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 is the first, and to date only, 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. 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 ofpractically all hotspots but also actively sequesters recombination away from functional genomic elements, such as promoters and enhancers, in mice. Previously we (see above) and others (Myers et al. Science (2005)) have identified meiotic recombination hotspots in the human genome using computational analysis of patterns of linkage disequilibrium (LD) in populations; however, this method only permits the evaluation of sex-averaged and population-averaged recombination rates. Furthermore, the resolution of this method is limited to approximately 2 Kb by the availability of informative single-nucleotide polymorphisms and the hotspots identified may not reflect present-day crossovers. In this work, we exploit a sequencing based method recently developed by us (Khil et al. Genome Res (2012), Brick et al. Nature (2012), Smagulova et al. Nature (2011)) to obtain the first direct high-resolution genome-wide map of meiotic recombination initiation hotspots in human males. 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. Finally, we explored the role of DSB hotspots in genomic rearrangements. We found that DSB hotspots were enriched at structural variants that arise via homology-mediated mechanisms and that meiotic DSBs occur at well known disease-associated chromosomal breakpoints. 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 interrogated the functions of the domains of PRDM9 in meiosis. PRDM9 contains three conserved domains typically involved in regulation of transcription; yet, the role of PRDM9 in gene expression control is not clear. Here, we analyze the germline transcriptome of Prdm9-/- male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. In aggregate, our data indicate that domains typically involved in regulation of gene expression do not serve that role in PRDM9, but are likely involved in setting the proper chromatin environment for initiation and completion of homologous recombination. 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.
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