"Genome-edited mouse missense and truncation alleles with cytological DSB and crossover assays","pmids":["37976262"],"confidence":"High","gaps":["Separation of timing control from PAR-targeting at the molecular level incomplete","Repair-defect mechanism downstream of delayed DSBs not detailed"]},{"year":2024,"claim":"Resolving how ANKRD31 relates to the canonical axis-seeding route, epistasis showed it acts in a complementary, partially redundant pathway to IHO1–HORMAD1, enhancing seeding and growth of DSB-machinery clusters.","evidence":"Compound mouse mutants combining IHO1-HORMAD1 and Ankrd31 mutations with quantitative immunofluorescence of DSB foci","pmids":["38580643"],"confidence":"High","gaps":["Biophysical basis of cluster condensation not characterized","Whether redundancy applies equally in oocytes not tested"]},{"year":2023,"claim":"Extending the DSB-complex membership, MEI1 variants from azoospermia patients were shown to lose interaction with ANKRD31, placing ANKRD31 within a MEI1-containing DSB-promoting complex.","evidence":"Co-immunoprecipitation of mutant MEI1 with ANKRD31 and other meiotic factors","pmids":["41706353"],"confidence":"Medium","gaps":["Single co-IP for the ANKRD31 interaction; primarily a MEI1-focused study","Direct versus indirect ANKRD31–MEI1 contact not established"]},{"year":null,"claim":"How ANKRD31 recognizes specific genomic features such as the PAR mo-2 arrays to nucleate DSB-machinery condensation, and the molecular basis of its epididymal barrier role, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No defined DNA/chromatin recognition determinant for PAR targeting","Epididymal junction partners and mechanism uncharacterized","No reconstituted biophysical model of cluster nucleation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,7]}],"localization":[{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[1,2,3]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,5,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,2]}],"complexes":["meiotic DSB-promoting complex (REC114-MEI4-IHO1)"],"partners":["REC114","IHO1","MEI4","MEI1","TOPOVIBL"],"other_free_text":[]}},"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":128,"is_preprint":false},{"pmid":"34794894","id":"PMC_34794894","title":"Genetics of ovarian insufficiency and defects of folliculogenesis.","date":"2021","source":"Best practice & research. 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Spermatocytes lacking ANKRD31 have altered DSB locations, fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes, and show delayed and/or fewer recombination sites but paradoxically more DSBs overall, indicating DSB dysregulation.\",\n      \"method\": \"Knockout mouse model (Ankrd31-deficient spermatocytes), cytological and molecular analysis of recombination\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotypes, replicated independently in two concurrent papers (PMID:31003867 and PMID:31000436)\",\n      \"pmids\": [\"31003867\", \"31000436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A crystal structure reveals that REC114 contains a pleckstrin homology (PH) domain that directly contacts ANKRD31 through intermolecular interactions. ANKRD31 stabilizes REC114 association with the PAR and elsewhere in vivo, functioning as a scaffold that anchors REC114 and other DSB-promoting factors to specific genomic locations.\",\n      \"method\": \"Crystal structure determination, in vivo chromatin association assays in mouse spermatocytes\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vivo functional validation, replicated in subsequent structural/biochemical work (PMID:37431931)\",\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 genome-wide delayed recombination initiation, reduced selectivity for DSB hotspot sites, and loss of a specialized PAR-axis domain highly enriched for DSB-promoting proteins.\",\n      \"method\": \"Knockout mouse model, immunofluorescence/cytology, ChIP, genome-wide DSB mapping\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with multiple orthogonal molecular readouts, replicated independently\",\n      \"pmids\": [\"31000436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MEI4 and ANKRD31 proteins are required for the hyperaccumulation of DSB-promoting factors in the PAR, elongation of PAR chromosome axes, and separation of sister chromatids prior to DSB formation — processes linked to heterochromatic mo-2 minisatellite arrays. These events make the PAR the hottest DSB segment in the male mouse genome.\",\n      \"method\": \"Knockout mouse models, super-resolution and conventional immunofluorescence, cytological axis measurements\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO in vivo with multiple orthogonal structural and molecular methods\",\n      \"pmids\": [\"32461690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ANKRD31 physically interacts with epithelial cell-cell junction proteins in the epididymis. Loss of ANKRD31 in knockout male mice disrupts the blood-epididymal barrier (BEB) due to cell-to-cell junction anomalies, resulting in oligo-astheno-teratozoospermia and infertility.\",\n      \"method\": \"Ankrd31 knockout mouse model, co-immunoprecipitation of ANKRD31 with junction proteins in wild-type epididymis, histology\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus KO phenotype in single lab; junction partner identities and mechanism not fully defined\",\n      \"pmids\": [\"34820371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pathogenic heterozygous variants in ANKRD31 identified in premature ovarian insufficiency (POI) patients disrupt the interaction between ANKRD31 and the DSB-formation factor REC114, exerting their pathogenic effect via haploinsufficiency, indicating dosage-dependent control of ovarian function.