In vitro studies of DNA binding by PRDM9 expressed in E. coli are being used to identify the genome-wide distribution of potential PRDM9 binding sites and to analyze the complex rules governing PRDM9’s DNA-binding specificity. This last has significance beyond the field of recombination biology as there are over 800 zinc finger protein genes in mammalian genomes; they serve as the most ubiquitous DNA recognition device in biology, and over half of them, like PRDM9, have eight or more fingers. The rules governing their DNA-binding specificities are poorly understood, and PRDM9 with its tens of thousands of potential binding sites in mammalian genomes, combined with the availability of multiple alleles of PRDM9, each with different binding specificities, makes this a favorable model system for understanding the rules governing this most common DNA-recognition device in biological systems.
Using ChIP-SEQ to identify sites of PRDM9-dependent H3K4 trimethylation, we have identified ~20,000 sites of PRDM9 action during early meiosis, showing that PRDM9 binding first serves to reorient nucleosomes locally, creating a nucleosome-free zone in which double-strand break formation can occur. The span of trimethylated nucleosomes then determines the migration limits of the Holliday junctions between recombining DNA strands and hence the locations of genetic crossovers. We are presently using this assay to characterize allelic variation in PRDM9 function, the competition that ensues when more than one PRDM9 allele is active in the same cell, and the influences of sex and of other chromatin-modifying enzymes on initiation of recombination.
Inferring that PRDM9 cannot act in isolation, we are searching for interacting partners using both physical and genetic strategies, testing physical interactions of PRDM9 with other proteins in vitro and in vivo, and using selected genetic crosses to search for modifiers of PRDM9 function.
Our long-term goals are to provide a more comprehensive understanding of how PRDM9 serves as the signaling device to initiate recombination and how the locations, relative activity and fate of recombination hotspots are determined.
Dr. Petko Petkov shares full responsibility for our research group. Other members include a bioinformatics specialist, three long-serving technicians and a group of rotating postdoctoral fellows.