Periodic Reporting for period 3 - URDNA (Origin and Protection of Unstable Repetitive DNA Elements During Sexual Reproduction)
Reporting period: 2018-05-01 to 2019-10-31
"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."
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
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. This analysis has revealed that Pch2 is associated with very specific regions in the budding yeast genome. Within euchromatin, we have found that Pch2 is often associated with regions of active, PolII-dependent transcription (i.e. highly-expressed ORFs) that lie in the relative vicinity of canonical ORC-binding sites (i.e. ARS sequences; origins). This result hints at a wide-spread role for ORC in the recruitment of Pch2 to defined genomic environments, in addition to its role in rDNA recruitment of Pch2. Within the rDNA array, we have mapped the binding of Pch2 to defined regions within a single rDNA repeat. Specifically, we have mapped Pch2 binding to the gene body of the 35S rRNA gene. This rRNA gene is transcribed by PolI RNA polymerase. Strikingly, we have not detected Pch2 binding to the highly-expressed 5S rRNA gene, transcribed by PolII RNA polymerase. Thus, Pch2 binding is associated with either PolI (in case of the rDNA array binding) or PolII (in case of euchromatic binding), but not with PolIII-dependent transcription. We are currently investigating what differentiates PolI/II from PolII-dependent transcription. It is important to note that within every rDNA repeat a single ORC binding site (ARS sequence) is encoded, but this ARS sequence is localized within the intergenic regions of the rDNA repeat (called the ITS region). The association of Pch2 within the 35S rRNA sequences that lie in the vicinity of a ORC-binding site is very reminiscent of the observed binding of Pch2 along chromosomes arms: it occurs at a highly-expressed ORF (in this case 35S) in the vicinity of a canonical ORC binding site (ITS-ARS). These findings strongly imply that the recruitment of Pch2 to the rDNA and to the euchromatin has shared underlying features, that are centered around the Pch2-Orc1-interaction. Corroborating this observation, we could indeed show that both rDNA and euchromatic recruitment of Pch2 critically relies on Orc1 function, as functional inhibition of ORC1 (via the use of a temperature-sensitive allele of ORC1; orc1-161) leads to a marked decrease in the association of Pch2 to both rDNA and euchromatic regions., as judged by ChIP analysis. In addition to the dependency of Pch2 binding on Orc1 function, we have shown that the recruitment of Pch2 to euchromatic regions also depends on active transcription. Chemical inhibition of PolII-dependent transcription leads to a marked reduction of Pch2 association with euchromatic regions. To further investigate the molecular basis by which Pch2 recruitment would be regulated within the rDNA array and within euchromatic regions, we have turned to factors previously shown to be involved Pch2 chromosome association. We analysed the role of the synaptonemal complex (SC) factor Zip1 in Pch2 recruitment. We found, in line with cytological observations, that Zip1 is required for euchromatic but not rDNA recruitment of Pch2. This finding suggests that, in currently unclear manner, Zip1 facilitates the association of Pch2 with (euchromatic) highly-expressed ORFs (in the vicinity of ORC sites). It also indicates that Zip1 per se is not required for this association (since within the rDNA array Pch2 recruitment is independent of Zip1), and it rather suggests that Zip1 functions an inhibitory signal that relives the inhibition of Pch2 association to its genomic location. In line with this model, we found that deleting the histone methyltransferase Dot1 allows association of Pch2 to euchromatic regions, even in the absence of Zip1 function. Interestingly, Dot1-activity (as a methyltransferase for H3K79) is associated with ongoing and active transcription, hinting at Dot1 as a potential mediator of the connection between Pch2 and active transcription. The connection between Dot1, Zip
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Using ChIp-seq, we have for the first time been able to determine the chromosomal recruitment of Pch2 during meiosis. Our results critically increase 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 are now in the ideal position to be able to reach a comprehensive understanding of the interplay between Pch2, transcription, ORC and chromatin. We expect to be able to reach important conclusions that are expected to impact our understanding of the molecular systems that are at play during meiotic recombination and sexual reproduction. 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.