Cataloging Human PRDM9 Allelic Variation Using Long-Read Sequencing Reveals PRDM9 Population Specificity and Two Distinct Groupings of Related Alleles

The PRDM9 protein determines sites of meiotic recombination in humans by directing meiotic DNA double-strand breaks to specific loci. Targeting specificity is encoded by a long array of C2H2 zinc fingers that bind to DNA. This zinc finger array is hypervariable, and the resulting alleles each have a potentially different DNA binding preference. The assessment of PRDM9 diversity is important for understanding the complexity of human population genetics, inheritance linkage patterns, and predisposition to genetic disease. Due to the repetitive nature of the PRDM9 zinc finger array, the large-scale sequencing of human PRDM9 is challenging. We, therefore, developed a long-read sequencing strategy to infer the diploid PRDM9 zinc finger array genotype in a high-throughput manner. From an unbiased study of PRDM9 allelic diversity in 720 individuals from seven human populations, we detected 69 PRDM9 alleles. Several alleles differ in frequency among human populations, and 32 alleles had not been identified by previous studies, which were heavily biased to European populations. PRDM9 alleles are distinguished by their DNA binding site preferences and fall into two major categories related to the most common PRDM9-A and PRDM9-C alleles. We also found that it is likely that inter-conversion between allele types is rare. By mapping meiotic double-strand breaks (DSBs) in the testis, we found that small variations in PRDM9 can substantially alter the meiotic recombination landscape, demonstrating that minor PRDM9 variants may play an under-appreciated role in shaping patterns of human recombination. In summary, our data greatly expands knowledge of PRDM9 diversity in humans.

Allelic diversity of the PRDM9 coding minisatellite in minke whales

The PRDM9 protein controls the reshuffling of parental genomes in most metazoans. During meiosis the PRDM9 protein recognizes and binds specific target motifs via its zinc-finger array, which is encoded by a hypervariable minisatellite. To date, PRDM9 diversity has been little studied outside humans, wild mice and some domesticated species where evolutionary constraints may have been relaxed. Here we explore the structure and variability of PRDM9 in samples of the minke whales from the Atlantic, Pacific and Southern Oceans. We show that minke whales possess all the features characteristic of organisms with PRDM9-directed recombination initiation, including complete KRAB, SSXRD and SET domains and a rapidly evolving array of C2H2-type-Zincfingers (ZnF). We uncovered eighteen novel PRDM9 variants and evidence of rapid evolution, particularly of DNA-recognizing positions that evolve under positive selection. At different geographical scales, we observed extensive PRDM9 diversity in Antarctic minke whales (Balaenoptera bonarensis), that conversely lack observable population differentiation in mitochondrial DNA and microsatellites. In contrast, a single PRDM9 variant is shared between all Common Minke whales and even across subspecies boundaries of North Atlantic (B. a. acutorostrata) and North Pacific (B. a. scammoni) minke whale, which do show clear population differentiation. PRDM9 variation of whales predicts distinct recombination initiation landscapes genome wide, which has possible consequences for speciation.

PRDM9 forms a trimer by interactions within the zinc finger array

PRDM9 is a trans-acting factor directing meiotic recombination to specific DNA-binding sites by its zinc finger (ZnF) array. It was suggested that PRDM9 is a multimer; however, we do not know the stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and characterized with electrophoretic mobility shift assays, mass spectrometry, and fluorescence correlation spectroscopy the stoichiometry of the PRDM9 multimer of two different murine PRDM9 alleles carrying different tags and domains produced with different expression systems. Based on the migration distance of the PRDM9–DNA complex, we show that PRDM9 forms a trimer. Moreover, this stoichiometry is adapted already by the free, soluble protein with little exchange between protein monomers. The variable ZnF array of PRDM9 is sufficient for multimerization, and at least five ZnFs form already a functional trimer. Finally, we also show that only one ZnF array within the PRDM9 oligomer binds to the DNA, whereas the remaining two ZnF arrays likely maintain the trimer by ZnF–ZnF interactions.

Formation of novel PRDM9 allele by indel events as possible trigger for tarsier-anthropoid split

PRDM9 is currently the sole speciation gene found in vertebrates causing hybrid sterility probably due to incompatible alleles. Its role in defining the double strand break loci during the meiotic prophase I is crucial for proper chromosome segregation. Therefore, the rapid turnover of the loci determining zinc finger array seems to be causative for incompatibilities. We here investigated the zinc finger domain-containing exon of PRDM9 in 23 tarsiers. Tarsiers, the most basal extant haplorhine primates, exhibit two frameshifting indels at the 5’-end of the array. The first mutation event interrupts the reading frame and function while the second compensates both. The fixation of this peculiar allele variant in tarsiers led to hypothesize that de‐ and reactivation of the zinc finger domain drove the speciation in early haplorhine primates. Moreover, the high allelic diversity within Tarsius point to multiple effects of genetic drift reflecting their phylogeographic history since the Miocene.