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Origin and Protection of Unstable Repetitive DNA Elements During Sexual Reproduction

Periodic Reporting for period 4 - URDNA (Origin and Protection of Unstable Repetitive DNA Elements During Sexual Reproduction)

Reporting period: 2019-11-01 to 2020-09-30

"The transmission of a stable genome is critical for the maintenance of genome stability throughout sexual reproduction in all eukaryotes. However, our current understanding of the mechanisms that guide how eukaryotic cells safeguard their genome during the meiotic division is limited. The main goal of this project is to investigate how unstable repetitive DNA elements are protected from instability during sexual reproduction. For this, we use the unicellular organism Saccharomyces cerevisiae (i.e. budding yeast) as a model system. We are focusing on a particular repetitive DNA elements, namely the ribosomal DNA element (rDNA), and we are trying to understand how a recently defined ""anti-DSB"" system, composed of the enzymes Pch2 and Orc1 is acting to protect these sequences agains meiotic DNA break formation and recombination. We have investigated, using cell biology, genome-wide analyses, and biochemical reconstitution to study this complex. In addition, we have developed systems to manipulate the behaviour of the repetitive DNA elements, in order to one able to elucidate how and why these regions are so at-risk during meiotic cell divisions. In general, the aim of this proposal is thus to illuminate our understanding of the maintenance of genome stability during sexual reproduction. Many human genetic disorders are caused by genome destabilisation during sexual reproduction. Therefore, deepening our understanding of the factors that safeguard sexual reproduction is expected to help us understand the aetiology of human disease."
We have invested in the elucidation of the molecular basis of the Pch2/ORC system as an “anti-DSB” system. We have established a global map of Pch2-chromatin association, with a focus on rDNA association. In Aim 2.1. we have used ChIP-seq to map the binding patterns of Pch2 along the rDNA chromatin and the entire genome, as proposed. This data has been used to show that 1) rDNA binding is dependent on ORC, and 2) rDNA binding is related to PolI-driven rRNA transcription. Likewise, the euchromatin binding patterns have been mapped, and show dependencies on ORC and PolII-driven transcription. A paper describing these finding was published (https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008905). In Aim 2.2. and 2.3. we determined that Pch2 interacts with the other ORC subunits (Orc2 and 5, specifically), confirming our hypothesis that the entire ORC complex interacts with Pch2. Using in vitro reconstitution, we created a Pch2-ORC hexamer-hexamer assembly, and we have used in vitro analysis to delineate the binding interfaces of this assembly. Surprisingly, we have found that Orc1 is a crucial interactor of Pch2, and that this interaction represents a non-canonical association of ORC to Pch2. This work was also published during this period (https://www.life-science-alliance.org/content/3/11/e201900630). We have slightly deviated from the work plan, as under Aim 2.2 we have already generated an analysis of the Pch2 interactome, using MS-analysis; this analysis was planned to be executed in Aim 2.5. Informed by the MS analysis, we have focused our attention in the interaction between Pch2 and Msh4/5, an important meiotic ATPase that we have found to interact with Pch2). Under Aim 2.4. we studied genetic and functional interaction between Pch2, Hop1 and Zip1, which is able to explain many of the roles that are ascribed to Pch2 in meiotic G2/prophase both at the rDNA and euchromatin. This work has yielded important insights into the wiring of the meiotic checkpoint and the role of Pch2 therein. This work was also published in this reporting period (https://www.cell.com/current-biology/fulltext/S0960-9822(20)31254-9).
We have developed a novel technology for ectopic targeting of selected proteins to defined regions. We have opted for a CRISPR/Cas-based targeting system. This system utilizes a nuclease-dead version of Cas9, that can be fused to proteins of interest to target these fusion proteins to genomic regions of choice. We have adapted this system to be used in meiosis, by generating dCas9-contructs that are meiosis-specifically expressed. These systems are combined with specific small guide RNAs (sgRNAs) of choice. We have shown that this system can successfully be used to target proteins of choice to defined regions within the genome. We have so far combined this system with a recently developed fluorescent-based recombination reporter that can reliably measure crossover frequencies within regions of choice. The advantage of this reporter is that is allows the use of microscopy-based analysis instead of the labour-intensive classical analysis of crossover frequencies via yeast tetrad dissection. In another project, my laboratory has shown that this approach (of combining dCas9-based targeting with the fluorescent reporter) can successfully be used to dissect control of local crossover recombination. For Aim 1.1 and 1.2. we have established a new system to interrogate DNA break and recombinational control, based on CRISPR/dCas9. These systems have been shown to be able to locally influence recombination patterns, and we have used this to delineate local DSB suppression. These findings were published recently (https://www.genetics.org/content/216/2/395.long).
Using ChIp-seq, we have for the first time been able to determine the chromosomal recruitment of Pch2 during meiosis. Our results critically increased the understanding of Pch2 function in several ways. First, we can now, based on the shared characteristics of Pch2 euchromatin and rDNA-chromatin binding start to build a comprehensive understanding of the biochemical underpinnings of Pch2 chromosome function. By coupling this kind of state-of-the-art approach with sophisticated biochemical reconstitution of the Pch2/ORC interaction, we have reached a comprehensive understanding of the interplay between Pch2, transcription, ORC and chromatin. In addition, we have developed a novel, meiosis-specific targeting system that is based on CRISPR/Cas9 dependent targeting, in order to study the effect of isolated components to meiotic DNA break formation and recombination. Finally, we used our observations of the interplay between Pch2 and Hop1 to establish a new framework that rationalizes how the meiotic checkpoint works.