Line Manceau

Le rhabdomyosarcome alvéolaire (RMSA) est un cancer pédiatrique rare des tissus mous causé par des réarrangements chromosomiques majeurs, incluant translocations, duplication du génome et perte ou amplification de pans de chromosomes. Le RMSA reste délétère pour les patients malgré l'utilisation de traitements complexes. Je me suis donc intéressé aux mécanismes moléculaires et cellulaires par lesquels les produits des translocations t(2,13) ou t(1,13) contribuent à la physiopathologie de ce cancer. Il s'agit de protéines chimères constituées des domaines de liaison à l'ADN de PAX3 ou PAX7, à savoir un paired (PD) et un homéodomaine (HD), et du domaine de transactivation de FOXO1. PAX3-FOXO1 et PAX7-FOXO1 sont des facteurs de transcription (FT) avec une capacité de transactivation significativement plus élevée que les PAX sauvages. Je me suis d'abord penchée sur un paradoxe révélé dans de nombreux types cellulaires. Malgré l'exceptionnel pouvoir de transactivation des PAX-FOXO1, leur potentiel de transformation maligne in vivo est faible. C'est également le cas dans le tube neural de l'embryon de poulet, modèle d'étude que nous avons utilisé. Dans ce modèle, les deux PAX-FOXO1 confèrent aux cellules neurales des caractéristiques moléculaires de RMSA, définies au préalable par des analyses transcriptomiques de biopsies et de lignées cellulaires de plusieurs types de RMS. Les PAX-FOXO1 conduisent également les cellules neurales épithéliales à adopter un phénotype mésenchymateux avec des propriétés invasives. En revanche, ils inhibent le cycle cellulaire. Cette inhibition peut être levée par surexpression d'activateurs des complexes CDK-cyclines. Cet impact négatif des PAX-FOXO1 sur la prolifération cellulaire fournit une explication assez simple de leur limitation apparente à induire une transformation maligne in vivo. J'ai ensuite entrepris une analyse structure-fonction de PAX3-FOXO1. Alors que les études génomiques montrent que son recrutement à l'ADN est principalement relayé par le PD, ce domaine n'est pas impliqué dans la délamination des cellules neurales induite par PAX3-FOXO1. Il est néanmoins requis pour l'induction de certains traits moléculaires associés au RMSA. A l'inverse, l'HD contribue à l'ensemble de l'activité de transformation de PAX3-FOXO1. Enfin, la transformation par PAX3-FOXO1 peut aussi être modulée en supprimant d'autres domaines protéiques, comme son extrémité N-terminale. Ainsi, notre étude fournit un faisceau de preuves montrant que la robustesse dans la génération d'un comportement oncogénique par un seul FT repose sur la complexité de sa structure protéique. Enfin, j'ai testé l'hypothèse d'une régulation différentielle de l'expression génique par PAX3-FOXO1 et PAX7-FOXO1, hypothèse qui pourrait expliquer des différences cliniques chez les patients portant la t(2,13) ou la t(1,13). À cette fin, j'ai créé des lignées inductibles de fibroblastes humains exprimant PAX3-FOXO1 ou PAX7-FOXO1. En plus d'une cohorte de gènes communément induits par ces deux FT, il existe des groupes de gènes dont l'expression est spécifiquement modulée par PAX3-FOXO1 ou PAX7-FOXO1, ce dernier induisant plus de gènes et à des niveaux plus forts. Grâce à la technique du 'CUT&Tag', nous avons montré que 30% des régions d'ADN sont spécifiquement liées par PAX3-FOXO1 ou PAX7-FOXO1 en raison d'une utilisation préférentielle respective de leur domaine PD ou HD. De plus, l'analyse du recrutement de la marque d'histone H3K27ac montre que PAX7-FOXO1 a une plus grande capacité à activer les régions enhanceurs que PAX3-FOXO1. Ces résultats à l'échelle du génome corrèlent avec des phénotypes de cycle cellulaire et de morphologie des fibroblastes spécifique à chaque PAX-FOXO1. Dans son ensemble cette étude soutient l'idée que les cellules à l'origine du RMSA interprètent de manière spécifique leur exposition à PAX3-FOXO1 ou à PAX7-FOXO1 ; ce qui pourrait sous-tendre l'hétérogénéité observée chez les patients.

