Tumors are composed of proliferating cells that invade healthy tissue and grow over time. Even though it is still unclear, it is a common opinion that the cells of origin should possess a proliferative capacity (Blanpain, 2013; Visvader, 2011). Particularly for brain cancers, the transition of neural progenitors to differentiated postmitotic neurons is considered irreversible in physiological and pathological conditions. Therefore, postmitotic neurons have not been considered as suitable cell of origin for brain cancer. Here, we show that neurons reprograming may occur upon Shh activation leading to medulloblastoma (MB) formation in vivo. Human SHH medulloblastoma (MB) is a brain tumor affecting adults and infants that is thought to originate from cerebellar granule neuron progenitors. Notably, several groups have shown that Shh pathway activation (SmoM2 overexpression) in mouse granule neuron progenitors is able to induce Shh MB (Schuller et al., 2008; Z.-J. Yang et al., 2008). These progenitors are present in infants and newborn mice, but they seem to be absent in adult humans and mice (Biran, Verney, & Ferriero, 2012; Marzban et al., 2014; Z.-J. Yang et al., 2008). Furthermore, it was recently discovered that the two different forms of SHH MB are distinguished by different transcriptome/methylome levels suggesting that the adult SHH MB may originate from a different cell of origin (Cavalli et al., 2017; Kool et al., 2014). Relying on these data, we take advantage of a conditional Cre-Lox recombination system to recapitulate the human adult medulloblastoma pathogenesis in mice, demonstrating that cerebellar post-migratory mature granule neurons upon SmoM2 overexpression can dedifferentiate and give rise to SHH MB in vivo. Moreover, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Notably, gene expression profiling on our samples allowed to associate “cerebellar granule progenitors-derived MB” with the human infant form while “truncated BRPF1-induced tumors” clustered with human adult SHH MB. Furthermore, as previously described by Kool et al., 2014, human adult SHH MB is characterised by the copresence of p-AKT and p-S6, compared to the human infant SHH MB that are positive for either p- AKT or p-S6 and always in a mutually exclusive way. Truncated BRPF1-induced tumors are double positive for p-AKT and p-S6, similarly to adult patients, while cerebellar granule progenitors derived MB present only p-S6. Furthermore, to define the contribution of chromatin changes in granule neurons dedifferentiation in response to Shh activation, we profiled changes in chromatin accessibility by ATAC-seq analysis on mice cerebella. SmoM2 overexpression changed the epigenetic landscape of granule neurons, enriching the number of open chromatin regions 12 associated with stem/progenitor-like genes. Moreover, the cooperation between truncated BRPF1 and SmoM2 in reshaping the chromatin arrangement of granule neurons was explored applying ATAC-seq on differentiated human cerebellar neurons derived from neuroepithelial cells. ATAC-seq analysis pointed out a synergistic mechanism between SmoM2 and truncated BRPF1 in modifying the epigenetic landscape of postmitotic neurons, increasing the chromatin accessibility of super-enhancers, associated with stemness and chromatin organization/modification genes. Our novel model of cancer development could explain the human SHH medulloblastoma onset in adult individuals where granule neuron progenitors are no more present. For these reasons, we strongly believe that our model configures as an important starting point for a new field in cancer and stem cell biology focusing on the study of mechanisms driving tumorigenesis in postmitotic cells.

