Chromatin alterations in response to UVC damage in human cells

Dans les noyaux cellulaires des organismes eucaryotes, l’organisation de l’ADN avec des protéines histones sous forme de chromatine est une source d’information dite épigénétique, qui dicte l’expression des gènes et l’identité cellulaire. Cependant, la chromatine est déstabilisée lors des activités de transcription, réplication et réparation de l’ADN. Ces évènements nucléaires sont donc susceptibles d’affecter l’information épigénétique. Pendant ma thèse, je me suis intéressée à la dynamique de la chromatine en réponse aux dommages à l’ADN générés par les UVC dans les cellules humaines. En particulier, j’ai cherché à comprendre comment le maintien de l’intégrité du génome et de l’épigénome sont coordonnés lors de la réparation des dommages de l’ADN. Pour répondre à cette question, j’ai développé des approches innovantes combinant l’irradiation locale de cellules humaines aux UVC et le suivi en temps réel des histones parentales, porteuses de l’information épigénétique d’origine. Ces méthodes m’ont permis de caractériser la dynamique des histones parentales dans les régions de chromatine endommagée aux UVC et d’en identifier les mécanismes sous-jacents. Ainsi, j’ai montré que les histones parentales sont rapidement redistribuées de manière conservative à la périphérie des zones de dommages à l’ADN puis recyclées en quasi-totalité. La contribution majeure des histones parentales à la chromatine réparée facilite vraisemblablement le maintien de l’intégrité de l’épigénome lors de la réparation des dommages à l’ADN. La réparation de la chromatine s’accompagne également de l’incorporation d’histones néo-synthétisées dont l’importance fonctionnelle n’est pas encore caractérisée. Pour explorer la fonction de ces nouvelles histones mises en place aux sites de dommages à l’ADN, j’ai étudié leur impact sur les modifications post-traductionnelles d’histones dans les régions endommagées. J’ai ainsi identifié leur contribution à un défaut local de phosphorylations d’histones associées à la condensation des chromosomes en mitose précoce. Ce mécanisme pourrait servir de base à un point de contrôle du cycle cellulaire retardant la ségrégation des chromosomes en réponse aux dommages à l’ADN. Ces travaux soulignent l’importance de la dynamique des histones accompagnant la réparation des dommages à l’ADN dans la coordination du maintien de l’intégrité du génome et de l’épigénome, et ouvrent de nouvelles perspectives quant au rôle de la plasticité de la chromatine dans le maintien de l’homéostasie cellulaireIn eukaryotic cell nuclei, DNA wrapping around histone proteins in the form of chromatin is a source of epigenetic information that specify gene expression and therefore, cell identity. However, chromatin is also the substrate of all DNA transactions such as transcription, replication and repair. These nuclear processes destabilize the chromatin structure and are thus susceptible to affect the epigenetic information that it carries. During my thesis, I was interested in chromatin dynamics in response to UVC-induced DNA damage in human cells. In particular, I sought to understand how the maintenance of both genome and epigenome integrity are coordinated during DNA repair. To tackle this question, I developed an innovative approach combining local UVC irradiation of human cells and real-time tracking of parental histones, which carry the original epigenetic information. These methods allowed me to characterize parental histone dynamics in UVC-damaged chromatin and to identify the underlying mechanisms. Thus, I show that parental histones are rapidly redistributed around the DNA damage site in a conservative manner, and then recycle within damaged chromatin regions. The major contribution of parental histone to repairing chromatin likely contributes to the epigenome maintenance during DNA damage repair. DNA damage repair is accompanied by new histone incorporation in damaged chromatin regions, which functional relevance is not elucidated yet. To investigate the function of new histone deposition at UVC damage sites, I studied their impact on histone post-translational modifications in damaged chromatin regions. I identified their contribution to a local defect in histone phosphorylations associated with chromosome condensation in early mitosis. This mechanism could serve as a chromatin-based DNA damage checkpoint in early mitosis, which would delay chromosome segregation in response to DNA damage. Altogether, this work put forward the importance of histone dynamics that accompany DNA damage repair in coordinating genome and epigenome maintenance, and open new avenues regarding the role of chromatin plasticity in maintaining cellular homeostasi

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors

International audiencePediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3′-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting

Health effects of ionising radiation in paediatrics undergoing either cardiac fluoroscopy or modern radiotherapy (The HARMONIC project)

The use of ionising radiation (IR) for medical diagnosis and treatment procedures has had a major impact on the survival of paediatric patients. Although the benefits of these techniques lead to efficient health care, evaluation of potential associated long-term health effects is required. HARMONIC aims to better understand the increased risk of cancer and non-cancer effects after exposure to medical IR in children with cancer treated with modern external beam radiotherapy (EBRT) – radiation energy in MeV range – and in children with cardiac defects diagnosed and treated with cardiac fluoroscopy procedures (CFP) – radiation energy in keV range. The project investigates, among survivors of paediatric cancer, potential endocrine dysfunction, cardiovascular and neurovascular damage, health-related quality of life and second (and subsequent) primary cancer (SPC). The cardiac component builds a pooled cohort of approximately 90 000 paediatric patients who underwent CFP during childhood and adolescence to investigate cancer risk following exposure to IR and explore the potential effects of conditions predisposing to cancer. HARMONIC develops software tools to allow dose reconstruction in both EBRT and CFP to enable epidemiological investigations and future optimisation of treatments. With the creation of a biobank of blood and saliva samples, HARMONIC aims to provide a mechanistic understanding of radiation-induced adverse health effects and identify potential biomarkers that can predict these effects