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A loss-of-function mutation in tetratricopeptide repeat domain 7A (TTC7A) is a recently identified cause of human intestinal and immune disorders. However, clues to related underlying molecular dysfunctions remain elusive. It is now shown based on the study of TTC7A-deficient and wild-type cells that TTC7A is an essential nuclear protein. It binds to chromatin, preferentially at actively transcribed regions. Its depletion results in broad range of epigenomic changes at proximal and distal transcriptional regulatory elements and in altered control of the transcriptional program. Loss of WT_TTC7A induces general decrease in chromatin compaction, unbalanced cellular distribution of histones, higher nucleosome accessibility to nuclease digestion along with genome instability, and reduced cell viability. Our observations characterize for the first time unreported functions for TTC7A in the nucleus that exert a critical role in chromatin organization and gene regulation to safeguard healthy immune and intestinal status.

The eukaryotic genome is divided into chromosomal domains of distinct gene activities. Transcriptionally silent chromatin chromatin is found in subtelomeric regions leading to telomeric position effect (TPE) in yeast, fly and man. Silent chromatin generally initiates at defined loci and tends to propagate from those sites by self-recruitment mechanisms implying the requirement for processes preventing ectopic spreading of silencing. Barrier elements that can block the spread of silent chromatin have been documented, but their relative efficiency is not known. Here we explore the dose-dependency of silencing factors for the extent of TPE in budding yeast. We characterized genome wide the impact of overexpressing the silencing factors Sir2 and Sir3 on the spreading of Sir3 and its impact on coding and non-coding transcription. We thus reveal that extension of silent domains can reach saturation. Analysis of published data sets enabled to uncover that the extension of Sir3 bound domains stops at zones corresponding to transitions of specific histone marks including H3K79 methylation that is deposited by the conserved enzyme Dot1. Importantly, DOT1 is essential for viability when Sir3 is in excess indicating that this transition actively block Sir3 spreading. Our work uncovers previously uncharacterized discrete chromosomal domains associated with specific chromatin features and demonstrates that TPE is efficiently restricted to subtelomeres by the preexisting chromatin landscape.

Background Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis. Results The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. Conclusions These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention. Electronic supplementary material The online version of this article (10.1186/s12864-018-4764-0) contains supplementary material, which is available to authorized users.

International audience

Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF-1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. Here, we investigated the role of CAF-1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. We depleted P180, the large subunit of CAF-1, in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I-element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, our work demonstrates that chromatin dynamics mediated by CAF-1 play an important role in both the regulation of stem cell identity and genome integrity.

Correction du nom : Céline Adam et non pas Cécile Adam; International audience; Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLb complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLb preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLb is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

During multi-cellular development, highly reproducible gene expression patterns determine cellular fates precisely in time and space. These processes are crucial during the earliest stages when the body plan and the future asymmetric body axes emerge at gastrulation. In some species, such as flies and worms, these early processes achieve near-single-cell spatial precision, even for macroscopic patterns. However, we know little about such accuracy in mammalian development, where quantitative approaches are limited. Using an in vitro model for mammalian development, i.e., gastruloids, we demonstrate that gene expression patterns are reproducible to within 20% in protein concentration variability, which translates to a positional error close to a single cell diameter at the tissue scale. In addition, 2-3 fold system size changes lead to scaled gene expression patterns again on the order of an individual cell diameter. Our results reveal developmental precision, reproducibility, and size scaling for mammalian systems. All three properties spontaneously arise in self-organizing cell aggregates and could thus be fundamental features of multicellularity.

International audience; The physical impact of the nucleus on cellular function becomes evident during migration in 3-D environments. With its large volume and relative rigidity, governed by the nuclear envelope proteins and chromatin organization, the nucleus acts as physical barrier, particularly relevant to immune cells and invading cancer cells. These must move through tissue pores and clefts often smaller than the size of the nucleus, which induce substantial compressive and shearing stresses, and may also lead to the temporary rupture of the nuclear envelope. Recent studies amply demonstrated that extreme nuclear deformations during confined migration can lead to DNA damage and increased genomic instability in cancer cells. Here we shed a first light on the molecular processes of stress transfer and relaxation down the scale of the individual chromatin units, the nucleosomes. In this talk, we will briefly outline our innovative experimental techniques aimed at measuring and biophysical signatures of cancer cells, notably single-cell MEMS nano-tweezers that provide high sensitivity to examine different biophysical properties (size, stiffness, viscosity, etc.), and high-throughput MEMS devices oriented at clinical applications. Then, by using molecular dynamics simulations of force-induced poly-nucleosome deformation under ideally controlled conditions, we show that external forces acting on the nucleosome core particle transmit a mechanical stress, which is mainly translated as elastic energy stored in the elastic and plastic response of DNA. The ability of the double-stranded DNA helix to absorb and release this stress, most notably in the form of bending and twisting deformations, may constitute a platform to elicit or repress the interaction with remodeler proteins, by controlling their access to active histone domains. The concerted actions of mechanical deformation and remodeler enzymes open the way for a new framework, to understand the microscopic control of chromatin organization by mechanical forces, and the attending modifications of gene expression and transcription factor activity.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level. ispartof: BMC Genomics vol:15 issue:1 pages:487-487 ispartof: location:England status: published

International audience; Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing minor roles for cross-links and topological effects and challenging the view that interphase chromatin is a gel-like material. Our technology opens avenues for future research in areas from chromosome mechanics to genome functions.

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an “epigenetic checkpoint” for tumor immunity.

International audience; In mammals, CENP-A, a histone H3 variant found in the centromeric chromatin, is critical for faithful chromosome segregation and genome integrity maintenance through cell divisions. Specifically, it has dual functions, enabling to define epigenetically the centromere position and providing the foundation for building up the kinetochore. Regulation of its dynamics of synthesis and deposition ensures to propagate proper centromeres on each chromosome across mitosis and meiosis. However, CENP-A overexpression is a feature identified in many cancers. Importantly, high levels of CENP-A lead to its mislocalization outside the centromere. Recent studies in mammals have begun to uncover how CENP-A overexpression can affect genome integrity, reprogram cell fate and impact 3D nuclear organization in cancer. Here, we summarize the mechanisms that orchestrate CENP-A regulation. Then we review how, beyond its centromeric function, CENP-A overexpression is linked to cancer state in mammalian cells, with a focus on the perturbations that ensue at the level of chromatin organization. Finally, we review the clinical interest for CENP-A in cancer treatment.

For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid’s partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

International audience; The primary functions of the eukaryotic nucleus as a site for the storage, retrieval, and replication of information require a highly dynamic chromatin organization, which can be affected by the presence of DNA damage. In response to double-strand breaks (DSBs), the mobility of chromatin at the break site is severely affected and, to a lesser extent, that of other chromosomes. The how and why of such movement has been widely studied over the last two decades, leading to different mechanistic models and proposed potential roles underlying both local and global mobility. Here, we review the state of the knowledge on current issues affecting chromatin mobility upon DSBs, and highlight its role as a crucial step in the DNA damage response (DDR).

