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The Evolution of PRDM9 Binding and Genomic Localization of Meiotic Recombination Events

Periodic Reporting for period 1 - PRDM9Recomb (The Evolution of PRDM9 Binding and Genomic Localization of Meiotic Recombination Events)

Reporting period: 2015-05-01 to 2017-04-30

Meiotic recombination, or genetic recombination, is an essential process for sexually reproducing organisms. The appropriate genomic distribution of recombination events is necessary for the accurate transmission of genetic from one generation to the next. Extensive research has demonstrated that a major determinant of recombination sites in mice and in humans is protein PRDM9. PRDM9 binds to DNA in a sequence-dependent manner, and also catalyses the methylation of histones, which, in addition to altering the recombination landscape, also changes the chromatin architecture at different genomic loci within the germline. Although meiotic recombination is an essential process, it is rapidly evolving, and it's evolution is also linked to the evolution of PRDM9, the chromatin landscape, and the evolution of the genome. The initial hypothesis of the project was that additional factors act in concert with PRDM9 to specify sites of meiotic recombination events. The overall objectives of the project is to understand the interplay between the evolution of the genome, the evolution of meiotic recombination, and the evolution of the chromatin landscape in the germline. Throughout the two-year duration of the fellowship, we have investigated and utilised different techniques to map recombination events in mammals, we have investigated the contribution of retrotransposons to meiotic recombination events, and we have mapped the evolution of the chromatin landscape during meiosis across mammals. These studies are ongoing, and will provide insights into the factors that influence recombination and genome evolution in mammals.
One of the goals of the project was to produce a high-resolution map of meiotic recombination events in order to compare to the binding of PRDM9 to investigate the relationship between the two events. In order to map meiotic recombination events genome wide with a method that could easily be adapted for different mouse strains and mammals, we optimised a protocol that would be complementary to the single-cell DNA sequencing approach that we initially proposed. This method relied on the chromatin immunoprecipitation of the meiotic recombination protein DMC1. DMC1 binds to sites of meiotic double-stranded breaks soon after meiotic recombination is initialised, and in this way it marks all recombination intermediates. The protocol is done in bulk, and can easily be performed on cross-linked tissue from different mammalian species. We initially optimised the protocol for different strains of mice, in order to produce high-resolution recombination maps and do a evolutionary comparison. Recent advances in single cell DNA sequencing will now facilitate the ongoing efforts to complement this bulk approach to map recombination events with a single cell view of meiotic recombination events.

In another aspect of the project, we have mapped the chromatin landscape during meiosis. We have used several different markers of chromatin structure, including H3K4me3, H3K4me1, and H3K27ac. H3K4me3 is a chromatin modification that marks promoters and enhancers of genes and regions of open chromatin, but also marks sites of recombination events during meiosis, and is catalysed in part by PRDM9. As H3K4me1 and H3K27ac are also markers of promoters and enhancers, including these additional marks allows us to focus on the H3K4me3 marks that are independent and are not associated with gene regulatory elements, for these are the regions that are most likely to be associated with PRDM9 and meiotic recombination. Furthermore, we have similarly mapped these chromatin markers in other somatic tissues, and have found that there is much more H3K4me3 that is unique to the testes and is not associated with gene regularity elements, and that this H3K4me3 is likely to be due to the activity of PRDM9. We will further investigate the evolution of these chromatin marks in different mammals where PRMD9 is not playing a role during meiotic recombination.

Furthermore, we wanted to investigate the role that retrotransposons may being playing in shaping meiotic recombination and genome evolution. During meiosis, the structure of the chromatin changes and many transposons that are normally suppressed become activated. This has severe implications for genome evolution, as any new insertions and mutations that get induced will be inherited by the next generation. Furthermore, as transposon activity causes breaks in the DNA, we were curious if there was an interplay between meiotic recombination and transposon activity. As LINE-1 is the only autonomous transposon in humans, and all active retrotransposons use LINE-1's machinery to transpose, we wanted to investigate the proteins encoded by LINE-1 to see if there was any association with meiotic recombinant hotspots. We carried out immunoprecipitation agains LINE-1 proteins to look for any enrichment in recombination sequences in the interacting nucleotides.

Progress on this project is ongoing. In particular we are investigating the relationship between the evolution of chromatin structure and organisation, and the evolution of meiotic recombination and PRDM9 association with chromatin. In the follow-up work that I will be carrying out now, we are expanding our study to look across 10 different species of mammals, rather than restricting the study only to mouse strains. In this study we will be able to look at the impact of PRDM9's activity on recombination landscape and also on chromatin structure and other aspects of genome function, such as gene expression. This study will give us insight into the interplay between genome evolution, chromatin structure, and the meiotic recombination landscape in mice and across mammals.
Although much progress has been made in understanding genome function in recent years, there still remains much to be understood. For meiotic recombination there is a complex combination of factors that influence where recombination events occur, which include underlying sequence, protein binding, chromatin modifications, and also three-dimentionsional architecture. The discovery of PRDM9 has shed a lot of light on how recombination sites are determined in some species of mammals, but it is still not as simple as wherever there is a PRDM9 motif there is always a recombination event at that sight. There are multiple layers of information involved. Furthermore, many species don't even use PRDM9 to determine recombination sites, and in these species other factors such as chromatin architecture are also important. Our study aims to advance the state of the art of knowledge by having a comprehensive study of various aspects of genome structure and function during meiosis to understand the interplay of multiple contributing factors. We are using cutting edge genomic technologies and are incorporating high-throughput pipelines into our protocols in order to process more samples so that we can look at various factors across various samples in a single comprehensive study.