Jumonji C (JmjC) domain containing proteins comprise a large family of enzymes that catalyse oxidative reactions. The jumonji domain containing protein 6 (JMJD6) has pleiotropic functions as a lysyl hydroxylase and arginyl demethylase. Previous studies have shown that Jmjd6 is involved in histone modification, mRNA splicing and regulation of polymerase II pause release. A constitutive knockout of Jmjd6 in mice is neonatal lethal and shows defects in macrophage host responses. Recently, JMJD6 was shown to support Foot-and-mouth disease virus replication through interactions with the dead-box RNA helicase Dhx9. This PhD thesis aims to further explore functions of Jmjd6 in macrophages and its roles during viral infections. The hypothesis is that through interactions with RNA helicases, Jmjd6 regulates host responses to foreign nucleic acids and/or has functions as a host factor for replication of DNA and/or RNA viruses. Testing of this hypothesis required the generation of Jmjd6-deficient cell recourses. A new conditional Jmjd6 mouse allele was characterised and a method optimised to knockout the gene in bone marrow-derived macrophages (BMDM) using TAT-Cre recombinase. To study vaccinia (VACV) and influenza A virus (IAV) infections in human cell lines, JMJD6 was depleted using RNA interference or CRISPR-Cas9 gene editing. In BMDM, JMJD6 expression was up-regulated in the late phase of lipopolysaccharide stimulation. The nuclear expression pattern of Jmjd6 in BMDM overlapped with that of DDX41 but not with DHX9, two RNA helicases that have been implicated in sensing of viral DNA and RNA, respectively. Deletion of Jmjd6 in BMDM reduced induction of type I interferon response genes after stimulation with synthetic analogs of viral RNA. To characterise the role of JMJD6 during infection with a DNA virus, Jmjd6-deficient cells were infected with VACV. Knockout of Jmjd6 reduced VACV growth in macrophages but not in HeLa cells. In contrast to HeLa cells, Jmjd6-deficient macrophages displayed abnormal localisations of viral factories and increased cell death, showing that Jmjd6 is specifically required for productive VACV infection in macrophages. To further analyse whether Jmjd6 has pro- or anti-viral functions during RNA virus infection, JMJD6 depleted A549 cells were infected with IAV. JMJD6 depletion in A549 drastically reduced IAV growth from an early stage of infection. Preliminary data indicate that this phenotype is related to a defect in nuclear import of IAV ribonucleoprotein complexes. In summary, this work has identified JMJD6 as a novel pro-viral host factor for VACV and IAV infection and has underpinned its importance for macrophage functions.

The finely tuned regulation of haematopoietic stem and progenitor cells (HSPCs) is crucial to sustain normal haematopoiesis. The disruption of the balance between the quiescence state of haematopoietic stem cells (HSCs) and the proliferation/differentiation programs that are necessary to meet daily haematopoietic demands and respond to external insults, can lead to malignant transformation, such as acute myeloid leukaemia (AML). Therefore, it is essential to investigate the players that are responsible to maintain haematopoietic homeostasis, so that novel therapeutic targets can be identified. HSCs reside in a hypoxic environment that is crucial for their maintenance, as it protects them from over-proliferation and exhaustion. The response to a limited availability of oxygen is critically mediated by a transcription factor - hypoxia inducible factor (HIF). HIF is predominantly regulated by prolyl hydroxylases (PHDs) that are 2-oxoglutarate (2OG) dependent oxygenases. This superfamily of oxygen-sensing enzymes has been assigned important roles ranging from hypoxia signaling, DNA repair, chromatin modifications and oncogenesis Following the data published by our group attesting that HIF is dispensable for HSC survival and maintenance, we focused our investigation on HIF-independent pathways. This manuscript describes the study of the role of an oxygen-sensor enzyme, member of the 2OG oxygenases and HIF negative regulator, jumonji domain-containing protein 6 (Jmjd6), in normal and malignant haematopoiesis. Our knockout studies deleting Jmjd6 specifically within the haematopoietic system (Jmjd6fl/fl;Vav-iCre) demonstrate that the homeostasis of HSPC pool was compromised and lymphopoiesis was attenuated in Jmjd6-deficient cohorts. Upon transplantation, HSCs lacking Jmjd6 exhibited a defective chimerism and impaired capacity to fully reconstitute haematopoiesis of recipient mice. Thus, Jmjd6 is essential for HSC self-renewal and maintenance. Our assessment of the impact of Jmjd6 deletion in the context of inflammatory response and recovery from treatment with a myelotoxic agent treatment revealed that Jmjd6 is a positive regulator of HSC homeostasis and recovery from cytotoxic stress. There are accumulating data on the importance of epigenetics in the development of haematological malignancies. Being an epigenetic regulator, clearly involved in RNA splicing, we investigated Jmjd6 as possible player in leukaemogenesis. The results from our leukaemic studies unravelled a new biological function for Jmjd6 as a tumour suppressor in Meis1/Hoxa9 murine model. Altogether, our findings offer important novel insights into the biological functions of Jmjd6 and pave the way for further studies to discover on the mechanism of action of this complex enzyme. Our observations add value to the idea that Jmjd6 might constitute a good candidate for cancer diagnosis, that can be use to ameliorate patient’s prognosis and that it can be used to help patient prognosis in the future.

Le cancer du sein représente une cause de mortalité élevée chez la femme. Le cancer du sein est un cancer hormono-dépendant. De ce fait, il est extrêmement important de définir le rôle joué par les différents acteurs protéiques de la signalisation hormonale, notamment la signalisation œstrogénique. Parallèlement aux effets nucléaires de ERa où l'hormone lie le récepteur nucléaire et régule la transcription génique, il existe une voie dite non génomique. L'équipe a montré que les œstrogènes induisent la méthylation de ERa, qui est un prérequis au recrutement de la Pl3K et de la tyrosine kinase Src, conduisant à l'activation de molécules de signalisation telles que les MAPK et Akt, induisant prolifération et survie cellulaire. Durant ma thèse, j'ai pu démontrer que le complexe mERa/Src/Pl3K existe in vivo et constitue un nouveau biomarqueur indépendant de mauvais pronostique. La recherche de nouveaux partenaires du complexe mERa/Src/Pl3K nous a permis d'identifier le suppresseur de tumeur LKB1 et l'arginine déméthylase JMJD6. De façon surprenante, l'étude de l'expression de LKB1 par immunohistochimie dans une cohorte de tumeurs mammaires a montré une dualité fonctionnelle selon sa localisation subcellulaire. De plus, nous avons démontré que JMJD6 s'associe à ERa méthylé lorsque le récepteur est complexé à Src et Pl3K, et permet ainsi la déméthylation de ERa et la dissociation du complexe mERa/Src/Pl3K. Ce travail a ainsi pu mettre en évidence que les différents acteurs de cette signalisation peuvent constituer des éléments clés au diagnostique mais également lors de la décision thérapeutique, puisque qu'il existe des drogues peuvant cibler cette voie de signalisation / Estrogen receptor a {ERa}, belonging to the superfamily of hormone nuclear receptors, regulates many physiological processes, notably the growth and survival of breast tumor cells, acting as a ligand-dependent transcription factor. Besides to the well described transcriptional effects, estrogen also mediate extranuclear events called non genomic signaling via its receptor. /n fact, team shows that ERa is methylated and that this event is a prerequisite for the recrutement of Src and P/3K and the activation of Akt which orchestrate cell proliferation and survival. During my PhD, / demonstrated that the non genomic signaling complex mERa/Src/P/3K exists in vivo and is operative. /n addition, the complex is found to be an independent prognostic factor for disease free survival. This is an emergent concept that estrogen non genomic pathway is operative in vivo and can constitute a new therapeutic targets. The search for new partners of the complex has allowed us to identify the tumor suppressor LKB1 and arginine demethylase JMJD6. Expression of LKB1 in immunohistochemistry revealed dual properties based on its subcellular localization. When LKB1 is complexed with mERa/Src/P/3K it may acquire oncogenic properties. /n addition, JMJD6 interacts with methylated ERa when the receptor is associated with Src and P/3K, and allows the demethylation of ERa and the dissociation of the complex mERa/Src/P/3K. This work showed that estrogenic non genomic players can constitute new therapeutic targets in Breast tumors

No description available.