Meiosis is a specialized form of cell division where homologous chromosomes are segregated in meiosis I before sister chromatids are segregated in meiosis II. To establish this pattern, a number of changes to the mitotic chromosome segregation machinery are put in place. Firstly, sister kinetochores orient towards the same pole in meiosis I (mono-orientation). Secondly, homologue recombination creates chiasmata, which link homologues together. And thirdly, cohesin, the molecule that holds sister chromatids together, is cleaved in a step-wise manner. This is achieved because the Shugoshin (Sgo1) protein recruits protein phosphatase 2A (PP2A) to centromeres to counteract cohesin phosphorylation, which is required for its cleavage. The work presented here has investigated two critical aspects of cohesin protection: firstly, how cohesin protection is deactivated in meiosis II and, secondly, how a meiosis-specific protein called Spo13 helps to set up cohesin protection in meiosis I. Previously, our lab had shown that Sgo1 is removed from chromosomes when sister chromatids come under tension during mitosis. I therefore sought to investigate whether sister kinetochore mono-orientation allows Sgo1 to stay on centromeres during meiosis I and carry out its protective function. To this end, I modified meiosis I chromosomes to lack both chiasmata and mono-oriented kinetochores. Under these conditions, where sister chromatids are forced to be under tension in metaphase I, Sgo1 is undetectable on chromosomes. As a consequence, centromeric cohesin is largely lost in anaphase I leading to the premature separation of sister chromatids in a fraction of cells. Since mono-orientation of sister kinetochores is exclusive to meiosis I, these findings suggest that Sgo1 localisation is influenced by sister kinetochore tension in both mitosis and meiosis. Therefore, our findings suggest a mechanism that could contribute to the deprotection of cohesin in meiosis II. However, loss of cohesin protection upon bi-orientation is not complete, suggesting that other factors are involved in the efficient protection and deprotection of cohesin. One such factor is the meiosis-specific protein Spo13, which had previously been shown to be required for cohesin protection as well as kinetochore monoorientation. Although it had been suggested that Spo13 regulates Sgo1 recruitment to centromeres, I could not find any evidence to support a loss of Sgo1, or PP2A, in spo13Δ cells. Additionally, even when Sgo1 is stabilised and clearly visible in anaphase I of spo13Δ mutants, pericentromeric cohesion is still defective. Therefore, I investigated the effect that polo kinase Cdc5, an interactor of Spo13, has on Sgo1. While cellular Sgo1 levels are increased in response to Cdc5 loss, this effect seems to be independent of Spo13. However, Spo13 is required for proper levels of Cdc5 at centromeres and the centromeric recruitment of Cdc5 by Spo13 is likely to be functionally important because tethering of Cdc5 to kinetochores rescued the mono-orientation phenotype of spo13Δ cells. In contrast, I found no evidence that the Spo13-Cdc5 interaction is required for cohesin protection. Meiotic overexpression of SPO13 enhances cohesin protection in meiosis I, apparently independent of its robust interaction with Cdc5, and causes increased Sgo1 enrichment at centromeres. This suggested that Spo13 might recruit Sgo1 to cohesin itself to facilitate its protection. Although I could not detect a loss of Sgo1-cohesin interaction in spo13Δ cells, tethering of Sgo1 to cohesin restores pericentromeric Rec8 to spo13Δ mutants in anaphase I. Surprisingly, sister chromatids still segregate in this case, suggesting that pericentromeric cohesion is defective, despite maintenance of Rec8. Furthermore, inhibition of either one of the cohesin kinases, DDK and Hrr25, restores sister chromatid cohesion to spo13Δ cells. Therefore, the findings in this study suggest that Spo13 is at the centre of a complex regulatory network that coordinates cohesin protection and sister chromatid cohesion in meiosis I.

Meiosis is the process by which haploid gametes are produced from a diploid cell. It is a specialised form of cell division which involves one round of DNA replication followed by two rounds of chromosome segregation. Errors in the segregation process can give rise to aneuploidy, which can result in miscarriages and birth defects. In the first meiotic division homologous chromosomes are segregated, and sister chromatids are segregated in the second division. This is coordinated with two rounds of spindle microtubule assembly and disassembly. How these two processes are coordinated is unknown. In my PhD, I study the role of the protein phosphatase 2A (PP2A) regulatory subunit, Cdc55, in budding yeast meiosis. PP2A is a conserved heterotrimeric enzyme that has important roles in mitosis and meiosis. These roles are dictated by binding to either of its two regulatory subunits, Rts1 and Cdc55, in budding yeast . I show that Cdc55 is required for the proper assembly of a meiotic spindle in meiosis I, through the maintenance of the Cdc14 phosphatase in the nucleolus early in meiosis. In addition, Cdc55 is also required to limit the formation of PP2A complexes with the Rts1 regulatory subunit, and this is essential for the timely dissolution of sister chromatid cohesion. Thus, Cdc55 couples spindle assembly with chromosome segregation through its interactions with Cdc14 and PP2ARts1. Finally, I show some preliminary studies looking at the possible downstream effectors of Cdc14 that are important in this mechanism.

