Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

Sonic hedgehog (Shh) is an important signalling protein expressed extensively in development, throughout tissues of the central nervous system, gut and the posterior of the limb bud. The complicated expression pattern of Shh is regulated by a series of long-range enhancers located flanking and dispersed throughout a 1 Mb genomic desert. Disruption of SHH as a result of mutations within the gene or its enhancers has been implicated in two developmental conditions. These are Holoproencephaly (HPE3) a common developmental defect of the forebrain and frequently the mid-face in humans, and preaxial polydactyly (PPD), a congenital limb abnormality encompassing a varied phenotype affecting the digits on the anterior side of the hands and feet that has been attributed to misexpression of Shh. In order to investigate the Shh regulatory landscape and survey regulatory activity, a transposon-based chromosomal engineering strategy known as the local hopping enhancer detection (LHED) system was employed. Using this method a targeting vector containing a LacZ reporter gene as well as LoxP sites was inserted within the Shh region. The ‘hopping’ nature of the transposable element was then exploited to scatter it throughout the region. Tetraploid complementation embryos derived entirely from ES cells were generated and examined in order to gain an insight into enhancer activity. The region was found to be in an open conformation over its length and is generally susceptible to all Shh enhancers. Genes within the regulatory domain, such as the widely expressed Rnf32 gene, were found to resist Shh enhancer activities by a process of regulatory evasion by the promoter, a mechanism that may be common in large regulatory domains. Finally, at the boundaries of the region Shh activity was found to be lost incrementally at a number of genomic positions. Mouse lines were also generated to look at both enhancer activity and loss of function effects and three deletions of increasing size were generated between Shh and the furthest enhancer, the Zrs. These in turn, delete firstly a gut and pharyngeal epithelial enhancer, secondly the gut, pharyngeal enhancers as well an oral epithelial enhancer and finally all three epithelial enhancers as well as three forebrain enhancers. Reporter gene expression was found to be lost incrementally from those enhancers deleted without disrupting the rest of the region. Previously unidentified notochord enhancer(s) were found to lie within the region 100-530 kb upstream of Shh. Examination of the resultant phenotypes showed that gut and craniofacial defects were found to occur as a result of the loss of enhancers which drive expression within these tissues. Variable phenotypes were found to occur potentially as a result of temporal changes to Shh expression or as a result of threshold levels of HH being required for normal development. Other enhancers within the Shh region and outwith the deletions were not found to be disrupted by these modifications suggesting the enhancers within the region act independently of each other. The largest deletion resulted in bringing the Zrs (which drives Shh limb expression) within 170 kb of the gene, however limb development; was not, found to be affected suggesting distance is not required for Zrs function. Overall, the LHED transposon system has been utilised in order to examine the Shh region in more detail, allowing mapping of enhancer function by reporter gene expression and examination of phenotypes generated by deletion of enhancers.

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

No description available.

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.

No description available.

Dans les cellules souches embryonnaires de souris (mESCs), le groupe de protéines Polycomb (PcG) répriment les gènes de développement en participant ainsi à la maintenance de l’état de pluripotence. Ce complexe dépose la H3K27me3au niveau des éléments régulateurs induisant une compaction de la chromatine. Cette marque forme en plus des marquesactives H3K4me3 présentes des domaines bivalents. Etrangement, des boucles d’ADN dites entre le promoteur et enhancer, généralement associé à l’activation du gènes, sont observées au niveau des gènes bivalents avant leur activation.On suppose que la fonction du PcG pourrait être de neutraliser l'enhancer conférant une future activation rapide des gènes.Au cours de ma thèse, j’ai identifié les boucles d’ADN formé par les réprimés par PcG dans les mESCs. Pour cela,j’ai effectué un profilage épigénomique de 4 marques d'histones et identifié près de 2500 promoteurs bivalents et 13000enhancers. En utilisant des données publiées de Hi-C à haute résolution, j’ai identifié toutes les boucles formées par les domaines bivalents. Etonnement, j’ai pu identifier que de nombreux gènes réprimés par PcG interagissent avec des enhancers actifs. Cette observation a été suivie d'une validation par le 4C-seq. De plus, j’ai effectué une caractérisation fonctionnelle des boucles en utilisant deux approches. Tout d'abord, j'ai mis en place, en collaboration avec D. Bourc'his(Institut Curie), un système de culture de mESCs (2i + VitC) où le taux de H3K27me3 est réduit. J'ai effectué un profilage épigénomique similaire révélant que les promoteurs réprimés par PcG ont perdu la marque H3K27me3. En RNA-seq, j’ai démontré que l’expression des gènes ne change pas après le PcG soit détacher des promoteurs.. Ensuite, par la réalisation de plusieurs validations en 4C-seq j’ai démontré que les interactions avec les enhancers ne sont pas affecté alors que la moitié des enhancers interagissant perdent leurs marques activatrices. Dans le système 2i+VitC, ces gènes semblent être réprimés par un autre mécanisme suite à la perte du PcG. De plus, j’utilise une approche ciblée pour enlever localement laH3K27me3 de deux gènes bivalents en utilisant le système Cette technique est en cours d’optimisation.Notre étude est la plus systématique au niveau génomique des boucles d'ADN dans le cadre de la régulation des gènes PcG. Notre étude révèle une nouvelle fonction du PcG qui est la répression de boucle d’ADN déjà établies entre promoteurs et enhancers. / In the mouse embryonic stem cells (mESCs), Polycomb Group Proteins (PcG) repress developmental genes and thereby participating in the maintenance of the pluripotency. PcG repress genes by depositing the H3K27me3 histone marks on their regulatory elements, followed by chromatin compaction. In addition to the H3K27me3 marks, those genes carry H3K4me3 active marks and were characterized as bivalent. Intriguingly, at many PcG repressed genes, DNA loops can be observed with enhancer elements, which are normally thought to have an activating function. The aim of my project is to both describe and mechanistically dissect the function of Polycomb repressed promoter – enhancer loops.During my PhD, I aimed firstly to identify all promoter–enhancer loops involved by PcG repressed genes in mESCs. I have performed ChIP-seq profiling of 4 histone marks and identified around 2500 PcG repressed promoters and 13000 enhancers. Using a recently published high-resolution Hi-C data in mESCs, I have identified all DNA loops that are formed by PcG repressed promoters. Surprisingly, a high percentage of bivalent promoters were found to contact active enhancers. The presence of those loops were validated by ultra-high 4C-seq on selected genes and imply a small significant increase of the gene expression without leading to a complete activation of the gene. I have established a more physiological ESC model (2i+VitC) where H3K27me3 is reduced at all promoters. I have performed ChIP-seq, where bivalent promoters were all classified as H3K27me3 negative. RNA-seq experiments have showed that those genes do not become activated. 4C-seq experiments have revealed that those loops do not disappear after PcG removal, whereas the half of interacted enhancer loose their H3K27ac active marks. Those genes seem to remain repressed by an unknown mechanism. These results argue for a possible role of PcG in preparing the gene for their activation by blocking the productivity of such DNA loops. Secondly, I aimed to functionally characterize those DNA loops by using a CRISPR/dCas9 approach to completely remove H3K27me3 from two PcG repressed genes that contact active enhancers Pax6 and Nkx1-1 genes. This system is still under optimization steps.My project revealed the most systematic characterization of DNA loops under the regulation of PcG, providing important insight how PcG function to inactivate such loops. I have highlighted an additional function of PcG which the involvement in the repression of already establish loops between active enhancers and promoters and thereby blocking the productivity of such activating loops. This function is an addition to the already described repressive function of PcG on both promoters and poised enhancers.

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.

La balance entre synthèse et dégradation des collagènes joue un rôle crucial dans le développement de pathologies de cicatrisation. Les collagènes fibrillaires sont synthétisés et sécrétés sous forme de précurseurs dans l'espace extracellulaire. Ils subissent alors une maturation protéolytique par des métalloprotéases, telles que les « BMP-1/Tolloid-like » (BTPs), permettant la formation de fibres de collagène. Ce clivage protéolytique est stimulé de façon spécifique par deux glycoprotéine, les « Procollagen C-Proteinase Enhancers » (PCPE-1 et -2). Ainsi, l'objectif de mon travail de recherche a été d'étudier les rôles de PCPE-1 et -2 dans la cicatrisation cutanée. De façon intéressante, nous avons montré que les BTPs et PCPE-1 étaient exprimés au niveau du derme et que leur expression augmentait pendant la première semaine de cicatrisation. PCPE-1 est principalement exprimée par les fibroblastes alors que PCPE-2 est retrouvée dans les kératinocytes. De plus, nous avons montré que PCPE-2 était abondante dans les cellules myéloides, suggérant un rôle pendant la phase inflammatoire de cicatrisation. Nos résultats montrent que PCPE-2 ne stimule pas efficacement la maturation des procollagènes fibrillaires et inhibe même l'activité de BMP-1 sur des substrats non-collagéniques, suggérant un rôle inattendu de PCPE-2. Enfin, aucune différence majeure n'a été observée dans la peau de souris déficientes en PCPE-2 au cours de la cicatrisation, suggérant que PCPE-1 serait le principal stimulateur impliqué dans la maturation et le dépôt des fibres de collagènes. Ainsi, ce travail suggère que PCPE-1 et -2 joueraient des rôles distincts dans la peau et pendant la cicatrisation cutanée / The imbalance between collagen assembly and degradation during skin wound healing is a major factor contributing to wound healing pathologies. Fibrillar collagen precursors are synthesized by fibroblasts and processed in the extracellular space by specific metalloproteases (e.g. BMP-1/Tolloid-like Proteinases, or BTPs) to form collagen fibrils. These proteolytic maturations can be efficiently stimulated by two glycoproteins, Procollagen C-Proteinase Enhancers (PCPE-1 and -2). The main goal of our study was to analyze the possible redundant and specific roles of the two PCPE proteins during wound healing.Interestingly, both BTPs and PCPE-1 were found to be expressed in the dermis and to be significantly increased during the first week after injury. Surprisingly, PCPE-1 is mainly expressed by dermal fibroblasts, whereas PCPE-2 is lowly expressed in fibroblasts and more abundant in basal keratinocytes. Moreover, PCPE-2 appears to be expressed by myeloid cells suggesting that PCPE-2 might rather play a role during the inflammatory phase of wound healing. In addition, our results indicate that recombinant PCPE 2 does not efficiently enhance their proteolytic maturation of fibrillar procollagens and can even inhibit the action of BMP-1 on other non-collagenous substrates, suggesting a differential and unexpected role of PCPE-2. Finally, the in vivo role of PCPE-2 was investigated using a Pcolce2 knockout model; skin morphology and wound healing were not affected by PCPE-2 loss, indicating that PCPE-1 is the main enhancer involved in collagen deposition during wound healing. Thus, this work suggests that PCPE-1 and -2 play distinct roles in the skin and during wound healing

Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l’activation oula répression des gènes. Les profils d’expression génique mis en place vont perdurer d’une générationcellulaire à l’autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d’expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l’activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d’identifier les partenaires de la protéineDSP1 dans l’embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j’aimis en place puis effectué l’immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d’identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j’aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1. / In multicellular organism, the identity of cell is determined by several factors playing on genesexpression. Once established, the gene expression pattern is transmitted to daughter cells through aprocess involving epigenetic mechanisms that locally reshape the structure of chromatin. In Drosophilamelanogaster, the Polycomb (PcG) and trithorax (trxG) group genes are involved in the maintenanceof gene expression profile during development. Inside multimeric complexes, PcG proteins maintaingenes in repressed state whereas TrxG maintain genes active. A third class of proteins, calledEnhancers of Trithorax and Polycomb, regulate PcG and TrxG activities. Dorsal Switch Protein 1(DSP1) is a High Mobility Group B protein acting as an ETP. But DSP1 has not yet been identified inPcG or TrxG complexes. On the basis of gel filtration analysis of protein complexes in embryo nuclearextracts, it appears that the majority of DSP1 is present in complex(es) from 100 kDa to 1MDa. Aimsof present work are the identification of DSP1 protein partners in drosophila embryo and thecharacterization of biochemical properties of DSP1. Firstly, I used immunopurification from drosophilaembryonic nuclear extracts. The proteins purified with DSP1 were characterized through sequencingof peptides from individual protein bands by mass spectrometry. Among identified proteins, wefocused on the DEAD Box RNA helicase, Rm62. The role of interaction between DSP1 and Rm62 hasbeen characterized. Secondly, I have identified a new physicochemical aspect of DSP1 protein.