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain., Author summary Multicellular development depends on a tight control of gene expression in each cell type. This relies on the coordinated activities of nuclear proteins that interact with DNA or its histone scaffold to promote or restrict gene transcription. For example, we previously showed that the histone modifying enzymes Lid and Sin3A/Rpd3 are required in Drosophila ovaries for the massive expression of deadhead (dhd), a gene encoding for a thioredoxin that is essential for fertility. In this paper, we have further identified two additional dhd regulators, Snr1 and Mod(mdg4) and dissected the mechanism behind hyperactivation of this gene. Using the epigenomic profiling method Cut&Run with a dedicated data analysis approach, we unexpectedly found that dhd is embedded in an unusual chromatin mini-domain featuring repressive histone modifications H3K27me3 and H3K9me3 and flanked by two regulatory elements. However, we further showed that Lid, Sin3A, Snr1 and Mod(mdg4) behave like obligatory activators of dhd independently of this mini-domain. Our study unveils how multiple broad-acting epigenomic effectors operate in non-canonical manners to ensure a critical and specialized gene activation event. These findings challenge our knowledge on these regulatory mechanisms and their roles in development and pathology.

How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process.

International audience; In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

International audience; Histone chaperones are key regulators of chromatin structure and function. Their frequent mis-regulation in various cancers can impact tumor initiation and progression. Here, we focus on H3-H4 histone chaperones to highlight recent studies concerning their roles in several cancers thereby expanding on previous reports illustrating their functions as tumor-promoting and/or as useful biomarkers for clinical applications. In particular, we discuss how imperfect compensation between H3-H4 histone chaperones favors tumor progression by stimulating the Epithelial mesenchymal transition (EMT) or the Alternative lengthening of telomeres (ALT) pathway. Finally, we present initial studies pointing towards therapies that target H3-H4 histone chaperones for cancer treatment.; Les chaperons d'histone sont des régulateurs clés de la structure et de la fonction de la chromatine. Leur mauvaise régulation fréquente dans divers cancers peut avoir un impact sur l'initiation et la progression de la tumeur. Ici, nous nous concentrons sur les chaperons d'histones H3-H4 pour mettre en évidence des études récentes concernant leurs rôles dans plusieurs cancers, développant ainsi des rapports antérieurs illustrant leurs fonctions de promotion de tumeurs et/ou de biomarqueurs utiles pour des applications cliniques. En particulier, nous discutons comment la compensation imparfaite entre les chaperons d'histones H3-H4 favorise la progression tumorale en stimulant la transition mésenchymateuse épithéliale (EMT) ou la voie d'allongement alternatif des télomères (ALT). Enfin, nous présentons des études initiales pointant vers des thérapies qui ciblent les chaperons d'histone H3-H4 pour le traitement du cancer.

The eukaryotic genome is highly organized in both space and sequence. From entire chromosomes to individual genes the 3D organization of the genome is linked to transcription and many regulatory mechanisms likely coexist at different scales. At the sub-megabase scale, the genome is physically organized into self-interacting topologically associating domains (TADs) that are thought to constrain the range of action of gene regulatory elements called ‘enhancers’. Current data suggest that TADs serve as ‘regulatory units’ to coregulate multiple genes by exposing them to the same enhancers. Genes from the same TAD indeed often display correlated expression across different tissues and cell types. Interestingly, correlated expression is seen between functionally related genes. However, how 3D organization at an individual locus plays a mechanistic role in coregulating functionally related genes is unknown. Using single-molecule imaging, I observed in single cells the spatial positions and transcription of three adjacent functionally related genes regulated by the same/different enhancers. I used estrogen stimulation in MCF7 cells as a model system to study hormone-responsive genes and enhancers. Using combined RNA-DNA FISH, I measured the coupling between genes as the correlation in cis of their transcription. I found, that stimulation with estrogen increases the correlation in cis between genes belonging to the same TAD. Perturbation of the TAD boundary revealed the contribution of contact insulation to gene coregulation. Together, this work lays the ground towards an understanding of how enhancers and genes communicate and coordinate their activity within the 3D genome., Le génome eucaryote est organisé dans l’espace et le long de sa séquence. Du chromosome aux gènes, son organisation 3D est étroitement liée à son activité transcriptionnelle. A une échelle inférieure à la mégabase, le génome est organisé physiquement en domaines d’auto-interaction (TAD, pour topologically associating domains), que l’on pense influencer l’action de séquences régulatrices des gènes, appelées ‘amplificateurs’. Les TAD sont parfois vus comme des ‘unités de régulation’ permettant de coréguler plusieurs gènes en les exposant aux mêmes amplificateurs. L’expression de gènes d’un même TAD est en effet souvent corrélée entre tissus et types cellulaires. On sait d’autre part que l’expression de gènes fonctionnellement liés est aussi souvent corrélée. Cependant, le rôle que joue l’organisation 3D d’un locus génomique dans la corégulation de gènes fonctionnellement liés reste mal compris. Par des techniques d’imagerie de molécules uniques, j’ai observé, dans des cellules individuelles, la position et la transcription de trois gènes adjacents et fonctionnellement liés, partageant –ou non– les mêmes amplificateurs. J’ai utilisé comme système modèle la réponse à l’œstrogène dans des cellules MCF7. En combinant les techniques de FISH ARN et ADN, j’ai mesuré le couplage transcriptionnel de paires de gènes en cis, mettant en évidence une augmentation de la corrélation entre gènes d’un même TAD en réponse à l’œstrogène. Une perturbation de la frontière entre TAD indique également le rôle des contacts génomiques dans la corégulation de gènes. Ces travaux posent les bases pour comprendre comment les amplificateurs et les gènes communiquent et coordonnent leur activité en 3D.

International audience; One striking feature of eukaryotic nuclei is the existence of discrete regions, in which specific factors concentrate while others are excluded, thus forming microenvironments with different molecular compositions and biological functions. These domains are often referred to as subcompartments even though they are not membrane enclosed. Despite their functional importance the physical nature of these structures remains largely unknown. Here, we describe how the Saccharomyces cerevisiae nucleus is compartmentalized and discuss possible physical models underlying the formation and maintenance of chromatin associated subcompartments. Focusing on three particular examples, the nucleolus, silencing foci, and repair foci, we discuss the biological implications of these different models as well as possible approaches to challenge them in living cells.

International audience; While major advances in single cell transcriptomics have occurred, the epigenomics arena has only more recently gained momentum. Indeed, methods to track changes in different molecules present in biological samples have developed both in scale – providing a global view in each case – and in terms of resolution, allowing us today to disentangle heterogeneity within samples and access at the level of a single cell. The application of omics methods, comprising analysis of genomes, epigenomes, transcriptomes, proteomes and metabolomes, among other functional genomics techniques, individually or in combination, provides a wealth of valuable datasets. The treatment of these data with the development of artificial intelligence tools opens unprecedented avenues. The possibility to gain a holistic view of cells functioning within organs and during their formation will allow us to tackle fundamental questions of normal organism development and also to reveal early changes associated with disease onset and progression. In this editorial, we discuss the place of epigenomics research in an era where mapping cellular trajectories and mechanisms offers unique opportunities to address fundamental questions related to cell identity of key importance for medical applications.

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we present a novel approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observe viscoelastic displacements over microns within minutes in response to near-picoNewton forces, which are well captured by a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing the minor role of crosslinks and topological effects and challenging the view that interphase chromatin is a gel-like material. Our new technology opens avenues for future research, from chromosome mechanics to genome functions.

