Essential roles of the ANKRD31-REC114 interaction in meiotic recombination and mouse spermatogenesis

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine if and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31- REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31- REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114, and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction. SignificanceHomologous recombination initiated by double-strand breaks (DSBs) during meiosis is a nearly universal feature of eukaryotic lifecycles, but is also dangerous because DSBs are potentially toxic or mutagenic. The vertebrate-specific protein ANKRD31 is an important regulator of DSB formation, proposed to be a scaffold protein that coordinates the activities of multiple DSB-promoting factors, including the widely conserved REC114. We test this hypothesis here through generation of targeted Ankrd31 mutations that specifically attenuate or eliminate the ANKRD31-REC114 interaction. Analysis of this allelic series demonstrates that the ANKRD31-REC114 interaction is essential for all ANKRD31 activities in vivo, providing insight into how ANKRD31 controls DSB locations, timing, and number.