PRDM9 has recently been identified as a likely trans-regulator of meiotic recombination hot spots in humans and mice1-3. The protein contains a zinc finger array that in humans can recognise a short sequence motif associated with hot spots4, with binding to this motif possibly triggering hot-spot activity via chromatin remodelling5. We now show that variation in the zinc finger array in humans has a profound effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Very subtle changes within the array can create hot-spot non-activating and enhancing alleles, and even trigger the appearance of a new hot spot. PRDM9 thus appears to be the preeminent global regulator of hot spots in humans. Variation at this locus also influences aspects of genome instability, specifically a megabase-scale rearrangement underlying two genomic disorders6 as well as minisatellite instability7, implicating PRDM9 as a risk factor for some pathological genome rearrangements.

PRDM9 is a major specifier of human meiotic recombination hotspots, probably via binding of its zinc-finger repeat array to a DNA sequence motif associated with hotspots. However, our view of PRDM9 regulation, in terms of motifs defined and hotspots studied, has a strong bias toward the PRDM9 A variant particularly common in Europeans. We show that population diversity can reveal a second class of hotspots specifically activated by PRDM9 variants common in Africans but rare in Europeans. These Africanenhanced hotspots nevertheless share very similar properties with their counterparts activated by the A variant. The specificity of hotspot activation is such that individuals with differing PRDM9 genotypes, even within the same population, can use substantially if not completely different sets of hotspots. Each African-enhanced hotspot is activated by a distinct spectrum of PRDM9 variants, despite the fact that all are predicted to bind the same sequence motif. This differential activation points to complex interactions between the zinc-finger array and hotspots and identifies features of the array that might be important in controlling hotspot activity.eiotic recombination is fundamentally important in ensuring correct chromosome disjunction at meiosis and in reshuffling haplotypes between generations, substantially increasing haplotype diversity within a population. Most recombination events in the human genome are clustered into narrow hotspots that can be identified indirectly from patterns of linkage disequilibrium (LD hotspots) (1), or directly through high-resolution linkage analysis in pedigrees (2) or by sperm typing (3). Genomewide comparison of LD hotspots has identified a sequence motif CCNCCNTNNCCNC associated with 40% of these hotspots; this motif appears to influence the initiation of meiotic recombination, because SNPs that disrupt the motif can down-regulate recombination (4).Recently, the meiosis-specific protein PRDM9 has been identified as a major specifier of hotspots in the human and mouse genome (5-7). PRDM9 contains a SET domain that might be responsible for activating hotspots by chromatin remodelling (8), plus a C-terminal tandem-repeat zinc-finger (ZnF) array encoded by a variable minisatellite. Evidence that PRDM9 regulates hotspots comes from the finding that the common European variant A has a ZnF array that binds, at least in vitro, to the 13-mer hotspot motif shared by many LD hotspots identified in Europeans (5, 6). Furthermore, association analyses in Hutterites (5) and Icelanders (2) have shown that individuals with variant non-A PRDM9 alleles can show genome-wide shifts in hotspot usage. These shifts suggest that ZnF variants that should not bind the PRDM9 A motif might trigger the appearance of new sets of hotspots (5), although it is possible that some of these shifts reflect additional hotspot-specification systems that only become manifest as the dosage of the PRDM9 A variant is reduced. The Icelandic study highlighted the PRDM9 C variant and its associated predicted motif CC...

Population diversity data have recently provided profound, albeit inferential, insights into meiotic recombination across the human genome, revealing a landscape dominated by thousands of cross-over hotspots. However, very few of these putative hotspots have been directly analyzed for cross-over activity. We now describe a search for very active hotspots, by using extreme breakdown of marker association as a guide for high-resolution sperm cross-over analysis. This strategy has led to the isolation of the most active cross-over hotspots yet described. Their morphology, sequence attributes, and cross-over processes are very similar to those seen at less active hotspots, but their activity in sperm is poorly predicted from population diversity information. Several of these hotspots showed evidence for biased gene conversion accompanying cross-over, in some cases associated with variation between men in cross-over activity and with two hotspots showing complete presence/absence polymorphism in different men. Hotspot polymorphism is very common at less active hotspots but curiously was not seen at any of the most active hotspots. This contrasts with the prediction that extreme hotspots should be the most vulnerable to attenuation by meiotic drive in favor of mutations that suppress recombination and should therefore show rapid rate evolution and thus variation in activity between men. Finally, these very intense hotspots provide a valuable resource for dissecting meiotic recombination processes and pathways in humans.conversion ͉ hotspot ͉ meiosis ͉ polymorphism ͉ recombination T he recombinational exchange of DNA between homologous chromosomes at meiosis is vital to ensure correct chromosome segregation and also plays a major role in increasing haplotype diversity within populations. In humans, the combination of low average cross-over frequency [Ϸ1% recombination frequency (RF) per Mb of DNA] and small numbers of informative meioses in pedigree studies has limited the resolution of current linkage maps to the megabase level (1, 2). Much higher resolution profiles of recombination can instead be obtained indirectly through examining patterns of marker association [linkage disequilibrium (LD)], established through population dynamic processes and eroded by recombination, or directly through labor-intensive screening of millions of sperm for recombinant DNA molecules within short DNA intervals (typically Ͻ10 kb).Population LD (3, 4) and sperm DNA (5-14) analyses have firmly established that most cross-over events in humans cluster into narrow hotspots spaced, on average, 50 kb apart. Recently, the International HapMap Project (15, 16) has mapped the LD landscape genome-wide at the kilobase level. These data allowed inference of the global recombination landscape at high resolution (4) by using coalescent analyses whereby observed haplotypes are explained through in silico reconstruction with variable historical recombination rates. These analyses have identified Ϸ33,000 putative cross-over hotspots (LD hotspots) thro...

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.

Minisatellite MS1 (locus D1S7) is one of the most unstable minisatellites identified in humans. It is unusual in having a short repeat unit of 9 bp and in showing somatic instability in colorectal carcinomas, suggesting that mitotic replication or repair errors may contribute to repeat-DNA mutation. We have therefore used single-molecule polymerase chain reaction to characterize mutation events in sperm and somatic DNA. As with other minisatellites, high levels of instability are seen only in the germline and generate two distinct classes of structural change. The first involves large and frequently complex rearrangements that most likely arise by recombinational processes, as is seen at other minisatellites. The second pathway generates primarily, if not exclusively, single-repeat changes restricted to sequence-homogeneous regions of alleles. Their frequency is dependent on the length of uninterrupted repeats, with evidence of a hyperinstability threshold similar in length to that observed at triplet-repeat loci showing expansions driven by dynamic mutation. In contrast to triplet loci, however, the single-repeat changes at MS1 exclusively involve repeat deletion, and can be so frequent--as many as 0.7-1.3 mutation events per sperm cell for the longest homogeneous arrays--that alleles harboring these long arrays must be extremely ephemeral in human populations. The apparently impossible existence of alleles with deletion-prone uninterrupted repeats therefore presents a paradox with no obvious explanation.