ANKRD31

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).

1. 2019 High

   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

   PMID:31000436 PMID:31003867

   Open questions at the time

   • Did not resolve the molecular basis of PAR-specific targeting
   • Mechanism behind the paradoxical increase in total DSBs unexplained
2. 2019 High

   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

   PMID:31003867

   Open questions at the time

   • Did not establish which other DSB factors are co-recruited
   • Structural basis of PAR enrichment versus generic axis sites not distinguished
3. 2020 High

   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

   PMID:32461690

   Open questions at the time

   • How mo-2 minisatellite arrays recruit the machinery not defined
   • Causal link between axis elongation and DSB hyperactivation not fully resolved
4. 2021 Medium

   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

   PMID:34257419

   Open questions at the time

   • Single-lab clinical cohort; broader genetic confirmation absent
   • Oocyte-level mechanistic consequences not directly examined
5. 2021 Medium

   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

   PMID:34820371

   Open questions at the time

   • Specific junction partner identities not defined
   • Single co-IP without reciprocal validation; molecular mechanism unresolved
6. 2023 High

   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

   PMID:37431931

   Open questions at the time

   • Did not define how competition is regulated in vivo
   • Temporal ordering of partner exchange unresolved
7. 2023 High

   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

   PMID:37976262

   Open questions at the time

   • Separation of timing control from PAR-targeting at the molecular level incomplete
   • Repair-defect mechanism downstream of delayed DSBs not detailed
8. 2024 High

   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

   PMID:38580643

   Open questions at the time

   • Biophysical basis of cluster condensation not characterized
   • Whether redundancy applies equally in oocytes not tested
9. 2023 Medium

   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

   PMID:41706353

   Open questions at the time

   • Single co-IP for the ANKRD31 interaction; primarily a MEI1-focused study
   • Direct versus indirect ANKRD31–MEI1 contact not established

• 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.
• No defined DNA/chromatin recognition determinant for PAR targeting
• Epididymal junction partners and mechanism uncharacterized
• No reconstituted biophysical model of cluster nucleation