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1. Rhabdomyosarcoma is a class of paediatric soft tissue cancers of genetic origin, but for which the causal links between genetic aberrations and tumour development remain to be deciphered. To answer this question, we focused on the products of two chromosomal translocations that generate the chimeric proteins PAX3-FOXO1 and PAX7-FOXO1. They are composed of the DNA-binding domains of the PAX3 or PAX7 proteins and a single portion of the FOXO1 protein. Several clinical parameters distinguish patients expressing PAX3-FOXO1 or PAX7-FOXO1 and we wondered if these differences could emanate from a different mode of action of the two chimeric proteins. Thus, we generated inducible human fibroblast cell lines expressing one or the other protein. We analysed some molecular and cellular characteristics of these cells 48 hours after induction of PAX3-FOXO1 or PAX7-FOXO1. As it was previously known for PAX3-FOXO1, we showed that PAX7-FOXO1 binds genomic DNA on cis-regulatory regions and activates them. Surprisingly, PAX7-FOXO1 only partially shares the DNA binding sites of PAX3-FOXO1 and its activation potential is stronger than that of PAX3-FOXO1. Thus, PAX3-FOXO1 and PAX7-FOXO1 generate partially divergent transcriptomic signatures, which include genes encoding regulators of cell morphology and cell cycle, two key oncogenic processes. In agreement, our data revealed specificities in these two processes that are either PAX3-FOXO1 or PAX7-FOXO1 dependent. Overall our results demonstrate a differential mode of action between the two chimeric proteins that could in turn participate in the heterogeneity of rhabdomyosarcoma manifestation.

Summary: Hox genes encode transcription factors that specify segmental identities along the Antero-Posterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, an evolutionary conserved feature known as collinearity. In Drosophila , the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive replacement of histone modifications, reorganization of 3D chromatin architecture and sequential activation of boundary elements and cis -regulatory regions. To dissect functional hierarchies, we compared chromatin organization in larvae and in cell lines, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary element for insulation between 3D domains marked by different histone modifications. Interestingly, we detected a non-canonical inversion of collinear chromatin dynamics at the Abd-B gene, with the active histone domain decreasing in size. This chromatin organization differentially instructed alternative Abd-B promoter use, thereby expanding the possibilities to regulate transcriptional output.

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma.

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformationin vivo. The reason for this is unclear. To address this, we developed anin ovomodel to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.

The chromosome translocations generating PAX3FOXO1 and PAX7FOXO1 chimeric proteins are the primary hallmarks of the paediatric cancer, Alveolar Rhabdomyosarcoma (ARMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo . The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAXFOXO1s. Our data demonstrate that PAXFOXO1s, but not wild-type PAX3 and PAX7, trigger the trans-differentiation of neural cells into ARMS-like cells with myogenic characteristics. In parallel expression of PAXFOXO1s remodels the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, gain for PAXFOXO1s, as for wild-type PAX3/7, reduces the levels of CDK-CYCLIN activity and arrests cells in G1. Introduction of CYCLIN D1 or MYCN overcomes PAXFOXO1s mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism underpinning the apparent limited oncogenicity of PAXFOXO1 fusion transcription factors and support a neural origin for ARMS.

Zebrafish are an emerging model in behavioral neuroscience. They display a wide range of measurable behaviors such as locomotion, aggression, anxiety, learning and memory, and social behavior. In addition, the relative ease of genetic manipulation and the increasing availability of disease models mean that zebrafish have gained in popularity as an animal model for various neurological and psychiatric diseases including autism spectrum disorder (ASD). In order to better characterize social behavior and behavioral abnormalities in zebrafish, we have developed the visually mediated social preference (VMSP) test, a novel assay to measure social preference and social novelty in two consecutive 5-min sessions. Using recording and video tracking, the time spent in different areas of the tank, the time spent immobile, swimming speed, and distance moved can be easily measured and analyzed. Untreated experimentally naive AB WT zebrafish typically show a strong preference for spending time near and interacting with a compartment containing unfamiliar conspecifics over the empty compartments during session 1 and a stronger preference for a group of unfamiliar zebrafish over familiar conspecifics from session 1, during session 2 of the test. Research in our lab has shown that the VMSP is suitable to measure the social behavior of individual zebrafish, to uncover social phenotypes of mutant strains, and to better understand animal models of disease that include impaired sociability such as ASD. The current paper provides a step-by-step guide on how to implement and perform this test and highlights important considerations for data acquisition, analysis, and interpretation.