Principles of the spatiotemporal control of meiotic recombination initiation in mice

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Programmed formation of several hundred DNA double-strand-breaks (DSBs) is essential in meiosis, as DSBs result in single-stranded DNA (ssDNA) ends which initiate homologous meiotic recombination. Recombination promotes pairing and synapsis between homologous chromosomes (homologs), and recombination-mediated repair of DSBs generates crossovers, which are essential for correct chromosome segregation in meiosis. Further, recombination creates new allele combinations on which selection acts. Anomalies in recombination cause aneuploidies and infertility, and persistent DSBs are potentially genotoxic. Hence, meiotic DSB formation is under tight spatiotemporal control.A poorly understood interplay of multiple factors regulates the numbers, the timing and the genomic locations of DSBs to ensure synapsis and crossovers between homologs. In particular, two distinct negative feedbacks are thought to control DSB activity: (1) DSBs limit further DSB formation in their vicinity, and (2) synapsis of homologs regionally shuts down DSB activity. Beyond these feedbacks, the cell cycle stage and interactions between DSB-promoting protein complexes and chromatin also affect the location of DSBs. A meiosis-specific histone methyltransferase, PRDM9, generates "open" chromatin sites in a sequence and meiosis stage-specific manner at several thousand sites in mammalian genomes. The DSB machinery preferentially targets these PRDM9-associated "open" sites, hence they act as DSB hotspots, where most meiotic recombination initiation occurs.Using an interdisciplinary approach combining time-resolved experiments and biophysical modelling the project aims to uncover how the positions of DSB activity are affected by feedback mechanisms, the stage of meiosis, and two key components of the DSB machinery, ANKRD31 and IHO1, which have distinct roles in recombination initiation. We will, for the first time, measure spatiotemporal dynamics of hotspot activity and underlying chromatin features during meiotic progression in mice. Specifically, we will use ChIP-seq to monitor genomic distribution of ssDNAs (DMC1), hotspot-associated open chromatin (tri-methylation of Lys 4 in histone H3) and unsynapsed regions (HORMAD1) in meiotic time-courses. To separate feedback- and meiosis stage-dependent effects on hotspot dynamics we will make measurements not only in wild-type but also in mutants defective in meiotic DSB and synapsis formation. These measurements will be used to generate a quantitative model of hotspot usage dynamics. The model, in the next step, will be used to interpret pleiotropic effects of IHO1- and ANKRD31-deficiencies on SC kinetics, DSB numbers, timing and positioning, to understand the differential roles of IHO1 and ANKRD31 in hotspot usage. Ultimately, the above measurements and their modelling will produce a comprehensive understanding of the mechanisms that govern the landscape of DSB formation and enable a successful passage of the genome to the next generation.