The kinetochore is a multiprotein complex that assembles on centromeric DNA and enables chromosome segregation during cell division. Essential for kinetochore assembly in most species is the histone variant CenH3 that initiates complex formation. Unexpected to this essential role, it was previously shown that CenH3 was lost in multiple insect orders including the Lepidoptera (butterflies, moths). Despite this loss, recent studies from my host lab revealed that other kinetochore homologs are present in Lepidoptera and must therefore be recruited in a different way. For my PhD I aimed to characterize the assembly of these CenH3-deficientt kinetochores in Bombyx mori cells. In order to address my aim, during the 1st and 2nd year of my PhD, I established tools and protocols in these cells with sparse experimental history. Those include the optimization of RNAI-mediated depletion, characterization of mitotic phenotypes, verification of antibodies and the generation of cell lines containing lacO arrays to able to study kinetochore assembly at ectopic sites. My studies focused on one particular kinetochore component, CENP-T that in other organisms makes important contributions to connect the kinetochore to DNA and to the spindle microtubules. Using the tools that I have generated, I could show that the B. mori CENP-T is essential for mitotic progression and both necessary and sufficient to recruit components to interact with the spindle.; Le kinétochore est un complexe multiprotéique qui s'assemble sur l'ADN centromérique et permet la ségrégation des chromosomes lors de la division cellulaire. Le variant d'histone CenH3 est essentiel pour l'assemblage des kinétochores dans la plupart des espèces où il initie leurs formation. De façon inattendue, compte tenu de leur rôle essentiel, il a été précédemment montré que CenH3 avait été perdu dans plusieurs ordres d'insectes dont les lépidoptères. Malgré cette perte, des études récentes au laboratoire ont révélé que d'autres composants du kinétochore ont des homologues chez les lépidoptères et doivent donc être recrutés de manière différente. Ma thèse a eu pour objectif de comprendre l'assemblage des kinétochores en absence de CenH3 dans des cellules de Bombyx mori. Pour cela, j'ai mis en place des outils dans ces cellules à l'histoire expérimentale sporadique. Par exemple: la déplétion d'un gène par ARNi et la validation des anticorps et la génération de lignées cellulaires contenant des répétitions lacO pour pouvoir étudier l'assemblage des kinétochores sur des sites ectopiques. Mes études se sont concentrées sur un composant particulier du kinétochore, CENP-T, qui dans d'autres organismes, contribue à la liaison du kinétochore à I'ADN et aux microtubules du fuseau. En utilisant les outils que j'ai générés, j'ai pu montrer que B. mori CENP-T est essentiel à la progression mitotique et à la fois nécessaire et suffisant pour recruter les composants interagissants avec le fuseau. Pris ensemble, les résultats de mon projet de doctorat généreront un nouveau système modèle pour l'étude de l'assemblage des kinétochores indépendants de CenH3.

The kinetochore is a multiprotein complex that assembles on centromeric DNA and enables chromosome segregation during cell division. Essential for kinetochore assembly in most species is the histone variant CenH3 that initiates complex formation. Unexpected to this essential role, it was previously shown that CenH3 was lost in multiple insect orders including the Lepidoptera (butterflies, moths). Despite this loss, recent studies from my host lab revealed that other kinetochore homologs are present in Lepidoptera and must therefore be recruited in a different way. For my PhD I aimed to characterize the assembly of these CenH3-deficientt kinetochores in Bombyx mori cells. In order to address my aim, during the 1st and 2nd year of my PhD, I established tools and protocols in these cells with sparse experimental history. Those include the optimization of RNAI-mediated depletion, characterization of mitotic phenotypes, verification of antibodies and the generation of cell lines containing lacO arrays to able to study kinetochore assembly at ectopic sites. My studies focused on one particular kinetochore component, CENP-T that in other organisms makes important contributions to connect the kinetochore to DNA and to the spindle microtubules. Using the tools that I have generated, I could show that the B. mori CENP-T is essential for mitotic progression and both necessary and sufficient to recruit components to interact with the spindle., Le kinétochore est un complexe multiprotéique qui s'assemble sur l'ADN centromérique et permet la ségrégation des chromosomes lors de la division cellulaire. Le variant d'histone CenH3 est essentiel pour l'assemblage des kinétochores dans la plupart des espèces où il initie leurs formation. De façon inattendue, compte tenu de leur rôle essentiel, il a été précédemment montré que CenH3 avait été perdu dans plusieurs ordres d'insectes dont les lépidoptères. Malgré cette perte, des études récentes au laboratoire ont révélé que d'autres composants du kinétochore ont des homologues chez les lépidoptères et doivent donc être recrutés de manière différente. Ma thèse a eu pour objectif de comprendre l'assemblage des kinétochores en absence de CenH3 dans des cellules de Bombyx mori. Pour cela, j'ai mis en place des outils dans ces cellules à l'histoire expérimentale sporadique. Par exemple: la déplétion d'un gène par ARNi et la validation des anticorps et la génération de lignées cellulaires contenant des répétitions lacO pour pouvoir étudier l'assemblage des kinétochores sur des sites ectopiques. Mes études se sont concentrées sur un composant particulier du kinétochore, CENP-T, qui dans d'autres organismes, contribue à la liaison du kinétochore à I'ADN et aux microtubules du fuseau. En utilisant les outils que j'ai générés, j'ai pu montrer que B. mori CENP-T est essentiel à la progression mitotique et à la fois nécessaire et suffisant pour recruter les composants interagissants avec le fuseau. Pris ensemble, les résultats de mon projet de doctorat généreront un nouveau système modèle pour l'étude de l'assemblage des kinétochores indépendants de CenH3.

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here, we investigate the well-conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3–5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing SIR complex composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long-range contacts between distant regions, including the rDNA, telomeres, and internal Sir3-bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach, we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, Sir4), Sir2 activity, or chromosome context. Altogether, these data suggest that Sir3 acts as a molecular bridge that stabilizes long-range interactions.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.; In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.

International audience; The lack of a consensus DNA sequence defining replication origins in mammals has led researchers to consider chromatin as a means to specify these regions. However, to date, there is no mechanistic understanding of how this could be achieved and maintained given that nucleosome disruption occurs with each fork passage and with transcription. Here, by genome-wide mapping of the de novo deposition of the histone variants H3.1 and H3.3 in human cells during S phase, we identified how their dual deposition mode ensures a stable marking with H3.3 flanked on both sides by H3.1. These H3.1/H3.3 boundaries correspond to the initiation zones of early origins. Loss of the H3.3 chaperone HIRA leads to the concomitant disruption of H3.1/H3.3 boundaries and initiation zones. We propose that the HIRA-dependent deposition of H3.3 preserves H3.1/H3.3 boundaries by protecting them from H3.1 invasion linked to fork progression, contributing to a chromatin-based definition of early replication zones.; L'absence d'une séquence d'ADN consensuelle définissant les origines de réplication chez les mammifères a conduit les chercheurs à considérer la chromatine comme un moyen de spécifier ces régions. Cependant, à ce jour, il n'y a pas de compréhension mécaniste de la façon dont cela pourrait être réalisé et maintenu étant donné que la rupture des nucléosomes se produit à chaque passage de fourche et avec la transcription. Ici, en cartographiant à l'échelle du génome le dépôt de novo des variants d'histone H3.1 et H3.3 dans les cellules humaines pendant la phase S, nous avons identifié comment leur mode de dépôt double assure un marquage stable avec H3.3 flanqué des deux côtés par H3.1. Ces frontières H3.1/H3.3 correspondent aux zones d'initiation des origines précoces. La perte du chaperon H3.3 HIRA conduit à la perturbation concomitante des frontières H3.1/H3.3 et des zones d'initiation. Nous proposons que le dépôt de H3.3 dépendant de HIRA préserve les frontières H3.1/H3.3 en les protégeant de l'invasion de H3.1 liée à la progression des fourches, contribuant ainsi à une définition chromatinienne des zones de réplication précoce.

Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer., L'inactivation de Polybromo 1 (PBRM1), une sous-unité spécifique du complexe de remodelage de la chromatine PBAF, se produit fréquemment dans le cancer, y compris dans 40% des carcinomes rénaux à cellules claires (ccRCC). Afin d'identifier de nouvelles approches thérapeutiques pour cibler les cancers déficients en PBRM1, nous avons utilisé une série de cribles génomiques fonctionnels orthogonaux qui ont identifié les inhibiteurs PARP et ATR comme étant synthétiquement létaux en cas de déficience en PBRM1. La létalité synthétique des inhibiteurs de PBRM1/PARP a été récapitulée en utilisant plusieurs inhibiteurs cliniques de PARP dans une série de systèmes modèles in vitro et in vivo dans un modèle de xénogreffe de ccRCC. En l'absence de lésions exogènes de l'ADN, les cellules déficientes en PBRM1 présentaient des niveaux élevés de stress de réplication, de micronoyaux et de boucles R. L'exposition à un inhibiteur de PARP a exacerbé la létalité synthétique. L'exposition à un inhibiteur de PARP a exacerbé ces phénotypes. La spectrométrie de masse quantitative a révélé que plusieurs facteurs de traitement des boucles R étaient régulés à la baisse dans les cellules tumorales défectueuses de PBRM1. L'expression exogène de l'enzyme de résolution des boucles R, la RNase H1, a inversé la sensibilité des cellules PBRM1 déficientes aux inhibiteurs de la PARP, ce qui suggère que des niveaux excessifs de boucles R pourraient être une cause de cette létalité synthétique. Les inhibiteurs de PARP et d'ATR ont également induit une signalisation immunitaire innée de type GMP cyclique-AMP synthase/stimulateur des gènes de l'interféron (cGAS/STING) dans les cellules tumorales déficientes en PBRM1. Dans l'ensemble, ces résultats fournissent une base préclinique pour l'utilisation des inhibiteurs de PARP dans les cancers déficients en PBRM1. SIGNIFICATION : Cette étude démontre que les inhibiteurs de PARP et d'ATR sont synthétiquement létaux en cas de perte de PBRM1, une sous-unité spécifique du PBAF, ce qui justifie l'évaluation de ces inhibiteurs chez les patients atteints d'un cancer déficient en PBRM1.

Despite their essentiality for chromosome segregation, centromeres are diverse among eukaryotes and embody two main configurations: mono- and holocentromeres, referring respectively to a localized or unrestricted distribution of centromeric activity. Previous studies revealed that holocentricity in many insects coincides with the loss of the otherwise essential centromere component CenH3 (CENP-A), suggesting a molecular link between the two events. In this study, we leveraged recently-identified centromere components to map and characterize the centromeres of Bombyx mori. This uncovered a robust correlation between centromere profiles and regions of low chromatin dynamics. Transcriptional perturbation experiments showed that low chromatin activity is crucial for centromere formation in B. mori. Our study points to a novel mechanism of centromere formation that occurs in a manner recessive to the chromosome-wide chromatin landscape. Based on similar profiles in additional Lepidoptera, we propose an evolutionarily conserved mechanism that underlies the establishment of holocentromeres through loss of centromere specificity.

The closely related replicative H3 and non-replicative H3.3 variants show specific requirement during development in vertebrates. Whether it involves distinct mode of deposition or unique roles once incorporated into chromatin remains unclear. To disentangle the two aspects, we took advantage of the Xenopus early development combined with chromatin assays. Our previous work showed that in Xenopus, depletion of the non-replicative variant H3.3 impairs development at gastrulation, without compensation through provision of the replicative variant H3.2. We systematically mutated H3.3 at each four residues that differ from H3.2 and tested their ability to rescue developmental defects. Surprisingly, all H3.3 mutated variants functionally complemented endogenous H3.3, regardless of their incorporation pathways, except for one residue. This particular residue, the serine at position 31 in H3.3, gets phosphorylated onto chromatin in a cell cycle dependent manner. While the alanine substitution failed to rescue H3.3 depletion, a phosphomimic residue sufficed. We conclude that the time of gastrulation reveals a critical importance of the H3.3S31 residue independently of the variant incorporation pathway. We discuss how this single evolutionary conserved residue conveys a unique property for this variant in vertebrates during cell cycle and cell fate commitment.; Les variantes H3 réplicative et H3.3 non réplicative, étroitement liées, présentent des besoins spécifiques au cours du développement chez les vertébrés. On ne sait pas encore s'il s'agit d'un mode de dépôt distinct ou de rôles uniques une fois incorporés dans la chromatine. Pour démêler ces deux aspects, nous avons tiré parti du développement précoce de Xenopus combiné à des tests de chromatine. Nos travaux antérieurs ont montré que chez le Xénopus, l'épuisement du variant non réplicatif H3.3 entrave le développement à la gastrulation, sans compensation par la fourniture du variant réplicatif H3.2. Nous avons systématiquement muté H3.3 à chaque quatre résidus qui diffèrent de H3.2 et avons testé leur capacité à remédier aux défauts de développement. De manière surprenante, toutes les variantes mutées H3.3 ont fonctionnellement complété le H3.3 endogène, quelle que soit leur voie d'incorporation, à l'exception d'un résidu. Ce résidu particulier, la sérine en position 31 dans H3.3, est phosphorylé sur la chromatine de manière dépendante du cycle cellulaire. Bien que la substitution par l'alanine n'ait pas permis de remédier à l'appauvrissement de H3.3, un résidu phosphomimique a suffi. Nous concluons que le moment de la gastrulation révèle une importance critique du résidu H3.3S31 indépendamment de la voie d'incorporation du variant. Nous discutons de la façon dont ce résidu unique conservé au cours de l'évolution confère une propriété unique à cette variante chez les vertébrés pendant le cycle cellulaire et l'engagement du destin cellulaire.

Polybromo 1 (PBRM1), a subunit of the PBAF chromatin-remodeling complex, is recurrently inactivated in cancer. To uncover novel synthetic lethal (SL) approaches for targeting PBRM1-defective cancers, we used a series of orthogonal functionalgenomic screens which identified SL between PBRM1 deficiency and inhibition of PARP1. This effect was elicited with clinical PARP inhibitors in vitro and in vivo and also operated when PARP inhibitors were combined with ATR inhibitors. These SL effects were characterized by pre-existing replication stress in PBRM1-defective cells and an increase in R-loops, micronuclei and tumor cell-autonomous cGAS/STING signaling in response to PARP inhibitors. Quantitative mass spectrometry indicated that PBRM1-defective cells exhibited downregulation of multiple R-loop processing factors, and exogenous expression of the R-loop resolution enzyme RNase H1 partially reversed PARP inhibitor sensitivity, providing a mechanistic rationale for the SL effects. Our data provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers.; Le polybromo 1 (PBRM1), une sous-unité du complexe PBAF remodelant la chromatine, est inactivé de manière récurrente dans le cancer. Pour découvrir de nouvelles approches létales synthétiques (SL) pour cibler les cancers défectueux PBRM1, nous avons utilisé une série de tests fonctionnels orthogonaux qui a permis d'identifier la SL entre la déficience en PBRM1 et l'inhibition de la PARP1.Cet effet a été obtenu avec des inhibiteurs cliniques de PARP in vitro et in vivo et a également fonctionné lorsque les inhibiteurs PARP ont été combinés avec les inhibiteurs ATR. Ces effets SL étaient caractérisés par un stress de réplication préexistant dans les cellules déficientes en PBRM1 et une augmentation des boucles R, des micronoyaux et des cellules tumorales - cGAS/STING autonomes en réponse aux inhibiteurs PARP. La spectrométrie de masse quantitative a indiqué que les cellules défectueuses PBRM1 présentaient une régulation à la baisse du traitement en boucle R multiple et l'expression exogène de l'enzyme de résolution de la boucle R, la RNase H1 a partiellement inversé la sensibilité aux inhibiteurs PARP, ce qui fournit une justification mécaniste des effets SL. Nos données fournissent la base préclinique de l'utilisation des inhibiteurs PARP dans les PBRM1- les cancers défectueux.

