What determines the location of meiotic crossovers in the mammalian genome?

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

In the last few years we have been able to generate a genome-wide map for hotspots for double-strand breaks in mouse meiosis by using Illumina ChIP-Seq for Dmc1 and Rad51 foci, both of which mark these sites (all in collaboration with the laboratory of Galina Petukhova at the Department of Biochemistry and Molecular Biology at the Uniformed Services University of Health Sciences). Depending on the level of statistical significance as many as 40,000 of these hotspots can be enumerated and the vast majority of both Rad51 and Dmc1 sites are identical. Compared to the LD recombination that has an accuracy of about 5 KB, our physical recombination map, has an accuracy of about 200 bp. This was the first high-resolution genome-wide map of recombination hotspots in a multicellular organism. Using such a map has allowed to identify novel structural features for recombination hotspots. For example, we determined that recombination hotspots share a centrally distributed consensus motif (in the vast majority of hotspots), possess a nucleotide skew that changes polarity at the center of the hotspots, and have both a calculated and experimental preference to be occupied by a nucleosome. Finally, we find that the vast majority of recombination hotspots in mice are associated with testis-specific H3K4 trimethylation that do not overlap transcription start sites even though these sites are well-known to be marked by H3K4 trimethylation. Thus, H3K4 trimethylation per se is not a sufficient mark for directing the meiotic double-strand break machinery. Recently, we developed a novel method that is a variant of chromatin immunoprecipitation followed by sequencing (ChIP-seq)single-stranded DNA sequencing (SSDS)- that specifically detects protein-bound single-stranded DNA. SSDS consists of a new sequencing library preparation procedure for the enrichment of fragments originating from ssDNA that creates a signature sequence that is computationally identified after high-throughput sequencing (Khil et al. Genome Res (2012))., Brick et al. Nature (2012. We have used this novel method to show that the product of the highly polymorphic and rapidly evolving gene Prdm9 not only determines the positions of practically all hotspots but also actively sequesters recombination away from functional genomic elements, such as promoters and enhancers, in mice (Brick et al. Nature (2012)). Subsequently, we used this method to obtain the first direct high-resolution genome-wide map of meiotic recombination initiation hotspots in individual human males (Pratto et al. Science (2014)). The meiosis specific methyltransferase PRMD9 has been shown to define the location of the vast majority of meiotic DSB hotspots (Brick et al. Nature (2012)). We mapped DSBs in several individuals: homozygous for the most common Prdm9 allele (A), heterozygous for the A allele and a closely related variant, the B allele, and heterozygous for the A allele and the C allele (a variant commonly found in African populations). We found that the A and B alleles of Prdm9 defined similar recombination initiation hotspots while we confirmed that the C allele defines a distinct set of hotspots. Approximately 60% of population LD hotspots are explained by A-allele hotspots, while C-allele hotspots explain an additional 10%. This demonstrates that relatively minor alleles significantly contribute to the LD map. We also found that the DSB distribution exhibits a strong telomeric bias which closely resembles that of male, but not female crossovers. This indicates that the recombination landscape is largely shaped at the level of initiation. We have also examined the relationship between speciation (how one species becomes two) and recombination (Smagulova et al. (2016) Genes and Development 30, 871 and Davies et al. (2016) Nature 530, 171). While PRDM9 was shown a few years ago to be, to date, the only known mammalian speciation gene it has been difficult to unravel how this function of PRDM9 relates to its role in determining the location of recombination initiation (DSB) hotspots. In one of these papers we mappedrecombination hotspots in several mouse subspecies with different Prdm9 alleles and in their F1 hybrids. We found an increase in sequence diversity specifically at new hotspots (not found in either parent) that become active in the hybrids. Finally, we showed that genetic exchanges are less frequent at such hotspots. Therefore, we proposed that sequence divergence might create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation. Recently, we have also examined sex differences at the initiation of genetic recombination. Meiotic recombination differs between males and females; however, when and how these differences are established is unknown. We have identified extensive sex differences at the initiation of recombination by mapping hotspots of meiotic DNA double-strand breaks in both male and female mice. Contrary to past findings in humans, few hotspots are used uniquely in either sex. Instead, grossly different recombination landscapes result from an up to fifteen-fold differences in hotspot usage between males and females. Indeed, the majority of recombination occurs at sex-biased hotspots. Sex-biased hotspots seem to be partly determined by long-range chromosome structure, and DNA methylation, which is absent in females at the onset of meiosis, has a major role. Most recently, we have studied the link between meiotic replication and recombination in both mice and humans.DSB formation is preceded by DNA replication in meiosis, and in simpler eukaryotes these two processes are intimately linked. We hypothesized that DNA replication could be imposing a constrain on DSB formation that would influence the broad-scale patterning of meiotic recombination. We sought to investigate this link in humans and mice where technical challenges have precluded such studies so far. Using a novel three-pronged approach we aimed to comprehensively describe meiotic DNA replication initiation in mammals (Pratto et al., in preparation). First, we leveraged our method that directly sequences single stranded DNA to sequence isolated nascent leading strands to map origins of replication in mouse testis. Our strand-specific signal allows for highly accurate, model-based origin detection, eliminating the problem of false positive calls. This method allowed for the identification of 12,000 high confidence origins from mouse testes. Second, using also a newly described by us to sort stage-specific nuclei from whole testes, we describe the meiotic replication timing (RT) landscape for the first time in meiotic S-phase nuclei. Origin density is highest in early replicating regions and intriguingly, early replicating regions are also enriched for the binding of PRDM9, for meiotic DSB formation and for genetic crossovers. Intriguingly, we found that the sub-telomeric regions in human male meiosis replicate consistently early. This strongly implicates meiotic replication as a key determinant of genetic recombination patterning in mammals. Third, to more precisely dissect the mechanisms that link meiotic replication and recombination, we developed an in-silico model of meiotic replication. Using only the locations of replication origins from testis this model accurately recapitulates experimentally determined RT. Furthermore, we can model the RT of mitotic cells remarkably well using origins from testes, indicating that mostly the same origins are used in meiotic and mitotic cells. Only half as many of these origins appear to fire in meiosis and this may prolong S-phase relative to that in mitosis. Finally, we are exploring how the interplay between meiotic replication and recombination shapes the genomes of mammals.