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Les contractions rythmiques résultent de l’activité stimulatrice du nœud sinusal dans le cœur et des cellules interstitielles de Cajal (CICs) dans les intestins. Nous avons découvert un nouveau syndrome résultant d’une combinaison de la maladie du nœud sinusal (MNS) et de la pseudo-obstruction intestinale chronique (POIC). Ce syndrome, que nous avons nommé Chronic Atrial and Intestinal Dysrhythmia (CAID), résulte d’une mutation récessive du gène SGO1 (K23E). Cependant, les rôles connus de SGO1 n'expliquent pas l'apparition postnatale du syndrome ni la pathologie spécifique, suggérant que des rôles non canoniques de SGO1 conduisent aux manifestations cliniques observées. Cette hypothèse est supportée par la comparaison de CAID avec les autres cohésinopathies qui présentent principalement des phénotypes développementaux sans ou avec des défauts légers du cycle cellulaire. Ce projet visait à une découverte non biaisée des mécanismes non canoniques expliquant le syndrome CAID en utilisant le dogme de la biologie moléculaire (ADN→ARNm→protéine) comme ligne directrice. Pour ce faire, nous avons effectué des criblages multi-omiques sur des fibroblastes de peau de patients CAID et de contrôles sains. Les résultats des criblages ont été validés par électrophysiologie, étude des voies de signalisation pertinentes, immunohistochimie, pyroséquençage des rétrotransposons LINE-1 et quantification des marques d’histones. Nos études multi-omiques ont confirmé des changements dans la régulation du cycle cellulaire, mais aussi dans la conduction cardiaque et la fonction des muscles lisses. Plus spécifiquement, plusieurs canaux potassiques étaient sous-régulés. L’électrophysiologie a confirmé une diminution du courant potassique rectifiant entrant (IK1). L'immunohistochimie des coupes intestinales de patients CAID a confirmé l’augmentation de l’expression de SGO1 et BUB1, un régulateur de la voie de signalisation TGF-β. De plus, la voie canonique de TGF-β est augmentée et est découplée de la voie non canonique. Au niveau épigénétique, une signature unique d’hyperméthylation et de fermeture de la chromatine a été observée. Ce qui est soutenu par l’augmentation de la méthylation de H3K9me3 et de H3K27me3. En conclusion, le syndrome CAID est associé à plusieurs changements ayant possiblement un effet cumulatif plutôt que d’une seule voie de signalisation dérégulée. Nos résultats désignent la perturbation du courant IK1, la dérégulation de la signalisation TGF-β, l’hyperméthylation de l’ADN et la compaction de la chromatine comme éléments conducteurs potentiels des manifestations cliniques observées. La voie TGF-β et les changements épigénétiques peuvent être ciblées par des médicaments existants, constituant ainsi des cibles thérapeutiques prometteuses pour le traitement du syndrome CAID. / Rhythmic contractions are driven by the pacemaker activity of the cardiac sinus node and the intestinal interstitial cells of Cajal (ICC). We have discovered a new syndrome resulting from a combination of sick sinus syndrome (SSS) and chronic intestinal pseudo-obstruction (CIPO). This syndrome, which we have named Chronic Atrial and Intestinal Dysrhythmia (CAID), results from a recessive mutation in the SGO1 gene (K23E). However, the known roles of SGO1 do not explain the postnatal onset of the syndrome nor the specific pathology, suggesting that non-canonical roles of SGO1 lead to the clinical manifestations observed. This hypothesis is supported by the comparison of CAID with other cohesinopathies which mainly exhibit developmental phenotypes without or with mild cell cycle defects. This project aimed towards an unbiased discovery of noncanonical mechanisms explaining CAID using the molecular biology dogma (DNA→mRNA→protein) as a guideline. We performed multi-omic screens on skin fibroblasts from CAID patients and healthy controls. Screening results were validated by electrophysiology, study of relevant signaling pathways, immunohistochemistry, LINE-1 retrotransposon pyrosequencing, and histone marks quantification. Our multiomics analyses confirmed changes in cell cycle regulation, but also in cardiac conduction and smooth muscle function. More specifically, several potassium channels were downregulated. Electrophysiology studies confirmed a decrease in the inward rectifier potassium current (IK1). Immunohistochemistry in CAID patient’s intestinal sections confirmed overexpression of SGO1 and BUB1, a regulator of TGF-β signaling pathway. Additionally, the canonical TGF-β signaling was increased and decoupled from noncanonical signaling. At the epigenetic level, CAID patient fibroblasts have a unique signature of hypermethylation and chromatin closure. This is supported by the increased methylation of H3K9me3 and H3K27me3. In conclusion, CAID syndrome is associated with several changes that, may have a cumulative effect rather than a single deregulated signaling pathway. Our results reveal the disturbance of the IK1 current, the deregulation of TGF-β signaling, DNA hypermethylation and chromatin accessibility changes as potential conductors of intestinal and cardiac manifestations of CAID syndrome. In particular, the TGF-β pathway and epigenetic changes, may be targeted by existing drugs, thus constituting promising therapeutic targets for the treatment of CAID syndrome.