\",\n      \"method\": \"Exome sequencing in POI patients, functional interaction assays (disruption of ANKRD31-REC114 interaction by variants)\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protein-interaction disruption assay plus clinical genetics, single lab\",\n      \"pmids\": [\"34257419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The REC114 PH domain interacts with ANKRD31 and with IHO1 and TOPOVIBL at the same surface, indicating mutually exclusive interactions. REC114 acts as a regulatory platform where ANKRD31 competes with other partners for binding.\",\n      \"method\": \"AlphaFold2 structural modeling combined with in vitro biochemical characterization and size exclusion chromatography-multi-angle light scattering (SEC-MALS)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural modeling validated by multiple biochemical assays in single lab\",\n      \"pmids\": [\"37431931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Complete disruption of the ANKRD31-REC114 interaction (by C-terminal truncation of ANKRD31) mimics the Ankrd31 null phenotype: delayed global DSB formation, defects in DSB repair, and failure to target DSBs to the PARs. Substantial but incomplete disruption (missense mutation) delays DSB formation but leaves recombination, repair, and DSB locations near normal. A dosage effect was observed when combining partial-loss and null alleles.\",\n      \"method\": \"Genome-edited mouse models carrying specific Ankrd31 missense and truncation mutations, cytological DSB assays, crossover analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple allelic series in mice with orthogonal molecular and cytological readouts, peer-reviewed\",\n      \"pmids\": [\"37976262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"When the IHO1-HORMAD1 axis-seeding pathway is disrupted, residual meiotic DSBs become dependent on ANKRD31, which enhances both the seeding and growth of DSB-machinery clusters on chromosome axes, demonstrating that ANKRD31 and the IHO1-HORMAD1 pathway act in complementary, partially redundant routes for DSB-machinery condensation.\",\n      \"method\": \"Compound mouse mutant analysis (IHO1-HORMAD1 interaction mutant combined with Ankrd31 mutant), immunofluorescence quantification of DSB-factor foci\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in vivo using clean compound mutants with quantitative cytological readouts, published in peer-reviewed journal\",\n      \"pmids\": [\"38580643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MEI1 variants identified in non-obstructive azoospermia patients disrupt MEI1 interactions with ANKRD31 (as well as IHO1, REC114, and MEI4) as detected by co-immunoprecipitation, consistent with ANKRD31 being a component of the MEI1-containing DSB-promoting complex.\",\n      \"method\": \"Co-immunoprecipitation assays of mutant MEI1 with ANKRD31 and other meiotic factors\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP result for the ANKRD31 interaction specifically; primarily a MEI1 study\",\n      \"pmids\": [\"41706353\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANKRD31 is a scaffold protein that localizes to meiotic chromosome axes and directly binds REC114 (via REC114's PH domain) to regulate the number, timing, and genomic distribution of meiotic DNA double-strand breaks, with a critical role in hyperactivating DSB formation at the pseudoautosomal regions of sex chromosomes; it also participates in the epididymal blood-epididymal barrier by interacting with cell-junction proteins, and its haploinsufficiency can cause premature ovarian insufficiency by impairing the ANKRD31-REC114 interaction in a dosage-dependent manner.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ANKRD31 is a meiotic scaffold protein that assembles with DSB-promoting factors on chromosome axes to control the number, timing, and genomic distribution of programmed meiotic DNA double-strand breaks (DSBs) [#0, #2]. Loss of ANKRD31 dysregulates DSBs genome-wide — delaying recombination initiation, reducing selectivity for hotspots, and paradoxically increasing total DSBs — while abolishing the specialized PAR-axis domain that normally makes the pseudoautosomal regions of the sex chromosomes the hottest DSB segment in the male genome [#0, #2, #3]. Mechanistically, ANKRD31 binds directly to the pleckstrin homology (PH) domain of REC114, anchoring REC114 and associated DSB machinery to the PAR and to axis sites in vivo; this same REC114 PH surface is engaged competitively by IHO1 and TOPOVIBL, casting REC114 as a regulatory platform for mutually exclusive partner binding [#1, #6]. The ANKRD31–REC114 interaction is functionally central: complete disruption phenocopies the null (delayed DSBs, repair defects, loss of PAR targeting), whereas partial disruption only delays DSB timing, defining a dosage-dependent requirement [#7]. ANKRD31 acts in a complementary, partially redundant route to the IHO1–HORMAD1 axis-seeding pathway, enhancing the seeding and growth of DSB-machinery clusters on axes [#8]. Heterozygous ANKRD31 variants cause premature ovarian insufficiency through haploinsufficiency by disrupting the ANKRD31–REC114 interaction [#5]. Beyond meiosis, ANKRD31 also interacts with epithelial cell-cell junction proteins in the epididymis, where its loss disrupts the blood-epididymal barrier and causes infertility [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Establishing whether a dedicated factor governs where and when meiotic DSBs form, knockouts showed ANKRD31 is required to direct DSBs to the sex-chromosome PARs and to set their number and timing.