The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.
Until recently the mechanisms controlling the localization of recombination hotspots in mammalian genomes were largely unknown. However, recent research has revealed that zinc-finger protein, PRDM9, binds to specific DNA motifs in the early stages of recombination initiation in order to direct such events [1]–[3]. PRDM9 trimethylates lysine 4 of histone H3 (H3K4me3), an epigenetic modification specifically enriched around recombination initiation sites [4]–[6]. The importance of PRDM9 for the localization of recombination events has been demonstrated in both humans [7], [8] and mice [2], [6], with recent results in mice suggesting that PRDM9 determines the location of virtually all recombination hotspots in these organisms [6]. Variation in the zinc-finger encoding domain of PRDM9 in humans can alter the DNA motif to which the protein binds, and in turn alter the activity of recombination hotspots [7]–[9]. High levels of variation in PRDM9 across species [2], [10], [11] may explain why humans and chimpanzees do not share recombination hotspots despite very high levels of overall sequence identity [10]. Indeed, PRDM9 shows clear evidence of rapid evolution across the metazoan taxa covering an era of roughly 700 million years [11]. Despite successful formation of double-strand breaks (DSBs) [6], the initiating event in meiotic recombination, knockout of Prdm9 results in infertility in both male and female mice with arrest of spermatogenesis and oogenesis at pachynema, impairment of DSB repair, chromosome asynapsis, and disrupted sex-body formation in males [12]. Intriguingly, Prdm9 has been shown to be involved with hybrid sterility, potentially implicating it in the process of speciation [12]. Given the wide-ranging importance of PRDM9, it was therefore surprising to note that dogs (Canis familiaris) and other canids are the only known mammals to carry functionally inert versions of PRDM9 with multiple disruptive mutations [11], [13]. This implies that either the function of PRDM9 is carried out by another gene, or that dogs have been able to avoid the loss of fertility associated with loss of PRDM9 while also ensuring that recombination continues to occur. In order to gain insight into recombination across the canine genome, we have constructed a genetic map using patterns of linkage disequilibrium (LD) estimated from next-generation sequencing of 51 dogs. Methods for estimating recombination rates from polymorphism data have been validated at both broad and fine scales [14]–[16], and have previously been used to obtain relatively broad-scale recombination rate estimates in dogs via the use of SNP microarray data [13]. A potential concern when using such methods in dogs is that breed-formation bottlenecks can lead to considerable levels of inbreeding. For this reason, we have utilized genetic polymorphism data collected primarily from free-ranging dogs from geographically diverse regions (Table S1) and largely lacking a history of excessive selective breeding following the original domestication event [17]. These non-breed dogs, which we term ‘village dogs’, show dramatically reduced levels of homozygosity, and a faster decay of LD when compared to inbred breed dogs (Figure S1).
In humans and mice, specific DNA motifs have been implicated as the binding sites for PRDM9 [1], [4], [21]. In order to investigate if DNA motifs could be identified within canine recombination hotspots, we selected 6,228 hotspot regions with no missing sequence data. For each hotspot we identified a region on the same chromosome showing no evidence for local recombination rate elevation (‘coldspots’), and with GC content within 0. 5% of that of the hotspot, and CpG content within 0. 1%. If more than one such region could be found, we selected the one that matched the hotspot most closely in terms of SNP density. In this way, we were able to identify 4,759 hotspots with matched coldspots. Using the sequences of the matched hotspots and coldspots, we performed a search of motifs showing enrichment in hotspot sequences. Our results indicate an extremely strong association with CpG-rich motifs (Table 1), with the most significant motif being the 7-mer CGCCGCG (p = 1. 1e-21, Fisher' s Exact Test after Bonferroni correction), which is found in 21. 3% of hotspots but only 13. 2% of coldspots, a relative enrichment of 61%. These highly CpG-rich motifs retain significantly high levels of enrichment in hotspots having masked either repeat or non-repeat DNA sequence. Both GC and CpG content show a strong association with canine recombination at fine scales (Figure S8A and B). However, CpG content shows a stronger correlation with recombination rate than GC content over multiple scales (r = 0. 37 vs r = 0. 25 respectively at 1 Mb; Figure S8C and D). If both measures are included as predictors in a multiple regression model, CpG content has a positive association, whereas GC content is negative (Table S3). The influence of GC and CpG content can also be seen when considering the average recombination rate around DNA repeats. The most recombinogenic repeats are low-complexity with high levels of GC and CpG content (Figure 2A). In contrast, the majority of LINE and SINE elements exhibit recombination rates close to the genome average, with a few such as Looper and L1_Canid2 showing weak suppression of recombination. The association between recombination and CpG-dense regions is suggestive of an association with gene promoter regions. Indeed, we observe highly elevated rates of recombination around transcription start sites (TSS; Figure 2B and Figure S9), dwarfing the elevation that has been observed around TSS in humans and chimps [10], [22]. Of the 7,677 called hotspots, 29% overlap with a TSS, and 50% are within 14. 7 kb. Only a small fraction (14%) of hotspots appear to be over 100 kb from a TSS. However, the elevation in recombination rate around TSS appears to be associated with CpG islands serving as promoter regions rather than the TSS themselves, as the recombination peak is shifted away from the TSS for genes with the nearest CpG island at some distance from the TSS (Figure 2C), with genes without a nearby CpG island not showing large peaks in local recombination. Conversely, CpG islands containing TSS show elevated recombination rates relative to CpG islands at some distance from TSS (Figure S10), although interestingly CpG islands >10 kb from the nearest TSS show higher rates than those near (but not containing) a TSS. PRDM9 is thought to have been disrupted early in canid evolution, as previous work has shown that the amino acid coding sequence contains multiple disruptive mutations across a diverse set of canid species [11], [13]. We have further investigated the extent of PRDM9 disrupting mutations within the Canidae family by sequencing within exon containing the zinc-finger domain of PRDM9 in the Lycalopex and Urocyon genera. Within in the Andean fox, Lycalopex culpaeus (6–7. 4 Mya divergence from dogs [23]), we found the same disrupting frameshift mutation as has observed in dog (Table S4 and Table S5), as well as an additional frameshift, and a premature stop codon. In the Island fox, Urocyon littoralis, (>10 Mya divergence from dogs [23]), while we do not observe the same mutations seen in dog, we do observe a distinct premature stop codon, indicating that PRDM9 has been disrupted in this species as well. As none of the identified mutations are common to all species, it would appear that the original disruptive mutation likely occurred outside of sequenced exon. Due to the early loss of PRDM9, it has been suggested that fine-scale patterns of recombination may be shared across species in the canid lineage [13], in contrast to other mammalian species in which hotspots are not shared [10]. Such inferences have been based on the effect of Biased Gene Conversion (BGC), in which a recombination-associated heteroduplex in the vicinity of an existing polymorphism can produce base-pair mismatches that are preferentially repaired with C/G alleles rather than A/T alleles (Figure S13). As such, BGC increases the probability of a C/G allele being transmitted to the next generation, and sustained BGC can ultimately alter the base composition of the genome [24]. To investigate if BGC is active around canine recombination hotspots, we consider the ratio of AT→GC polymorphisms to GC→AT polymorphisms within local regions of the genome (see Supplementary Text S1). In order to polarize polymorphisms in dog, we sequenced a female Andean Fox, which, as described above, is diverged from dogs by approximately 6–7. 4 million years [23] and also lacks a functional version of PRMD9. The fox sample was sequenced to approximately 11× coverage using 100 bp paired-end Illumina sequencing. In the absence of a reference genome for this species, reads were mapped to the dog genome (canFam3. 0). Using this data, we polarized the ancestral and derived alleles for polymorphisms observed in dog by assuming that shared alleles represent the ancestral allele. Using this data, we were able to polarize 3. 2 million polymorphisms on the dog lineage with high confidence. Likewise, we performed SNP discovery in the fox data, and were able to identify and polarize 1. 2 million polymorphisms on the fox linage. Around canine recombination hotspots, we observe a localized skew in the rate of AT to GC acquisition in the dog genome (Figure 3A), which is stronger for hotspots with higher peak recombination rates (Figure 3B; Figure S11A). The effect remains visible even after excluding all polymorphisms within putative CpG sites (Figure S11B). The dog genome is notable for its high density of CpG-rich regions [25], [26]. Given CpG-rich regions are highly recombinogenic in the dog genome, it is plausible that BGC is the mechanism by which the dog genome has acquired its high density of CpG rich regions. Specifically, if CpG regions are promoting recombination, and are thereby acquiring increasing levels of GC content via BGC, this would in turn further increase the CpG content of the region and hence further increase recombination in a self-reinforcing process. If dogs and foxes shared the same hotspots, a similar pattern would be expected for polymorphisms observed on the fox linage. However, we do not see evidence of a skew in fox linage polymorphisms around canine recombination hotspots (Figure 3A), which would imply that these two species do not share recombination hotspots. The role of PRDM9 is to trimethylate histone H3K4, and studies in mice have shown that nearly 95% of hotspots overlap an H3K4me3 mark [4]. It is therefore interesting to ask if canine recombination maintains an association with H3K4me3, especially given the apparent elevation of recombination around gene promoter regions in dogs. We have used ChIPseq to identify regions of H3K4me3 in dog spermatocytes during the leptotene/zygotene (L/Z) and pachytene phases of prophase I of meiosis. We identified 28,349 autosomal ChIPseq peaks in L/Z and 32,830 for pachynema. Of these, 8,721 (31%) peaks were unique to L/Z and 13,613 (41%) unique to pachynema. While recombination rates do appear highly elevated around H3K4me3 marks, the effect is almost entirely explained by the presence or absence of a putative CpG island overlapping the mark (Figure 4A and B). The pattern is very similar for both L/Z and pachytene cells, albeit with a small increase in rate for pachytene-specific marks in the absence of CpG islands (Figure S12). Conversely, while CpG islands overlapping H3K4me3 marks are ∼60% more recombinogenic than those without marks, a strong elevation in local recombination rate remains visible for islands without H3K4me3 marks. Notably, putative CpG islands without H3K4me3 marks also appear to have elevated background rates (Figure 4C), and this effect persists even after extensive thinning CpG islands to ensure lack of clustering. This could reflect background sequence context, as islands with H3K4me3 marks have lower levels of CpG content in the flanking 50 kb than those without marks (2. 2% vs 1. 2%, p≪1e-16), or may be indicative of other epigenetic factors such as DNA methylation.
