PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice

Abstract

Meiotic recombination events cluster into narrow segments of the genome, defined as hotspots. Here, we demonstrate that a major player for hotspot specification is the Prdm9 gene. First, two mouse strains that differ in hotspot usage are polymorphic for the zinc finger DNA binding array of PRDM9. Second, the human consensus PRDM9 allele is predicted to recognize the 13-mer motif enriched at human hotspots; this DNA binding specificity is verified by in vitro studies. Third, allelic variants of PRDM9 zinc fingers are significantly associated with variability in genome-wide hotspot usage among humans. Our results provide a molecular basis for the distribution of meiotic recombination in mammals, in which the binding of PRDM9 to specific DNA sequences targets the initiation of recombination at specific locations in the genome.

Figure 1. Mouse wm7 and b Prdm9 alleles are polymorphic at residues involved in specifying DNA targets in the zinc finger array

A. The tandem repeat structure of the mouse PRDM9 zinc finger array Upper panel: The structure of the mouse b allele is shown, with the Krueppel-associated box (KRAB), the PR/SET domain and the zinc fingers (Zn) shaded in blue, yellow and green, respectively. Lower panel: The sequences of the C-terminal tandem arrays of zinc fingers of the b allele (left) and the wm7 allele (right) are shown. The coordinate of the first residue of each repeat on the protein sequence is indicated. The residues identical to the second repeat are represented by stars (except for the first, incomplete zinc finger). The C and H residues, characteristic of the C2H2 zinc fingers, are in red. The residues at positions −1, 3 and 6 of every zinc finger, which are of special importance for specifying the DNA target, are in blue. B. PRDM9 wm7 and b alleles are predicted to recognize distinct DNA sequences. The amino-acids at position −1, 3 and 6 of the zinc finger alpha helices, used for the prediction, are indicated under the corresponding bases of each DNA motif.

Figure 2

A. Human PRDM9 major alleles (A and B) are predicted to bind the 13-mer hotspot motif, whereas the I allele is predicted to bind a distinct motif. The LD-based hotspot consensus identified by Myers et al. (13) is shown above. The amino-acids at position −1, 3 and 6 of the zinc finger alpha helices are indicated as in figure 1B, with the residues predicted to recognize the LD-hotspot consensus motif shown in red. B. Allelic diversity of the human PRDM9 zinc finger tandem array. Interspersion patterns of variant repeats (colored boxes) of alleles from unrelated individuals was established either by Minisatellite Variant Repeat mapping (105 CEPH unrelated parents or grand-parents and 351 Hutterite parents) or by sequencing clones from a testis cDNA library made from 39 donors. Major allele A and minor allele B were found in all three sets of unrelated individuals and other rare alleles only in one or two sets. The structures of some rare alleles (I, C, E and F) differ strongly from A and B in the region encoding the critical domain (red bar) for recognition of the 13-mer hotspot motif.

Figure 3

Association of human Prdm9 alleles with genome-wide (LD-based) hotspot usage. The different genotypes for variants in the zinc finger array are indicated by different colors. A) In each individual, the percentage of recombination events that occurred in LD-based hotspots. The maximum likelihood estimate (MLE) for each individual is shown as a point, and the 95% confidence intervals (asymptotic cutoff) are indicated by the lengths of the horizontal lines. Individuals are ordered by their MLE. The black vertical line shows the joint MLE for all individuals. B) and C) The relative log-likelihood surfaces of the percentage of recombination events that occurred in LD-based hotspots for the three genotypes (AA,AB and AI), for males and females respectively. The curve for the BI genotype is left out due to low sample size (n=1). The grey line is provided as a visual guide, to indicate where the asymptotic cutoff is for the 95% confidence interval.

Figure 4

Human PRDM9 zinc finger domains of alleles A (ZA) and I (ZI) interact specifically with double stranded oligonucleotides containing the extended motif associated with LD-based hotspots (13) (HM) and the predicted binding motif for hPRDM9 I allele (IM) respectively. (A–D) Left panels: South-Western blotting experiment performed with His-tagged ZI and ZA proteins from total E. coli extracts, probed with HM. Right panels are mirror image blots obtained after diffusion transfer to a membrane placed on the other side of the same protein gel (25).(A) Immunoblotting experiment using monoclonal α-polyhistidine antibody. (B) South-Western blotting using IM probe. (C) South-Western blotting using the HM* probe, which contains multiple mutations in the 13-mer motif. (D) Electrophoretic mobility shift assays with in vitro translated GST-hPRDM9 zinc finger domain fusions of alleles A (ZA) or I (ZI). The probes on the left and right panels are respectively HM and IM. Cold competitor, in molar excess of 20 and 200 fold over the probe, has been added as mentioned.