Tumor-infiltrating CD8+ T cells progressively lose functionality and fail to reject tumors. Theunderlying mechanism and re-programing induced by checkpoint blockers are incompletelyunderstood. We show that genetic ablation or pharmacological inhibition of H3K9-methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In theabsence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival anddifferentiation of IFN-γ and GZMb-producing effector cells that express negativecheckpoints, but do not reach final exhaustion. Their transcriptional program correlates withthat of melanoma patients responding to immune-checkpoint blockade and identifies theemergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinicalresponse. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory andpluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effectorloci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combinedwith anti-PD-1, suggesting that Suv39h1 is an “epigenetic checkpoint” for tumor immunity.; Les cellules T CD8+ qui infiltrent les tumeurs perdent progressivement leur fonctionnalité et ne parviennent pas à rejeter les tumeurs. Le sitele mécanisme sous-jacent et la reprogrammation induite par les bloqueurs de points de contrôle sont incompletscompris. Nous montrons que l'ablation génétique ou l'inhibition pharmacologique du H3K9-méthyleLa transférase Suv39h1 retarde la croissance de la tumeur et potentialise le rejet de la tumeur par l'anti-PD-1. Dans lel'absence de Suv39h1, l'anti-PD-1 induit des voies d'activation alternatives permettant la survie etla différenciation des cellules effectrices productrices d'IFN-γ et de GZMb qui expriment desmais n'atteignent pas l'épuisement final. Leur programme de transcription est en corrélation aveccelle des patients atteints de mélanome répondant au blocage des points de contrôle immunitaire et identifie lesl'émergence de lymphocytes infiltrant les tumeurs à effecteur cytolytique comme biomarqueur desréponse. L'anti-PD-1 favorise l'ouverture de la chromatine dans les loci liés à l'activation des cellules T, à la mémoire etpluripotence, mais en l'absence de Suv39h1, les cellules acquièrent l'accessibilité dans l'effecteur cytolytiqueloci. Globalement, l'inhibition du Suv39h1 renforce les réponses immunitaires anti-tumorales, seules ou combinéesavec l'anti-PD-1, ce qui suggère que le Suv39h1 est un "point de contrôle épigénétique" pour l'immunité aux tumeurs.

Tumor-infiltrating CD8+ T cells progressively lose functionality and fail to reject tumors. Theunderlying mechanism and re-programing induced by checkpoint blockers are incompletelyunderstood. We show that genetic ablation or pharmacological inhibition of H3K9-methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In theabsence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival anddifferentiation of IFN-γ and GZMb-producing effector cells that express negativecheckpoints, but do not reach final exhaustion. Their transcriptional program correlates withthat of melanoma patients responding to immune-checkpoint blockade and identifies theemergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinicalresponse. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory andpluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effectorloci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combinedwith anti-PD-1, suggesting that Suv39h1 is an “epigenetic checkpoint” for tumor immunity.; Les cellules T CD8+ qui infiltrent les tumeurs perdent progressivement leur fonctionnalité et ne parviennent pas à rejeter les tumeurs. Le sitele mécanisme sous-jacent et la reprogrammation induite par les bloqueurs de points de contrôle sont incompletscompris. Nous montrons que l'ablation génétique ou l'inhibition pharmacologique du H3K9-méthyleLa transférase Suv39h1 retarde la croissance de la tumeur et potentialise le rejet de la tumeur par l'anti-PD-1. Dans lel'absence de Suv39h1, l'anti-PD-1 induit des voies d'activation alternatives permettant la survie etla différenciation des cellules effectrices productrices d'IFN-γ et de GZMb qui expriment desmais n'atteignent pas l'épuisement final. Leur programme de transcription est en corrélation aveccelle des patients atteints de mélanome répondant au blocage des points de contrôle immunitaire et identifie lesl'émergence de lymphocytes infiltrant les tumeurs à effecteur cytolytique comme biomarqueur desréponse. L'anti-PD-1 favorise l'ouverture de la chromatine dans les loci liés à l'activation des cellules T, à la mémoire etpluripotence, mais en l'absence de Suv39h1, les cellules acquièrent l'accessibilité dans l'effecteur cytolytiqueloci. Globalement, l'inhibition du Suv39h1 renforce les réponses immunitaires anti-tumorales, seules ou combinéesavec l'anti-PD-1, ce qui suggère que le Suv39h1 est un "point de contrôle épigénétique" pour l'immunité aux tumeurs.

Polybromo 1 (PBRM1), a subunit of the PBAF chromatin-remodeling complex, is recurrently inactivated in cancer. To uncover novel synthetic lethal (SL) approaches for targeting PBRM1-defective cancers, we used a series of orthogonal functionalgenomic screens which identified SL between PBRM1 deficiency and inhibition of PARP1. This effect was elicited with clinical PARP inhibitors in vitro and in vivo and also operated when PARP inhibitors were combined with ATR inhibitors. These SL effects were characterized by pre-existing replication stress in PBRM1-defective cells and an increase in R-loops, micronuclei and tumor cell-autonomous cGAS/STING signaling in response to PARP inhibitors. Quantitative mass spectrometry indicated that PBRM1-defective cells exhibited downregulation of multiple R-loop processing factors, and exogenous expression of the R-loop resolution enzyme RNase H1 partially reversed PARP inhibitor sensitivity, providing a mechanistic rationale for the SL effects. Our data provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers.; Le polybromo 1 (PBRM1), une sous-unité du complexe PBAF remodelant la chromatine, est inactivé de manière récurrente dans le cancer. Pour découvrir de nouvelles approches létales synthétiques (SL) pour cibler les cancers défectueux PBRM1, nous avons utilisé une série de tests fonctionnels orthogonaux qui a permis d'identifier la SL entre la déficience en PBRM1 et l'inhibition de la PARP1.Cet effet a été obtenu avec des inhibiteurs cliniques de PARP in vitro et in vivo et a également fonctionné lorsque les inhibiteurs PARP ont été combinés avec les inhibiteurs ATR. Ces effets SL étaient caractérisés par un stress de réplication préexistant dans les cellules déficientes en PBRM1 et une augmentation des boucles R, des micronoyaux et des cellules tumorales - cGAS/STING autonomes en réponse aux inhibiteurs PARP. La spectrométrie de masse quantitative a indiqué que les cellules défectueuses PBRM1 présentaient une régulation à la baisse du traitement en boucle R multiple et l'expression exogène de l'enzyme de résolution de la boucle R, la RNase H1 a partiellement inversé la sensibilité aux inhibiteurs PARP, ce qui fournit une justification mécaniste des effets SL. Nos données fournissent la base préclinique de l'utilisation des inhibiteurs PARP dans les PBRM1- les cancers défectueux.

