Final Report Summary - MEIOREP (Interplay between the program of DNA replication and meiotic recombination)
For our studies, we have developed several innovative approaches. Notably, we have implemented methodologies that allow us to analyze the impact of changing the organization of genome duplication on meiotic recombination in live cells. In particular, we aimed to induce alterations in DNA replication as well as synchronous meiosis through the addition of different chemical compounds. We therefore worked to produce the only microfluidic devices available to date that do not absorb the small molecules required in our assays while being compatible with high-resolution fluorescence microscopy. The development of this technology gives us the unrivaled capacity to monitor the effects of changing the program of genome duplication on meiotic recombination in single cells in real-time, opening new avenues for our research. In addition, we have established single-molecule methods to visualize the patterns of DNA replication and meiotic recombination along individual chromosomes. Collectively, these approaches provide us with an unprecedented view of the coordination between replication and recombination at the single-cell and single-molecule levels.
To investigate the molecular links between replication initiation and meiotic recombination, we have taken diverse strategies. First, as replication and DSB formation may interact at different steps in these processes, we have assessed one of the earliest events in the regulation of meiotic recombination: the establishment of meiosis-specific chromosome structures. Our results suggest that the organization of DNA replication may play a role in controlling the binding of the factors that set up these structures, thereby affecting the subsequent generation of DSBs. Next, we aimed to identify the steps in genome duplication that are important for meiotic DSB formation. To this end, we adapted a system using inducible barriers to DNA replication that allows us to determine whether the progression of the replication machinery along the chromosomes contributes to DSBs. This approach enables us to ascertain the molecular mechanisms by which DNA replication modulates meiotic recombination. Finally, as recent studies have indicated that chromosome architecture may be important for regulating genome duplication in proliferating cells, we have investigated whether changes in chromosomal structure and context alter the organization of DNA replication and recombination during meiosis. We thus generated a series of chromosomal rearrangements and discovered distinct changes in the pattern of genome duplication in these conditions. Importantly, our ongoing analyses of the accompanying alterations in DSB formation will further elucidate the relationship between replication and recombination. All together, our studies will bring new insight to our understanding of the interplay between genome duplication, chromosomal architecture, and the exchange of genetic material during meiosis.
Beyond advancing our comprehension of the mechanisms that underlie the duplication of the genetic material and its exchange during sexual reproduction, our findings are relevant for human health and disease. Meiosis is essential to sexual reproduction in eukaryotes, and errors in meiosis can result in chromosomal translocations or aneuploidy. In particular, the consequences of meiotic aneuploidy in humans are severe: it is a key cause of infertility, miscarriage, and congenital birth defects. An estimated up to 70% of human conceptions result in aneuploid embryos, most of which are spontaneously lost early in pregnancy. In cases where gains or losses of certain chromosomes are tolerated, this can then lead to problems such as infertility and learning disabilities. Understanding how the genome is duplicated and how this affects the products of meiosis is therefore indispensable for our comprehension of the congenital diseases that arise through dysfunctions in these processes.
Furthermore, genome duplication is integral to cell growth and proliferation, and maintenance of genome integrity is critical for preventing pathologies such as cancer. Cancer cells often show alterations of the replication pattern that can potentially lead to the formation of DSBs and gross chromosomal rearrangements. While DSB formation is highly deleterious for genome stability in proliferating cells, this process is stimulated and regulated during meiosis: meiotic cells generate DSBs to induce the exchange of genetic material, and these breaks are then repaired via specific mechanisms. Interestingly, a number of studies have now shown that key meiotic genes, including recombination factors involved in DSB formation, are overexpressed in primary tumors and metastatic cancers, including melanoma, non-small cell lung carcinoma and cervical cancer. Our investigation of the molecular mechanisms that couple replication with recombination will thus provide us with insight into how the deregulation of these processes may contribute to genome instability and tumorigenesis. Moreover, as studies have associated poor prognosis with the expression of germline factors involved in meiotic recombination, our findings may eventually lead to the development of new diagnostic markers or therapeutic targets.