MODELING THE MULTIPLE ZINC FINGER PROTEIN PRDM9 BINDING WITH AFFINITY-SEQ

Mammalian genomes contain over 700 zinc finger proteins (ZFPs), but most have unknown functions. One example, PR Domain Containing 9 (PRDM9), regulates location of meiotic homologous recombination by binding to the DNA, causing epigenetic modifications and allowing for a double strand break (DSB); however its targeting mechanism is not fully understood. Knockouts in mice PRDM9 have been associated with infertility, which is suspected to be caused by mislocation of DSBs during meiosis. Over 100 alleles of PRDM9 are known in mice, each of which contains a unique zinc finger array and therefore selects different DNA binding sites for PRDM9. To detect and quantify all binding sites of PRDM9 without interference by additional regulatory effects (protein abundance, chromatin accessibility), we used a novel, in vitro assay called Affinity-Seq. We found over 39,000 significant binding sites for the PRDM9^Dom2 allele in C57BL/6J mouse DNA. Quantification of the binding frequency at each sequence enabled estimation of binding affinity at each site in addition to standard nucleotide frequencies. To gauge the contribution of each nucleotide, we built a linear regression model that includes additive and interactive effects on the binding preference of PRDM9. We identified a few core nucleotides required for binding and additional bases that quantitatively modify binding affinity. We tested this model by performing Affinity-Seq on the CAST/EiJ genome providing various natural polymorphisms for quantitative validation. Our work yields a detailed view of the targeting mechanism of PRDM9 in meiosis and can be broadly applied to any ZFP.