International audience; Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns., The boundaries of topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. Here the authors find that CTCF positions cohesin through its N-terminus but does not control its overall binding dynamics on chromatin, and show how the orientation of CTCF binding sites translates into genome folding patterns.

International audience; Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells. We uncovered a transcription-dependent loss of old histone variants H3.1 and H3.3. At transcriptionally active domains, H3.3 enrichment reflected both old H3.3 retention and new deposition. Mechanistically, we found that the histone regulator A (HIRA) chaperone is critical to processing both new and old H3.3 via different pathways. De novo H3.3 deposition is totally dependent on HIRA trimerization as well as on its partner ubinuclein 1 (UBN1), while antisilencing function 1 (ASF1) interaction with HIRA can be bypassed. By contrast, recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows absolute dependency on ASF1–HIRA interaction. We propose a model whereby HIRA coordinates these distinct pathways during transcription to fine-tune chromatin states.; Les nucléosomes représentent un défi en ce qui concerne la transcription. L'éviction des histones permet la progression de l'ARN polymérase II (RNAPII) dans l'ADN, mais compromet l'intégrité de la chromatine. Ici, nous avons utilisé le système SNAP-tag pour distinguer les nouvelles et les anciennes histones et surveiller le réassemblage de la chromatine couplé à la transcription dans les cellules humaines. Nous avons découvert une perte dépendante de la transcription des anciens variants d'histones H3.1 et H3.3. Au niveau des domaines transcriptionnellement actifs, l'enrichissement en H3.3 reflétait à la fois la rétention de l'ancien H3.3 et le nouveau dépôt. Sur le plan mécanique, nous avons constaté que le chaperon du régulateur d'histone A (HIRA) est essentiel pour traiter à la fois le nouveau et l'ancien H3.3 par des voies différentes. Le dépôt de novo de H3.3 dépend totalement de la trimérisation de l'HIRA ainsi que de son partenaire l'ubinucléine 1 (UBN1), tandis que l'interaction de la fonction antisilencing 1 (ASF1) avec l'HIRA peut être contournée. En revanche, le recyclage du H3.3 nécessite l'AERH mais se fait indépendamment de la trimérisation de l'UBN1 ou de l'AERH et montre une dépendance absolue de l'interaction ASF1-AERH. Nous proposons un modèle dans lequel l'ARHI coordonne ces voies distinctes pendant la transcription pour affiner les états de la chromatine.

Despite their essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes and embody two main configurations: mono- and holocentromeres, referring respectively to a localized or unrestricted distribution of centromeric activity. Previous studies revealed that holocentricity in many insects strongly coincides with the loss of the otherwise essential centromeric marker CenH3 (CENP-A), suggesting a molecular link between the two events. In this study, we leveraged recently- identified centromere components and genomics approaches to map and characterize the centromeres of the silk moth Bombyx mori, a holocentric insect representative lacking CenH3. This uncovered a robust correlation between centromere profiles and regions of low chromatin dynamics found anywhere along the chromosome. Transcriptional perturbation experiments showed that centromeres become excluded from active chromatin regions but can form de novo in regions where chromatin activity is low. The identified link to chromatin dynamics helps to discuss the plasticity of centromere identity. In this context, our study points to a novel mechanism of centromere formation that occurs in a manner recessive to the chromosome-wide chromatin landscape rather than being defined by the presence of CenH3. Based on similar profiles observed in additional Lepidoptera, we propose an evolutionarily conserved mechanism that underlies the establishment of holocentromeres through loss of a specified centromere., Malgré leur caractère essentiel pour une ségrégation fidèle des chromosomes, il existe diverses architectures de centromères parmi les eucaryotes représentés en deux principales configurations : mono- et holocentromères pour, respectivement une distribution localisée ou non localisée de l’activité centromérique. De précédentes études ont révélé que l’holocentrisme chez de nombreux insectes coïncide fortement avec la perte du marqueur centromérique – par ailleurs essentiel – CenH3 (CENP-A), suggérant un lien moléculaire entre les deux évènements. Nous avons exploité des éléments de centromères récemment identifiés ainsi que des approches génomiques pour cartographier et caractériser les centromères du vers à soie Bombyx mori, un insecte représentatif holocentrique dépourvu de CenH3. Cela a révélé une corrélation robuste entre les profils de centromères et les régions de faible dynamique de la chromatine trouvée n’importe où le long du chromosome. Des expériences de perturbation transcriptionnelle ont montré que les centromères sont exclus des régions actives de la chromatine, mais peuvent se former de novo dans les régions où l’activité de la chromatine est faible. Le lien identifié avec la chromatine aide à discuter de la plasticité de l’identité du centromère. Dans ce contexte, notre étude pointe vers un nouveau mécanisme de formation des centromères qui se produit de manière récessive sur le chromosome – large paysage chromatinien plutôt que d’être défini par la présence de CenH3. Sur la base de profils similaires observés chez d’autres lépidoptères, nous proposons un mécanisme conservé qui sous-tend l’établissement d’holocentromères par la perte d’un centromère précis.

Tumour evolution is driven by both genetic and epigenetic changes. CENP-A, the centromeric histone H3 variant, is an epigenetic mark that directly perturbs genetic stability and chromatin when overexpressed. Although CENP-A overexpression is a common feature of many cancers, how this impacts cell fate and response to therapy remains unclear. Here, we established a tunable system of inducible and reversible CENP-A overexpression combined with a switch in p53 status in human cell lines. Through clonogenic survival assays and single-cell RNA-sequencing over time, we uncover the tumour suppressor p53 as a key determinant of how CENP-A impacts cell state, cell identity and therapeutic response. If p53 is functional, CENP-A overexpression promotes senescence and radiosensitivity. But, when we inactivate p53, CENP-A overexpression instead promotes epithelial-mesenchymal transition, an essential precursor for tumour cell invasion and metastasis. Thus, CENP-A overexpression drives distinct cell fates depending on p53 status, with important implications for tumour evolution.; L'évolution des tumeurs est déterminée par des changements génétiques et épigénétiques. CENP-A, le variant centromérique de l'histone H3, est une marque épigénétique qui perturbe directement la stabilité génétique et la chromatine lorsqu'elle est surexprimée. Bien que la surexpression de la CENP-A soit une caractéristique commune à de nombreux cancers, l'impact de cette surexpression sur le sort des cellules et la réponse à la thérapie reste incertain. Nous avons donc établi un système accordable de surexpression inductible et réversible de la CENP-A, combiné à un changement de statut de p53 dans les lignées cellulaires humaines. Grâce à des tests de survie clonogénique et au séquençage d'ARN unicellulaire dans le temps, nous découvrons le suppresseur de tumeur p53 comme un déterminant clé de l'impact de la CENP-A sur l'état des cellules, l'identité cellulaire et la réponse thérapeutique. Si p53 est fonctionnel, la surexpression de CENP-A favorise la sénescence et la radiosensibilité. Mais, lorsque nous désactivons p53, la surexpression de CENP-A favorise plutôt la transition épithélio-mésenchymateuse, un précurseur essentiel de l'invasion des cellules tumorales et de la métastase. Ainsi, la surexpression du CENP-A entraîne des destins cellulaires distincts selon le statut de p53, avec des implications importantes pour l'évolution des tumeurs.