Le neuroblastome est cancer du jeune enfant se développant au sein du système nerveux périphérique sympathique. Cette tumeur est caractérisée par sa grande hétérogénéité clinique : allant de formes régressant spontanément aux tumeurs de haut-risque, réfractaires aux traitements les plus agressifs. La survie à long terme des patients présentant un neuroblastome de haut-risque reste par ailleurs inférieure à 50%, ce qui souligne la nécessité de trouver de nouveaux traitements afin d’améliorer leur prise en charge thérapeutique.Récemment, en définissant le paysage épigénétique des cellules de neuroblastome, nous avons observé la présence de super-enhancers (SE). La caractérisation du paysage des SE dans les lignées de neuroblastome nous a permis de révéler l’hétérogénéité cellulaire du neuroblastome, composée de deux identités distinctes : noradrénergique et mésenchymateuse. Chacune des identités cellulaires est caractérisée par un circuit de régulation transcriptionnelle (CRC) : les facteurs PHOX2B, HAND2 et GATA3 définissent l’identité noradrénergique alors que les facteurs de la famille AP-1 gouvernent l’identité mésenchymateuse. Nous avons par ailleurs montré la différence de sensibilité aux chimiothérapies classiquement utilisées en clinique entre ces deux types cellulaires, avec une résistance accrue des cellules mésenchymateuses.Mon travail de thèse porte sur la caractérisation du rôle des facteurs de transcription PHOX2B et GATA3 dans l’établissement et le maintien de l’identité noradrénergique des cellules de neuroblastome. J’ai réalisé leur knock-out par CRISPR-Cas9 dans la lignée noradrénergique SH-SY5Y. L’inactivation de PHOX2B ne modifie ni le programme transcriptionnel ni le phénotype des cellules, arborant une identité noradrénergique. En revanche, les cellules inactivées pour GATA3 possèdent un phénotype cellulaire mésenchymateux ainsi que des capacités de migration, d’invasion et de résistance aux chimiothérapies. Le knock-out de PHOX2B et GATA3 entraine une diminution de la prolifération cellulaire, traduisant le phénomène d’addiction transcriptionnelle des cellules cancéreuses. La caractérisation du paysage épigénétique des cellules inactivées pour GATA3 démontre leur reprogrammation de l’identité noradrénergique vers l’identité mésenchymateuse avec l’effondrement des SE noradrénergiques ainsi que l’acquisition de SE mésenchymateux. GATA3 est donc indispensable pour le maintien de l’identité noradrénergique in vitro.Les résultats générés lors de ma thèse montrent que les facteurs de transcription impliqués dans un même CRC possèdent des rôles distincts dans l’identité cellulaire. La caractérisation de la dynamique de reprogrammation ainsi que des facteurs impliqués dans ce processus nous permettrons de mieux comprendre les phénomènes de plasticité cellulaire à l’origine de la progression tumorale et de la rechute thérapeutique des patients. / Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system characterized by its diversity of clinical presentations from spontaneous regression to highly aggressive tumors. Currently, the overall survival of high-risk neuroblastoma patients remains under 50% which highlight the need to find new therapeutic approaches to improve patient outcome.Recently, we defined the epigenetic landscape of neuroblastoma cell lines and observed the presence of super-enhancers (SE). The characterization of the SE landscape let us to define the heterogeneity of neuroblastoma cell identity with the presence of noradrenergic and mesenchymal cells. Both cell identities are governed by a core regulatory circuitry (CRC), composed by PHOX2B-HAND2-GATA3 in the noradrenergic cells and by AP-1 transcription factors in the mesenchymal cells. We also demonstrate the different behaviors of the cells regarding chemotherapy treatments with a higher resistance of the mesenchymal cells.My thesis aimed at deciphering the role of PHOX2B and GATA3 transcription factors in the establishment and the maintenance of the noradrenergic identity of neuroblastoma cells. To do this, PHOX2B and GATA3 were knock-out by CRISPR-Cas9 in the noradrenergic SH-SY5Y cell line. PHOX2B knock-out has no major impact neither on the transcriptomic profile nor the phenotype of the cells. PHOX2B knock-out cells still maintain their noradrenergic identity. In contrast, GATA3 knock-out cells harbor a mesenchymal phenotype showing higher ability to migrate, invade and being pore resistant to chemotherapy than control SH-SY5Y cells. Both PHOX2B and GATA3 knock-out decrease the SH-SY5Y cell proliferation in vitro and in vivo, which highlight the transcriptional dependency of the noradrenergic cells for their identity-related transcription factors. The characterization of the epigenetic landscape of GATA3 knock-out cells revealed their reprograming from the noradrenergic to the mesenchymal identity with the loss of noradrenergic SE and the acquisition of mesenchymal SE. These results demonstrate that GATA3 is essential for the maintenance of the noradrenergic identity in vitro.Altogether, these results show that transcription factors involved in a CRC can have distinct role in the cell identity. The characterization of the reprogramming dynamics as well as the factors involved in this process will allow us to better understand the cellular plasticity involved in the tumor progression and patient relapse.

No description available.

Chromatin ist ein Makromolekül, dessen Genregulation innerhalb des räumlich eingeschränkten Zellkerns organisiert werden muss. Die Genomorganisation ist eng mit Genaktivierung und Genrepression verknüpft. In den vergangenen Jahren wurde gezeigt, dass die DNA hierarchisch organisiert ist. Die Faltung läuft in aufeinander folgenden Schritten ab, wobei jede Organisationsebene sowohl zur räumlichen Komprimierung, als auch zur Genregulation beiträgt. In dieser Dissertation wurden mit Hilfe von hochauflösender Mikroskopie verschiedene Ebenen der 3D Chromatinorganisation auf Einzelzell-Basis untersucht. Auf der kleinsten Organisationsebene wurde die Struktur zweier, nebeneinander liegender topologischer Domänen (TADs) am Sox9-Lokus erforscht. Mit Hilfe von Fluoreszenz in situ Hybridisierung (FISH) in 3D Zellen, sowie Cryoschnitten in embryonalen Stammzellen von Mäusen konnten Interaktionen zwischen den benachbarten TADs festgestellt werden. FISH in Zellen mit genomischen Duplikationen, zeigte das Entstehen von zwei unterschiedlichen, durch die Duplikation entstandenen, Konformationen. Unter Verwendung von FISH wurden long-range Kontakte, die zuvor mit GAM entdeckt wurden, untersucht und es zeigte sich, dass sie häufig zwischen TADs die regulatorischen Domänen enthalten auftreten. Zudem zeigte sich die Bildung von Clustern zwischen mehreren, weit auseinander liegenden, regulatorischen Elementen. Dies lässt unter Umständen auf das Entstehen von regulatorischen Zentren zwischen diesen Enhancer-reichen Regionen schließen. Weitere Untersuchungen zeigten Veränderung der sogenannten Super-Enhancer Cluster in unterschiedlichen Zelltypen. Des Weiteren sind Super-Enhancer TADs sehr dekondensiert und wurden häufig an Splicing-Speckle Regionen vorgefunden. / Chromatin needs to organize gene regulation whilst fitting into the confined space of the nucleus. Chromatin organization is therefore intertwined with gene activation and silencing. In recent years many advances in the field of chromatin architecture have been made showing that chromatin is organized hierarchically. Folding occurs in subsequent units, where each level of organization contributes to the spatial compaction of DNA and gene regulation. In this dissertation different levels of 3D chromatin organization were analysed using single-cell, high-resolution imaging. On the smallest scale, the 3D organization of two neighbouring Topologically Associating Domains (TADs) at the Sox9 locus was investigated. Performing Fluorescence in situ Hybridization (FISH) in 3D and cryosectioned mouse embryonic stem cells, extensive contacts between the two neighbouring TADs across the TAD boundary were detected. Applying FISH in a cell line bearing a genomic duplication within the Sox9 locus, the occurrence of two different conformations that result from the duplication was shown. Recent evidence from GAM showed the formation of long-range, multimer contacts between distal regulatory elements. Investigating the occurrence of long-range contacts between super-enhancer TADs in single cells by FISH, showed that they establish frequent interactions at close spatial distances. Furthermore the formation of clusters containing distal super-enhancer TADs could be demonstrated, indicating the possibility of higher-order regulatory hubs between these enhancer-rich regions. Further investigation showed that super-enhancer regions form different clusters in different cell types. Finally, it was shown that super-enhancers are highly decondensed and preferentially located at splicing speckles.