In Drosophila oocytes, chromosomes undergo dynamic reorganisation during the prophase of the first meiotic division. This is essential to prepare chromatin for synapsis, recombination and consequent chromosome segregation. The progression of meiotic prophase I is well described, while the molecular mechanisms and regulation of these dramatic chromosomal reorganisations are not well understood. Histone modifying enzymes are major regulators of chromatin structure, however, our knowledge of their roles in meiotic prophase I is still limited. In this work, I investigated the role of the histone demethylase Kdm5/Lid, which removes one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). I showed that Kdm5/Lid is important for the assembly of the synaptonemal complex, pairing of homologous centromeres, and the karyosome formation. Additionally, Kdm5/Lid promotes crossing over and therefore ensures accurate chromosome segregation. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid rescued the above cytological defects. Thereby, I found that Kdm5/Lid regulates chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity. To further identify the regulators of meiotic chromatin organisation during prophase I, I carried out a small-scale RNAi screen for karyosome defects. I found that depletion of ubiquitin ligase components, SkpA, Cul-3 and Ubc-6, disrupted the karyosome formation and the assembly of the synaptonemal complex. The success of the small-scale screen motivated me to initiate the genome-scale RNAi screen for karyosome defects. I found 40 new genes that, when depleted, strongly impaired karyosome morphology. Further studies are required to confirm and elucidate their role in chromatin organisation in oocytes. Overall, my findings have advanced our understanding of the regulation of chromatin reorganisation during oocyte development. Because of the conservation between Drosophila and human meiosis, this study provides novel insights into the regulation of meiotic progression in human oocytes.

<p>KDM5b acts as a transcriptional repressor through its ability to demethylate tri-methylated lysine (K) 4 on histone H3 (H3K4me3). Demethylation of this histone modification leads to transcriptional repression and downstream biological effects on gene expression. KDM5b is involved in the regulation of differentiation and can exert an oncogenic and a tumour suppressive role depending on cellular context, making it an attractive future target for pharmaceutical intervention. Work from our group has shown that KDM5b expression is linked to differentiation, and that recruitment of the enzyme does not always result in an alteration of H3K4me3. Additionally, work from our group, as well as others, has failed to observe H3K4me3 demethylation by KDM5b in nucleosomal preparations. We therefore hypothesized that KDM5b may exert its demethylase potential on alternative histone targets and that KDM5b requires enzymatic co-factors to demethylate nucleosomes, similar to what is observed for other histone-modifying proteins. In this thesis, we describe KDM5b as having an alternate histone target, di-methylated histone H2B lysine 43 (H2BK43me2). We show that this methyl mark is the primary target for KDM5b, and that the expression level of H2BK43me2 is directly related to the process of differentiation. We additionally present a novel co-factor for KDM5b, the co-repressor TLE4 of the Groucho/TLE family. The presence of TLE4 is required and sufficient to confer nucleosomal demethylase activity to KDM5b, a novel discovery for any of the KDM5 family members. Overall, this work has described both an additional KDM5b target, and detailed requirements for KDM5b nucleosomal demethylation, advancing our understanding of how this enzyme is regulated <em>in vivo</em>. The novel aspects of KDM5b regulation presented within this thesis provide a framework from which future studies can be designed. This work contributes to our overall understanding of epigenetic regulation and will potentially aid in the development of novel anti-cancer therapeutic strategies.</p> / Doctor of Philosophy (PhD)

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It is increasingly recognized by the scientific community that the field of epigenetics is a key step for a better understanding of human biology in both normal and pathological states. Its implication in cancer, and in particular in breast cancer, is now well accepted. Breast cancer, responsible for more than 450,000 deaths worldwide yearly, is a heterogenous disease at the histological and clinical levels as well as at the molecular level. Despite considerable efforts to develop new treatments and improve patient management, patients with a same “profile” of breast cancer can respond differently to therapies and have completely different clinical outcomes. There is therefore a critical need to improve our understanding of breast cancer biology and diversity, in order to find new markers that should provide a better management of patients and the development of new therapies. An increasing number of biologists, pathologists as well as clinicians are currently working towards these goals. During my PhD, we have conducted two studies in order to gain new insights into the contribution of epigenetics in breast cancer biology.<p>In the first study, by performing large genome-scale DNA methylation profiling of numerous breast tumors as well as of normal breast tissues, we first revealed the existence of six groups of breast tumors based on their DNA methylation profiles. Three of these groups showed a strong association with the basal-like, HER2 and luminal A breast cancer subtypes, previously identified by gene expression profiling. Interestingly, the three other groups were found to be a mixture of several gene expression-based subtypes, thus revealing the capacity of DNA methylation profiling to improve breast tumor taxonomy. Second, our study suggests that the establishment of DNA methylation patterns of breast tumors might help to determine their cell type of origin. Finally, we also showed that DNA methylation profiling can reflect the cell type composition of the tumor microenvironment and that a signature of T cell tumoral infiltration is associated with a good prognosis in particular categories of breast cancer patients. <p>In the second study, we revealed the clinical relevance of the KDM5 histone demethylases in breast cancer. The expression of these histone demethylases was deregulated in the analyzed breast tumors as well as in the pre-invasive samples as compared to normal breast samples. This suggests that KDM5 enzymes might be good markers for early diagnosis of breast cancer. Moreover, we showed a prognostic value of the KDM5C histone demethylase.<p>In conclusion, the above data should provide a better understanding of breast cancer biology and diversity, and this should bring new insights to improve breast cancer patient management.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished