The epidermal differentiation complex (EDC) encodes co-ordinately regulated genes critically involved in epidermal differentiation, however knowledge of the molecular mechanisms involved in co-ordinating EDC gene expression is limited. Recent findings indicate p63 dependent changes in the nuclear localisation and higher-order chromatin folding the EDC coincide with the onset of epidermal stratification during embryonic development. Here it is demonstrated that a direct transcription target of p63, the chromatin-remodelling enzyme Brg1, modulates the specific nuclear positioning of the EDC and transcription of differentiation-specific gene encoded at the EDC. In addition, the results of high-resolution 5C-based analyses of the spatial chromatin interactome at a 5.3Mb region spanning the murine EDC in epidermal keratinocytes, and the silenced EDC in thymocytes, are presented. Chromatin interactions at the EDC region in keratinocytes include long-range interactions between multiple proximal and distal candidate gene regulatory regions. Many candidate regulatory elements involved in looping chromatin interactions at the EDC region are enriched for both active (H3K4me1, H3K27ac) and repressive (H3K27me3) chromatin marks and are bound by Sin3a and RBP2 co-repressor complexes. The chromatin interactome at the EDC in epidermal progenitor cells is enriched for bound chromatin architectural proteins Satb1, Satb2, and the cohesin subunit Rad21. Further, a substantial degree of co-localisation is observed between these chromatin architectural proteins, transcription factors and co-factors. Findings presented here suggest that a functional chromatin interactome, mediated by Satb proteins and cohesin, acts in conjunction with transcriptional repressor complexes to facilitate co-ordinated gene expression at the EDC in epidermal progenitor cells upon differentiation. These results provide a foundation for further study of the mechanisms controlling EDC gene expression in health and disease.

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The epidermal differentiation complex (EDC) encodes co-ordinately regulated genes critically involved in epidermal differentiation, however knowledge of the molecular mechanisms involved in co-ordinating EDC gene expression is limited. Recent findings indicate p63 dependent changes in the nuclear localisation and higher-order chromatin folding the EDC coincide with the onset of epidermal stratification during embryonic development. Here it is demonstrated that a direct transcription target of p63, the chromatin-remodelling enzyme Brg1, modulates the specific nuclear positioning of the EDC and transcription of differentiation-specific gene encoded at the EDC. In addition, the results of high-resolution 5C-based analyses of the spatial chromatin interactome at a 5.3Mb region spanning the murine EDC in epidermal keratinocytes, and the silenced EDC in thymocytes, are presented. Chromatin interactions at the EDC region in keratinocytes include long-range interactions between multiple proximal and distal candidate gene regulatory regions. Many candidate regulatory elements involved in looping chromatin interactions at the EDC region are enriched for both active (H3K4me1, H3K27ac) and repressive (H3K27me3) chromatin marks and are bound by Sin3a and RBP2 co-repressor complexes. The chromatin interactome at the EDC in epidermal progenitor cells is enriched for bound chromatin architectural proteins Satb1, Satb2, and the cohesin subunit Rad21. Further, a substantial degree of co-localisation is observed between these chromatin architectural proteins, transcription factors and co-factors. Findings presented here suggest that a functional chromatin interactome, mediated by Satb proteins and cohesin, acts in conjunction with transcriptional repressor complexes to facilitate co-ordinated gene expression at the EDC in epidermal progenitor cells upon differentiation. These results provide a foundation for further study of the mechanisms controlling EDC gene expression in health and disease.

CCCTC-binding factor (CTCF) binds DNA, thereby helping to partition the mammalian genome into discrete structural and regulatory domains. In doing so, it insulates chromatin and fine-tunes gene activation, repression, and silencing. Complete removal of CTCF from mammalian cells causes catastrophic genomic dysregulation, most likely due to widespread collapse of 3D chromatin looping within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction in CTCF expression are viable but have an increased incidence of spontaneous multi-lineage malignancies. In addition, CTCF is mutated in many human cancers and is thus implicated as a tumour suppressor gene. This study aimed to interrogate the genome-wide consequences of a reduced genomic concentration of Ctcf and its implications for carcinogenesis. In a genetically engineered mouse model, Ctcf hemizygous cells showed modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these were enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, several hundred genes concentrated in cancer-related pathways were dysregulated due to changes in transcriptional regulation. Global chromatin structure was preserved but some loop interactions were destabilised, often around differentially expressed genes and their enhancers. Importantly, these transcriptional alterations were also seen in human cancers. These findings were then examined in a hepatocyte-specific mouse model of Ctcf hemizygosity with diethylnitrosamine-induced liver tumours. Ctcf hemizygous mice had a subtle liver-specific phenotype, although the overall tumour burden in Ctcf hemizygous and wild-type mice was the same. Using whole genome sequencing, the highly reproducible mutational signature caused by DEN exposure was characterised, revealing that Braf(V637E), orthologous to BRAF(V600E) in humans, was the predominant oncogenic driver in these liver tumours. Taken together, while Ctcf loss is partially physiologically compensated, chronic CTCF depletion dysregulates gene expression by subtly altering transcriptional regulation. This study also represents the first comprehensive genome-wide and histopathological characterisation of this commonly used liver cancer model.

Dans les cellules eucaryotes, la réponse aux lésions de l'ADN s'accompagne d'une réorganisation de la chromatine. Cette structure, associant l'ADN aux protéines histones, est porteuse de l'information épigénétique, qui définit l'identité cellulaire. Cependant, nos connaissances concernant les mécanismes impliqués dans la réorganisation de la chromatine dont l'intégrité structurale et fonctionnelle a été menacée par un stress génotoxique sont encore limitées, en particulier dans les cellules humaines. Au cours de ma thèse, je me suis donc intéressée à cette thématique en me concentrant sur l'étude de la dynamique des variants de l'histone H3 et de leurs chaperons associés après dommages UVC. En combinant une technologie innovante de suivi spécifique des histones parentales ou néo-synthétisées à des techniques de pointe d'induction de dommages locaux dans l'ADN, j'ai ainsi mis en évidence que le chaperon HIRA (Histone Regulator A) est recruté tôt aux sites de lésions où il stimule l'incorporation locale de nouveaux variants H3.3 et assure la reprise de la transcription après réparation des dommages UVC. Nous avons aussi démontré que les anciennes histones sont initialement redistribuées dans la chromatine autour des sites de lésions par un mécanisme faisant appel au facteur de détection des dommages DDB2 (DNA Damage Binding protein 2). A plus long terme, des histones parentales " reviennent " dans les régions de chromatine en cours de réparation où elles se mélangent aux nouvelles histones incorporées. Le " retour " d'histones préexistantes contribuerait ainsi au maintien de l'intégrité de l'information épigénétique véhiculée par la chromatine avant stress génotoxique. / In eukaryotic cells, the DNA damage response involves a reorganization of chromatin structure. This structure, in which DNA is associated with histone proteins, conveys the epigenetic information, which is critical for cell identity. However, we are still far from understanding the mechanisms underlying chromatin dynamics in response to DNA damage, which challenges both the structural and functional integrity of chromatin architecture. During my PhD, I thus decided to explore this issue in human cells, by deciphering the dynamics of histone H3 variants and their dedicated chaperones in response to UVC lesions. By combining local UVC irradiation with an innovative technology that allows specific tracking of parental and newly synthesized histones, I revealed that the histone chaperone HIRA (Histone Regulator A) is recruited early to UVC-damaged chromatin regions, where it promotes local deposition of new histone H3.3 variant and facilitates transcription recovery upon repair completion. We also demonstrated that old H3 histones are initially redistributed around the damaged chromatin zone, this conservative redistribution requiring the UVC damage sensor DDB2 (DNA Damage Binding protein 2). Later in the repair process, most parental histones recover and mix with newly deposited histones in repairing chromatin regions. The recovery of pre-existing histones may contribute to preserve the integrity of the epigenetic information conveyed by chromatin before genotoxic stress.

Le nucléole est le site de biogenèse des ribosomes, qui commence par la transcription des gènes d’ARN ribosomique (ARNr). Cependant, le nucléole est également impliqué dans d'autres processus cellulaires, comme l’organisation 3D du génome. Ainsi, des régions génomiques appelées NADs pour Nucleolus-Associated chromatin Domains, ont été identifiées dans des cellules animales et végétales. Ces régions sont surtout hétérochromatiques et les gènes associés ont tendance a être peu ou pas transcrits. Un des objectifs de ma thèse a été d’étudier l’implication du nucléole dans l’organisation de la chromatine au sein du noyau et la régulation transcriptionnelle de gènes transcrits par l’ARN Polymérase II chez Arabidopsis thaliana. Par ailleurs, parmi les centaines de copies de gènes d’ARNr, uniquement une fraction participe au processus de biogenèse des ribosomes. Dans un second temps, j’ai donc étudié le rôle de ces copies inactives. On a pu démontrer que l’absence des gènes d’ARNr inactifs n’engendre pas de changements majeurs dans la fonction nucléolaire. Par contre, ces copies participent à la stabilité du génome. En effet, en leur absence, des duplications génomiques allant jusqu’à plusieurs centaines de kilobases s’accumulent, entraînant des duplications de gènes et des différences du niveau d’expression de ces derniers. Finalement, les effets de ces changements structuraux sur la biologie de la plante sont discutés. / The nucleolus is the site of ribosome biogenesis, which begins with the transcription of ribosomal RNA (rRNA) genes. However, the nucleolus is also involved in other cellular processes, such as the 3D genome organization. Thus, genomic regions called NADs for Nucleolus-Associated chromatin Domains, have been identified in animal and plant cells. These regions are mostly heterochromatic and the associated genes tend to be poorly transcribed. One of the objectives of my thesis was to study the involvement of the nucleolus in the 3D genome organization and the transcriptional regulation of genes transcribed by RNA Polymerase II in Arabidopsis thaliana. In addition, only a fraction of rRNA gene copies participates in the process of ribosome biogenesis. In a second time, I studied the role of the inactive rRNA gene copies. We show that in their absence, there is no major changes in the nucleolus function. However, these copies contribute to genome stability. Indeed, in their absence, up to several hundred of kilobases long duplication events accumulate, resulting in the duplication and the differential expression of hundreds of genes. Finally, the impact of these structural changes on the plant biology are discussed.