\",\n      \"evidence\": \"Ankrd31-knockout mouse spermatocytes with cytological and molecular recombination analysis, replicated in two concurrent papers\",\n      \"pmids\": [\"31003867\", \"31000436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not resolve the molecular basis of PAR-specific targeting\",\n        \"Mechanism behind the paradoxical increase in total DSBs unexplained\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"To define how ANKRD31 acts at the molecular level, a crystal structure and in vivo assays showed it binds the REC114 PH domain directly and anchors REC114 to specific genomic sites, defining its scaffold function.\",\n      \"evidence\": \"Crystal structure determination plus in vivo chromatin association assays in mouse spermatocytes\",\n      \"pmids\": [\"31003867\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not establish which other DSB factors are co-recruited\",\n        \"Structural basis of PAR enrichment versus generic axis sites not distinguished\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Addressing why the PAR is the hottest DSB region, ANKRD31 (with MEI4) was shown to drive hyperaccumulation of DSB factors, PAR axis elongation, and sister separation linked to mo-2 minisatellite arrays.\",\n      \"evidence\": \"Knockout mouse models with super-resolution immunofluorescence and cytological axis measurements\",\n      \"pmids\": [\"32461690\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How mo-2 minisatellite arrays recruit the machinery not defined\",\n        \"Causal link between axis elongation and DSB hyperactivation not fully resolved\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Testing dosage-dependent human relevance, heterozygous ANKRD31 variants in POI patients were found to act by disrupting the ANKRD31–REC114 interaction via haploinsufficiency.\",\n      \"evidence\": \"Exome sequencing in POI patients with functional interaction-disruption assays\",\n      \"pmids\": [\"34257419\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab clinical cohort; broader genetic confirmation absent\",\n        \"Oocyte-level mechanistic consequences not directly examined\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Probing a meiosis-independent role, ANKRD31 was found to interact with epididymal cell-junction proteins and to be required for blood-epididymal barrier integrity.\",\n      \"evidence\": \"Ankrd31-knockout mice with co-immunoprecipitation of junction proteins and histology\",\n      \"pmids\": [\"34820371\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific junction partner identities not defined\",\n        \"Single co-IP without reciprocal validation; molecular mechanism unresolved\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Clarifying how REC114 integrates multiple inputs, structural and biochemical work showed ANKRD31, IHO1, and TOPOVIBL bind the same REC114 PH surface, establishing mutually exclusive, competitive interactions.\",\n      \"evidence\": \"AlphaFold2 modeling with in vitro biochemistry and SEC-MALS\",\n      \"pmids\": [\"37431931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not define how competition is regulated in vivo\",\n        \"Temporal ordering of partner exchange unresolved\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Dissecting the functional importance of the binding interface, an allelic series showed full ANKRD31–REC114 disruption phenocopies the null while partial disruption only delays DSB timing, defining a dosage-graded requirement.\",\n      \"evidence\": \"Genome-edited mouse missense and truncation alleles with cytological DSB and crossover assays\",\n      \"pmids\": [\"37976262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Separation of timing control from PAR-targeting at the molecular level incomplete\",\n        \"Repair-defect mechanism downstream of delayed DSBs not detailed\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolving how ANKRD31 relates to the canonical axis-seeding route, epistasis showed it acts in a complementary, partially redundant pathway to IHO1–HORMAD1, enhancing seeding and growth of DSB-machinery clusters.\",\n      \"evidence\": \"Compound mouse mutants combining IHO1-HORMAD1 and Ankrd31 mutations with quantitative immunofluorescence of DSB foci\",\n      \"pmids\": [\"38580643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Biophysical basis of cluster condensation not characterized\",\n        \"Whether redundancy applies equally in oocytes not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extending the DSB-complex membership, MEI1 variants from azoospermia patients were shown to lose interaction with ANKRD31, placing ANKRD31 within a MEI1-containing DSB-promoting complex.\",\n      \"evidence\": \"Co-immunoprecipitation of mutant MEI1 with ANKRD31 and other meiotic factors\",\n      \"pmids\": [\"41706353\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single co-IP for the ANKRD31 interaction; primarily a MEI1-focused study\",\n        \"Direct versus indirect ANKRD31–MEI1 contact not established\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ANKRD31 recognizes specific genomic features such as the PAR mo-2 arrays to nucleate DSB-machinery condensation, and the molecular basis of its epididymal barrier role, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No defined DNA/chromatin recognition determinant for PAR targeting\",\n        \"Epididymal junction partners and mechanism uncharacterized\",\n        \"No reconstituted biophysical model of cluster nucleation\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [1, 2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 5, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [\n      \"meiotic DSB-promoting complex (REC114-MEI4-IHO1)\"\n    ],\n    \"partners\": [\n      \"REC114\",\n      \"IHO1\",\n      \"MEI4\",\n      \"MEI1\",\n      \"TOPOVIBL\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}