Cernunnos/Xlf Deficiency Results in Suboptimal V(D)J Recombination and Impaired Lymphoid Development in Mice

Xlf/Cernunnos is unique among the core factors of the non-homologous end joining (NHEJ) DNA double strand breaks (DSBs) repair pathway, in the sense that it is not essential for V(D)J recombination in vivo and in vitro. Unlike other NHEJ deficient mice showing a SCID phenotype, Xlf−/− mice present a unique immune phenotype with a moderate B- and T-cell lymphopenia, a decreased cellularity in the thymus, and a characteristic TCRα repertoire bias associated with the P53-dependent apoptosis of CD4+CD8+ DP thymocytes. Here, we thoroughly analyzed Xlf−/− mice immune phenotype and showed that it is specifically related to the DP stage but independent of the MHC-driven antigen presentation and T-cell activation during positive selection. Instead, we show that V(D)J recombination is subefficient in Xlf−/− mice in vivo, exemplified by the presence of unrepaired DSBs in the thymus. This results in a moderate developmental delay of both B- and T-lymphocytes at key V(D)J recombination dependent stages. Furthermore, subefficient V(D)J recombination waves are accumulating during TCRα rearrangement, causing the typical TCRα repertoire bias with loss of distal Vα and Jα rearrangements

Evolution from XIST-Independent to XIST-Controlled X-Chromosome Inactivation: Epigenetic Modifications in Distantly Related Mammals

X chromosome inactivation (XCI) is the transcriptional silencing of one X in female mammals, balancing expression of X genes between females (XX) and males (XY). In placental mammals non-coding XIST RNA triggers silencing of one X (Xi) and recruits a characteristic suite of epigenetic modifications, including the histone mark H3K27me3. In marsupials, where XIST is missing, H3K27me3 association seems to have different degrees of stability, depending on cell-types and species. However, the complete suite of histone marks associated with the Xi and their stability throughout cell cycle remain a mystery, as does the evolution of an ancient mammal XCI system. Our extensive immunofluorescence analysis (using antibodies against specific histone modifications) in nuclei of mammals distantly related to human and mouse, revealed a general absence from the mammalian Xi territory of transcription machinery and histone modifications associated with active chromatin. Specific repressive modifications associated with XCI in human and mouse were also observed in elephant (a distantly related placental mammal), as was accumulation of XIST RNA. However, in two marsupial species the Xi either lacked these modifications (H4K20me1), or they were restricted to specific windows of the cell cycle (H3K27me3, H3K9me2). Surprisingly, the marsupial Xi was stably enriched for modifications associated with constitutive heterochromatin in all eukaryotes (H4K20me3, H3K9me3). We propose that marsupial XCI is comparable to a system that evolved in the common therian (marsupial and placental) ancestor. Silent chromatin of the early inactive X was exapted from neighbouring constitutive heterochromatin and, in early placental evolution, was augmented by the rise of XIST and the stable recruitment of specific histone modifications now classically associated with XCI

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

International audienc

The role of CTCF in regulating V(D)J recombination

International audienc

A New Take on V(D)J Recombination: Transcription Driven Nuclear and Chromatin Reorganization in Rag-Mediated Cleavage

International audienc

Qualité de vie et conditions de travail des chirurgiens-dentistes français en milieu rural et urbain

CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

L'inactivation du chromosome X chez la souris (étude du rôle des modifications épigénétiques et de l'organisation nucléaire dans la régulation des gènes au cours du développement)

Chez les femelles mammifères, l'inactivation d'un des deux chromosomes X au cours du développement embryonnaire précoce permet d'assurer la compensation de dose entre les sexes. Si l'accumulation en cis de l'ARN non-codant Xist sur le chromosome est essentielle à la mise en place de l'inactivation, les mécanismes qui aboutissent à la répression transcriptionnelle sont encore peu compris. Dans ce contexte, je me suis attachée à étudier les évènements précoces sous-jacents à l'induction de l'inactivation, au cours de la différenciation des cellules souches embryonnaires murines. Dans un premier temps, j'ai approfondi la caractérisation de la combinaison de modifications des histones, connues pour leur rôle dans la régulation transcriptionnelle, qui se met en place très vite après l'accumulation de l'ARN Xist. Si leur stabilité au cours du cycle cellulaire suggère que certaines d'entre elles pourraient faire partie des marques épigénétiques responsables du maintien de l'état inactif, des études récentes ont montré qu'elles n'étaient pas nécessaires à l'induction de l'inactivation. J'ai ensuite montré que l'ARN Xist crée de manière très précoce un compartiment répressif dépourvu de facteurs de transcription dans lequel les gènes sont relocalisés au moment de leur répression. L'ARN Xist semble donc avoir plusieurs fonctions, remplies par différentes régions du transcrit, d'une part de création d'un compartiment répressif et d'autre part d'extinction génique liée à la réorganisation de la chromatine. Ces résultats apportent des données importantes sur le rôle de l'organisation nucléaire dans la régulation transcriptionnelle et la fonction des ARNs non-codants dans ce mécanisme.During early development, mammalian female embryos inactive one of their two X chromosomes to ensure dosage compensation between sexes. Although coating of the X chromosome by Xist RNA is essential for the initiation and propagation of X inactivation, little is known about how this signal is transformed into transcriptional silencing. In this context, I investigated the precise timing of early events during X inactivation, using differentiating female embryonic stem cells as a model system. I first focused on the characterisation of histone modifications, known to be involved in transcription regulation and appear rapidly after Xist RNA coating. Although their stability throughout cell cycle and mitosis suggests that they could be part of epigenetic marks involved in the maintenance of the inactive state, recent studies have shown that they are not crucial for induction of X inactivation. I then showed that the exclusion of the transcription machinery from the Xist RNA-coated X chromosome represents the earliest event following Xist RNA accumulation described so far, several days before complete gene silencing. Examination of the 3D organization of X-linked genes reveals that their repression involves their relocation within Xist RNA domain. Based on these results, we propose that Xist RNA has dual silencing functions, accomplished by different regions of the transcript. On the one hand, it seems to create a repressive nuclear compartment and, on the other hand, it silences genes through X chromatin reorganization. Thus, these results provide important findings on the role of nuclear organization and non coding RNAs on transcription regulation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Contribution à la connaissance d' une maladie métabolique orpheline (l'homocystinurie classique)

PARIS-BIUP (751062107) / SudocSudocFranceF

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

International audienc

Epigenetic regulation of V(D)J recombination

International audienceChromosome breaks are dangerous business, carrying the risk of loss of genetic information or, even worse, misrepair of the break, leading to outcomes such as dicentric chromosomes or oncogenic translocations. Yet V(D)J recombination, a process that breaks, rearranges and repairs chromosomes, is crucial to the development of the adaptive immune system, for it gives B- and T-cells the capacity to generate a virtually unlimited repertoire of antigen receptor proteins to combat an equally vast array of antigens. To minimize the risks inherent in chromosomal breakage, V(D)J recombination is carefully orchestrated at multiple levels, ranging from DNA sequence requirements all the way up to chromatin conformation and nuclear architecture. In the present chapter we introduce various regulatory controls, with an emphasis on epigenetic mechanisms and recent work that has begun to elucidate their interdependence