REC114 partner ANKRD31 controls number, timing and location of meiotic DNA breaks

Abstract

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation.Here we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to sex chromosomes' pseudoautosomal regions (PAR). They also have delayed/fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines direct ANKRD31-REC114 molecular contacts and reveals a surprising pleckstrin homology domain in REC114. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model that ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, promoting efficient and timely DSB formation but possibly also suppressing formation of clustered DSBs. We thank Attila Tóth and Bernard de Massy for discussions and sharing unpublished information. We thank Keeney lab members C. Claeys Bouuaert, N. Mohibullah, M. van Overbeek, and S. Kim for experimental advice. We thank MSKCC core facilities, supported by NIH cancer center core grant P30 CA008748: Mouse Genetics (P. Romanienko and W. Mark) for generating the Ankrd31 knockout; Molecular Cytology (K. Manova) for histology and expansion microscopy; the Integrated Genome Operation (A. Viale and N. Mohibullah) for sequencing; Bioinformatics (N. Socci) for analysis; and the Laboratory of Comparative Pathology (S. Monette) for necropsy. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). X-ray diffraction studies were

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region

“…We found that the PAR was highly enriched for DSB-promoting factors REC114, MEI4, MEI1, and IHO1 [proteins essential for genomewide DSB formation (22-24, 29)] as well as ANKRD31, a REC114 partner essential for PAR DSB formation (30,31). All five proteins (hereafter RMMAI for simplicity) colocalized in several bright, irregular "blobs" for most of prophase I (Fig.…”

“…5D). [Ankrd31 -/mutants form substantially fewer RPA2 foci and other cytological DSB markers at leptonema and early zygonema, but not from mid-zygonema on (30,31).] As a consequence, the X and Y chromosomes were paired in only 6% of mid-pachytene spermatocytes ( Fig.…”

“…As a consequence, the X and Y chromosomes were paired in only 6% of mid-pachytene spermatocytes ( Fig. 5F), whereas most Ankrd31 -/cells pair and synapse all autosomes (30,31).…”

“…To test more directly whether autosomal mo-2 regions experience PAR-like DSB formation, we used maps of ssDNA bound by the strandexchange protein DMC1 (ssDNA sequencing, or SSDS) (9,41,42). Separate studies showed that ANKRD31 is critical for high-level DSB formation in the PAR (30,31). We used our SSDS maps from wholetestis samples of juvenile mice (12 days post partum (30)) and other SSDS maps to test for PAR-like (i.e., ANKRD31-dependent) DSB signal at autosomal mo-2 regions.…”

“…Separate studies showed that ANKRD31 is critical for high-level DSB formation in the PAR (30,31). We used our SSDS maps from wholetestis samples of juvenile mice (12 days post partum (30)) and other SSDS maps to test for PAR-like (i.e., ANKRD31-dependent) DSB signal at autosomal mo-2 regions.…”