International audience; Stem cells can be maintained through symmetric cell divisions (SCDs) and asymmetric cell divisions (ACDs). How and when these divisions occur in vivo in vertebrates is poorly understood. Here, we developed a clonogenic cell tracing method that demonstrates the asymmetric distribution of transcription factors along with old and new DNA in mouse muscle stem cells during skeletal muscle regeneration. Combining single-cell tracking and artificial niches ex vivo, we show how cells switch from ACDs to SCDs, suggesting that they are not engaged in an obligate mode of cell division. Further, we generated SNAP-tagged histone H3-reporter mice and find that, unlike fly germline stem cells, differential fate outcomes are associated with a symmetric distribution of the H3.1 and H3.3 histone variants in mouse muscle stem cells. This versatile and efficient H3-SNAP labeling system will allow an investigation of mechanisms underlying the maintenance of epigenomic identity and plasticity in a variety of tissues.; Les cellules souches peuvent être maintenues par des divisions cellulaires symétriques (DCS) et des divisions cellulaires asymétriques (DCA). On comprend mal comment et quand ces divisions se produisent in vivo chez les vertébrés. Ici, nous avons développé une méthode de traçage cellulaire clonogène qui démontre la distribution asymétrique des facteurs de transcription ainsi que de l'ADN ancien et nouveau dans les cellules souches de muscles de souris pendant la régénération des muscles squelettiques. En combinant le traçage d'une seule cellule et des niches artificielles ex vivo, nous montrons comment les cellules passent des ACD aux SCD, ce qui suggère qu'elles ne sont pas engagées dans un mode de division cellulaire obligatoire. En outre, nous avons généré des souris indicatrices d'histone H3 marquées par SNAP et nous avons découvert que, contrairement aux cellules souches germinales de mouches, les résultats différentiels du destin sont associés à une distribution symétrique des variantes d'histone H3.1 et H3.3 dans les cellules souches musculaires de souris. Ce système de marquage H3-SNAP polyvalent et efficace permettra d'étudier les mécanismes sous-jacents au maintien de l'identité et de la plasticité épigénomiques dans une variété de tissus.

Background Maintaining a proper supply of soluble histones throughout the cell cycle is important to ensure chromatin and genome stability. Following their synthesis, histones undergo a series of maturation steps to prepare them for deposition onto chromatin. Results Here, we identify the lysine demethylase JMJD1B as a novel player in the maturation cascade that contributes to regulate histone provision. We find that depletion of JMJD1B increases the protein levels of the histone chaperone tNASP leading to an accumulation of newly synthesized histones H3 and H4 at early steps of the histone maturation cascade, which perturbs chromatin assembly. Furthermore, we find a high rate of JMJD1B mutations in cancer patients, and a correlation with genomic instability. Conclusions Our data support a role for JMJD1B in fine-tuning histone supply to maintain genome integrity, opening novel avenues for cancer therapeutics.

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis., Epigenetic modifications are a key contributor to cell identity, and their propagation is crucial for proper development. Here the authors use a super-resolution microscopy approach to reveal how histone variants are faithfully transmitted during genome duplication, and reveal an important role for the histone chaperone ASF1 in the redistribution of parental histones.

International audience; During cell division, maintenance of chromatin features from the parental genome requires their proper establishment on its newly synthetized copy. The loss of epigenetic marks within heterochromatin, typically enriched in repetitive elements, endangers genome stability and permits chromosomal rearrangements via recombination. However, how histone modifications associated with heterochromatin are maintained across mitosis remains poorly understood. KAP1 is known to act as a scaffold for a repressor complex that mediates local heterochromatin formation, and was previously demonstrated to play an important role during DNA repair. Accordingly, we investigated a putative role for this protein in the replication of heterochromatic regions. We first found that KAP1 associates with several DNA replication factors including PCNA, MCM3 and MCM6. We then observed that these interactions are promoted by KAP1 phosphorylation on serine 473 during S phase. Finally, we could demonstrate that KAP1 forms a complex with PCNA and the histone-lysine methyltransferase Suv39h1 to reinstate heterochromatin after DNA replication.

International audience; Introduction: A mission-oriented approach to cancer care in Europe was proposed by Julio Celis and Dainius Pavalkis in 2017. The major proposed objective is to achieve long-term survival of 3 out of 4 cancer patients by 2030. Background: The authors are president or president-elect of Organization of European Cancer Institutes (OECI) EU Life or European Cancer Community Organization (ECCO). Results: The goal is laudable and ambitious. However, it can only be successful if it is coordinated with active involvement of all stakeholders and interacts with an already well-organized, recognized, and certified European Accreditation and Designation quality approach to identify and select the potential candidates to participate in such a virtual network. Conclusion: The building of a virtual European Cancer Institute based on coordinated networks should refer to unquestioned criteria using a solid and proven methodology operated by an independent arbitration body. The cancer community must share a fundamental responsibility to act collectively and in today's era of the World Wide Web, it is time to think out of the box and consider the possibility of establishing networks of networks.; Introduction : Une approche de la prise en charge du cancer en Europe axée sur la mission a été proposée par Julio Celis et Dainius Pavalkis en 2017. Le principal objectif proposé est de parvenir à la survie à long terme de 3 patients sur 4 atteints de cancer d'ici 2030. Contexte : Les auteurs sont président ou président élu de l'Organisation des instituts européens du cancer (OECI) EU Life ou de l'Organisation communautaire européenne du cancer (ECCO). Résultats : L'objectif est louable et ambitieux. Toutefois, il ne peut être atteint que s'il est coordonné avec la participation active de toutes les parties prenantes et s'il interagit avec une approche de qualité déjà bien organisée, reconnue et certifiée de l'accréditation et de la désignation européennes pour identifier et sélectionner les candidats potentiels à participer à un tel réseau virtuel. Conclusion : La construction d'un Institut européen du cancer virtuel basé sur des réseaux coordonnés devrait se référer à des critères incontestables en utilisant une méthodologie solide et éprouvée gérée par un organisme d'arbitrage indépendant. La communauté de lutte contre le cancer doit partager la responsabilité fondamentale d'agir collectivement et, à l'ère du World Wide Web, il est temps de sortir des sentiers battus et d'envisager la possibilité d'établir des réseaux de réseaux.

International audience; Treating metastatic cancers is an ongoing challenge in oncology. A recent paper by Gomes and colleagues proposes histone H3 variant dynamics as major regulator of cell fate transition during metastasis and suggests histone chaperones as therapeutic targets for invasive carcinoma.; Le traitement des cancers métastatiques est un défi permanent en oncologie. Un article récent de Gomes et ses collègues propose la dynamique du variant de l'histone H3 comme régulateur majeur de la transition du destin cellulaire pendant la métastase et suggère les chaperons d'histones comme cibles thérapeutiques pour les carcinomes invasifs.

