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. 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 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. 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). More recently, we have also examined sex differences at the initiation of genetic recombination (Brick et al. (2018) Nature 561, 338) and found that the majority of recombination occurs at sex-biased hotspots. 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. (2021) Cell 184, 4251). 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 our newly described method (Lam et al., 2019)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. More recently (Alleva et al., (2022)), we have cataloged human PRDM9 variability utilizing long-read sequencing technologies to reveal PRDM9 population-specificity. Assessment of PRDM9 diversity is important for understanding the complexity of human population genetics, the inheritance of linkage patterns, and the predisposition to genetic disease. Last, we analyzed the propensity for rearrangements to occur at a repetitive 40kb locus predicted to contain multiple PRDM9-A and -C hotspots. This region contains CYP2D6, a gene that encodes a protein metabolizing 25% of all prescription drugs, and two pseudogenes with high degrees of homology. Using a long PCR assay and CRISPR-Cas9 targeted enrichment, we examined 144 individuals and found 9 different possible rearrangements that can occur at this locus. Of those 144 individuals, 22% were found to have at least one gross rearrangement within this region. More recently, we set out to study how a few DSBs are ultimately selected to become crossovers (CO) and how this process is regulated. To study the factors affecting this process we set out to characterize the intermediate steps between when the initiating DSBSs are introduced and CO formation. In this regard, we have accomplished the first genome-wide mapping of recombination intermediates in a higher eukaryote (MSH4 in mouse spermatocytes) (Lam et al., in preparation). Most recently, to gain insights into chromatin folding during spermatogenesis, we isolated nuclei populations on a temporal trajectory from spermatogonia to the end of MPI and performed in-situ Hi-C and Micro-C (Cheng et al., in preparation). We found that extended loops were formed immediately after cells escaped mitotic cycles; the loop density was associated with the abundance of meiotic specific cohesins. The presence and formation of extended pre-meiotic loops set up the foundation for the loop-axis configuration, a majority of which were extended continually during meiotic prophase I and grew in size about 5 to 10 fold.Our study provides the first direct evidence demonstrating that CTCF binding sites preferentially associate with meiotic chromosome axes and mark the base of meiotic loops. Furthermore, the unprecedented temporal resolution of our study refines the point at which the chromosomes organize into a meiotic configuration. This transition occurs before the typically defined meiotic entry point, underscoring the complex, multi-step process of meiotic commitment and initiation. Notably, we captured a chromatin folding intermediate mirroring the structure found upon mitotic exit. More generally, we find that although meiotic chromosomes lose the typical A/B compartments and topologically associated domains (TADs), the contacts between regulatory elements remain intact. This provides a natural system to assess how the loss of higher-order chromosome structure affects the regulation of transcription.