The apparent loss of PRDM9 in canids makes dogs of particular interest for the study of meiotic recombination. Our study reveals that while broad scale patterns of recombination appear superficially typical of mammals, dogs appear to have a quite different landscape compared to other mammals so far studied at the fine scale. Of particular note is the strong association of recombination with CpG-rich features of the genome, particularly around promoter regions, which is reminiscent of the double-strand break localization around H3K4me3 marks that has been observed around promoter regions in Prdm9-knockout mice [6]. However, in contrast to these results, we find that the elevation in canine recombination rate around promoters appears to be primarily associated with CpG content, and shows little association with H3K4me3 marks. The association with promoter regions is also superficially reminiscent of the elevated recombination rates observed around promoter in yeast, which has been related to nucleosome spacing [27], [28]. The biased conversion of A/T to G/C alleles at already CpG enriched recombination hotspots may help explain the 2–3-fold increase in putative CpG islands in the dog genome compared to human and mouse [25]. Despite the relative high density of CpG islands in the dog genome, it has also been noted that dogs have fewer promoter-associated CpG islands than humans or mice, especially near essential and highly expressed genes [25]. If the role of PRDM9 is to deflect DSBs away from functional elements, as has been suggested in other species [6], then the preferential loss of recombinogenic CpG islands near promoters in dogs may indicate that selection is acting to deflect recombination from genic regions since the loss of PRDM9. PRDM9-knockout mice are infertile due to the failure to properly repair DSBs [29]. How canids escaped this infertility is unknown, but it must have occurred in the common ancestor of dogs, wolves, foxes and jackals, but after the split from Panda ∼49 Mya [13]. The stable recombination landscape of canids resulting from the loss of PRMD9 may contribute to the ability to successfully hybridize relatively divergent canine species (e. g. dog and jackal [30]). Our comparison of substitution patterns in dogs and Andean foxes does not support the hypothesis that the death of PRDM9 has resulted in the evolutionary stability of recombination along the canid lineage, at least at the fine scale. It is therefore possible that while PRDM9 is dysfunctional, an unknown ortholog of PRDM9 could have assumed a similar role. Nonetheless, the observed recombination landscape in dogs does appear to have some unusual features, and it is plausible that these result directly from the loss of PRDM9. However, it is worth noting that in addition to the loss of PRDM9, canines are considerably diverged from other species that have so far been studied for fine-scale recombination rate variation, and it is plausible that the observed differences in the recombination landscape have also been influenced by other factors such as genomic structure. In order to fully understand the dynamics of recombination rate evolution, it will be necessary to obtain high-quality and fine-scale genetic maps across a wide range of species. As such maps become available, it will be possible to place the canine map into a proper evolutionary context, and thereby identify the factors that determine the forces that shape the distribution of recombination in the genome.
Canine blood samples were collected under Cornell Institutional Animal Care and Use Committee (IACUC) approval (#2005-0151,2007-0076 and 2011-0061) in accordance with applicable federal, state, and local laws, regulations, policies, and guidelines. For each animal, approximately 3–5 ml of blood was drawn from the cephalic vein into K2-EDTA blood collection tubes. Dogs were unsedated to minimize handling time and overall distress during the short blood drawing procedure. Culpeo blood was extracted from a captive female fox using a similar procedure during a routine physical by trained veterinary personnel at the Universidad de San Antonio Abad Zoo in Cusco.
Recombination in mammalian genomes tends to occur within highly localized regions known as recombination hotspots. These hotspots appear to be a ubiquitous feature of mammalian genomes, but tend to not be shared between closely related species despite high levels of DNA sequence similarity. This disparity has been largely explained by the discovery of PRDM9 as the gene responsible for localizing recombination hotspots via recognition and binding to specific DNA motifs. Variation within PRDM9 can lead to changes to the recognized motif, and hence changes to the location of recombination hotspots thought the genome. Multiple studies have shown that PRDM9 is under strong selective pressure, apparently leading to a rapid turnover of hotspot locations between species. However, uniquely amongst mammals, PRDM9 appears to be dysfunctional in dogs and other canids. In this paper, we investigate how the loss of PRDM9 has affected the fine-scale recombination landscape in dogs and contrast this with patterns seen in other species.
Genetic Recombination Is Targeted towards Gene Promoter Regions in Dogs