Interest in epigenetics has taken on considerable importance with the general public and the media in recent years. However, cover pages with striking headlines often display conclusions that are not necessarily based on empirically verified research data. For example, on the front page of the New Scientist in 2009, the headline reads: "Darwin was wrong!1" And the text goes on to say: "Darwin was wrong!continued: "DNA is not everything, the environment modulates gene expression and certain traits can be modified and transmitted without altering the genome sequence. This is what the field of epigenetics touches upon and which leads us to reflect on the Darwinian conception of evolution by adding a touch of Lamarckism.If Jean-Baptiste Lamarck had seen evolution as a theoretical necessity, Charles Darwin puts it forward as the product of chance and contingency. Much of the debate amounts to opposing "innate" and "acquired" or "nature versus nurture"; that is, to contrast the contributions of epigenetics and more traditional approaches to genetics concerning phenotypic variations, and even to contrast the theories of evolution between neo-Lamarckism and Darwinism. In reality, such an opposition has no real foundation.biological, because epigenetic mechanisms operate in the context of genetics, as we will see below. Thus, there is considerable interaction between genetic variation and epigenome. Finally, the notion of transgenerational heritability remains a subject of discussion, and sometimes even controversy. Moreover, the effectspossible over a few generations of these processes are not necessarily transposable to the time scale needed for evolution. It is probably important to remain cautious and to avoid epigenetics falling victim to fashionable effects such as those that have prevailed in the past, like the beginnings of genetic epidemiology, which saw the slippage of the "everything is genetic" and a simplistic vision according to which each individual would be the sum of his or her genes. It is therefore important for researchers in this scientific community to be careful to manage public expectations and avoid the pitfalls of sometimes hasty communication., L’intérêt pour l’épigénétique a pris une importance considérable auprès du grand public et des médias au cours de ces dernières années. Cependant les pages de couvertures aux titres fracassants affichent souvent des conclusions qui ne reposent pas nécessairement sur des données de recherches empiriquement vérifiées. Ainsi pouvait-on lire, à la une du New Scientist en 2009 : « Darwin was wrong !1 » Et le texte sepoursuivait : « L’ADN n’est pas tout, l’environnement module l’expression des gènes et certains caractères peuvent être modifiés et transmis sans altération de la séquence du génome ». C’est ce à quoi le domaine de l’épigénétique touche et qui amène à réfléchir sur la conception darwinienne de l’évolution en y ajoutant une touche de lamarckisme.Si Jean-Baptiste Lamarck avait vu l’évolution comme une nécessité théorique, Charles Darwin la met en avant comme le produit du hasard et de la contingence. Une grande partie du débat revient à opposer « inné » et « acquis » ou encore « nature versus nurture » ; c’est-à-dire à mettre en opposition les apports de l’épigénétique et les approches plus traditionnelles de la génétique concernant les variations phénotypiques, et même à opposer les théories de l’évolution entre le néolamarckisme et le darwinisme. En réalité, une telle opposition, n’a pas vraiment de fondementbiologique, car les mécanismes épigénétiques opèrent dans le contexte de la génétique, comme nous le verrons plus loin. Il y a donc une interaction considérable entre variation génétique et épigénome. Enfin, la notion d’héritabilité transgénérationnelle reste matière à discussion, et suscite même parfois des controverses. De plus, les effetspossibles sur quelques générations de ces processus ne sont pas nécessairement transposables dans l’échelle de temps nécessaire à l’évolution. Il est vraisemblablement important de rester prudent et d’éviter que l’épigénétique ne fasse les frais des effets de mode comme ceux qui ont prévalus auparavant, à l’instar des débuts de l’épidémiologie en génétique, qui a vu naître les dérapages du « tout est génétique » et une vision simpliste selon laquelle chaque individu serait la somme de ses gènes. Il importe donc, pour les chercheurs de cette communauté scientifique, de veiller à bien gérer les attentes du public et de se soustraire aux pièges d’une communication parfois hâtive.

International audience; Epigenetic dysregulation has long been recognized as a key factor contributing to tumorigenesis and tumour maintenance that can influence all of the recognized hallmarks of cancer. Despite regulatory approvals for the treatment of certain haematological malignancies, the efficacy of the first generation of epigenetic drugs (epi-drugs) in patients with solid tumours has been disappointing; however, successes have now been achieved in selected solid tumour subtypes, thanks to the development of novel compounds and a better understanding of cancer biology that have enabled precision medicine approaches. Several lines of evidence support that, beyond their potential as monotherapies, epigenetic drugs could have important roles in synergy with other anticancer therapies or in reversing acquired therapy resistance. Herein, we review the mechanisms by which epi-drugs can modulate the sensitivity of cancer cells to other forms of anticancer therapy, including chemotherapy, radiation therapy, hormone therapy, molecularly targeted therapy and immunotherapy. We provide a critical appraisal of the preclinical rationale, completed clinical studies and ongoing clinical trials relating to combination therapies incorporating epi-drugs. Finally, we propose and discuss rational clinical trial designs and drug development strategies, considering key factors including patient selection, tumour biomarker evaluation, drug scheduling and response assessment and study end points, with the aim of optimizing the development of such combinations.; La dysrégulation épigénétique est depuis longtemps reconnue comme un facteur clé contribuant à la tumorigénèse et à la maintenance des tumeurs qui peut influencer toutes les caractéristiques reconnues du cancer. Malgré les approbations réglementaires pour le traitement de certaines malignités hématologiques, l'efficacité de la première génération de médicaments épigénétiques (épi-drugs) chez les patients atteints de tumeurs solides a été décevante ; cependant, des succès ont maintenant été obtenus dans certains sous-types de tumeurs solides, grâce au développement de nouveaux composés et à une meilleure compréhension de la biologie du cancer qui ont permis des approches médicales de précision. Plusieurs sources de preuves soutiennent que, au-delà de leur potentiel en tant que monothérapies, les médicaments épigénétiques pourraient jouer un rôle important en synergie avec d'autres thérapies anticancéreuses ou pour inverser la résistance acquise à la thérapie. Nous passons ici en revue les mécanismes par lesquels les médicaments épigénétiques peuvent moduler la sensibilité des cellules cancéreuses à d'autres formes de thérapie anticancéreuse, notamment la chimiothérapie, la radiothérapie, l'hormonothérapie, la thérapie moléculaire ciblée et l'immunothérapie. Nous fournissons une évaluation critique du raisonnement préclinique, des études cliniques achevées et des essais cliniques en cours concernant les thérapies combinées incorporant des médicaments d'épinéphrine. Enfin, nous proposons et discutons des plans d'essais cliniques rationnels et des stratégies de développement de médicaments, en tenant compte de facteurs clés tels que la sélection des patients, l'évaluation des biomarqueurs tumoraux, la programmation des médicaments et l'évaluation de la réponse et les résultats des études, dans le but d'optimiser le développement de ces combinaisons.

International audience; The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.

International audience; A number of studies across different model systems revealed that chromatin undergoes significant changes in dynamics in response to DNA damage. These include local motion changes at damage sites, increased nuclear exploration of both damaged and undamaged loci, and directed motions to new nuclear locations associated with certain repair pathways. These studies also revealed the need for new analytical methods to identify directed motions in a context of mixed trajectories, and the importance of investigating nuclear dynamics over different time scales to identify diffusion regimes. Here we provide an overview of the current understanding of this field, including imaging and analytical methods developed to investigate nuclear dynamics in different contexts. These dynamics are essential for genome integrity. Identifying the molecular mechanisms responsible for these movements is key to understanding how their misregulation contributes to cancer and other genome instability disorders.

International audience; Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3 while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components, including a divergent CENP-T homolog, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN homologs in other independently derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.