Les télomères sont des structures nucléoprotéiques à l’extrémité des chromosomes linéaires qui préservent la stabilité et la fonction du génome. Les variations de structure des télomères sont associées à sénescence des cellules, la biologie des cellules souches et le développement du cancer. Notre équipe étudie chez les levures S. cerevisiae et S. pombe  la réplication, les mécanismes de maintenance des télomères, et les réponses cellulaires à l’érosion des télomères. Nos travaux et projets actuels chez  la levure se concentrent sur la dynamique de la réparation des télomères pendant la sénescence réplicative et sur le rôle joué par le pore nucléaire dans ces processus.  Nous étudions également les mécanismes de maintenance des télomères pendant la quiescence en utilisant la levure fiissipare comme modèle. D’autre part, nous avons initié de nouveaux programmes visant à déchiffrer comment l’expression ectopique de la télomérase chez la souris favorise en même temps l’échappement à la sénescence et dérégule les voies de signalisation. Ce programme a des implications fortes à la fois en médecine régénératrice et en cancérologie. Enfin, nous étudions les mécanismes de l’ALT dans le cancer humain d’origine mésenchymateuse.

TELOMERES
TEMPORAL AND SPATIAL REGULATION OF TELOMERIC FORK REPAIR PATHWAYS

The nuclear pore complex (NPC) are composed of 30 individual nucleoporins to form highly conserved macromolecular structures in the nuclear envelope. Its core function is the nucleo-cytoplasmic transport and RNA export, but several individual nucleoporins have been involved in DNA repair. Hard‐to‐repair DNA lesions, including irreparable double strand breaks (DSBs), eroded telomeres and collapsed replication forks relocate to NPC at which alternative repair pathways take place. We investigate in budding and fission yeast how eroded telomeres and replication forks stalled at telomere repeats are processed and the role of the NPC in promoting telomere repair and fork‐restart.

THE p21-mTERT KNOCK-IN MOUSE: AN IN VIVO MODEL OF SENESCENCE BYPASS AND MUCH MOR

A key regulator of cellular arrest in response to telomere shortening and DNA damage is the cyclin-dependent kinase inhibitor p21. p53-dependent upregulation of p21 is thought to be the primary event inducing replicative senescence. We asked whether aging could be delayed by abrogating telomere shortening in senescent cells by expressing telomerase “only when it is needed” and what would be the consequences of this conditional ectopic expression of telomerase. Indeed, previous studies revealed that overexpression of telomerase promotes cell proliferation and inflammation independently of its activity at telomeres. To this purpose, we have created a knock-in mouse model in which a cassette encoding mCherry-2A-mTert (telomerase) has been inserted after the first exon of p21 (p21-mTert mouse). While p21-driven expression of telomerase suppresses senescence in several tissues, we observed unexpected phenotypes associated to this ectopic telomerase expression. Our project aims to understand how the p21-driven expression of telomerase at the same time promotes the escape of senescence and deregulates signalling and metabolic pathways

ALTERNATIVE LENGTHENING OF TELOMERES IN OSTEOSARCOMAS

Osteosarcomas are highly aggressive bone tumours that mainly occur in children and adolescents. Genetically, they are characterized by complex structural and numerical aberrations. Osteosarcomas are known for exhibiting a high frequency of ALT activation. Previous reports showed that ATRX gene mutation and/or loss of protein expression is detectable in only 30% of them. This discrepancy between a high level of ALT and a low proportion of ATRX inactivation led us to the hypothesis that ATRX-mediated ALT inhibition could be overridden in certain conditions. We investigate the mechanisms underlying ALT osteosarcomas.

CHROMATINE
The MANY FACES OF SET1

By the same time as the group of Loraine Pillus (UCSD), we identified SET1 as the Saccharomyces cerevisiae gene encoding the yeast most closely related to SET domain proteins of multicellular organisms. Deletion of SET1 alleviated telomeric position effect, resulted in a mild shortening of telomeres and increased viability after DNA damage of checkpoint sensor mutants. Few years after, it was discovered by several other teams that Set1 was the catalytic subunit of a protein complex called Set1C or COMPASS (for Complex of Proteins Associated with Set1) that mediates H3 methylation at lysine 4 (H3K4). We had then an important contribution in collaboration with Francis Stewart’s Team in defining how each subunit of the Set1C was bound to the docking platform made by the catalytic Set1 subunit. Others and we showed that loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation patterns, and H3K4 methylation along active genes. After having characterized the RNA Recognition Motifs of Set1, we uncovered in collaboration with Jaehoon Kim and Domenico Libri that Set1 directly binds RNA in vitro an in vivo. We discovered that Set1 binding to nascent transcripts is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. We also reported in collaboration with Frank Holstege that H3K4 trimethylation loss on its own had little effect on steady-state mRNA expression levels and that the combined loss of H3K4me3 and H3K4me2 results in steady-state upregulation of a group of genes associated with Set1-mediated repression of 3’-end antisense transcription.

15 years ago, we demonstrated that inactivation of Set1 reduces the number of DSBs and impairs meiotic replication. This seminal paper was the first observation linking Set1 and H3K4 methylation to replication and to meiosis. In collaboration with Alain Nicolas and Valérie Borde, it was further shown that meiotic DSBs occur in regions showing higher H3K4me3 occupancy, although no higher transcript levels were detected near these DSB sites. We further reported that tethering of the PHD-containing protein, Spp1 to recombinationally cold regions was sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacted with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus Spp1, by interacting with H3K4me3 and Mer2, promotes recruitment of potential meiotic DSB sites to the chromosomal axis. In collaboration with Lóránt Székvölgyi, we recently reported that spatial interactions of Spp1 and Mer2 occurred independently of Set1C. Our work led us to propose an enriched chromatin loop-axis model for the regulation of DSB formation that addresses how the meiotic DSB sites are mechanistically selected.

 

Our team has therefore a unique expertise to provide an integrative view of the function of the Set1C. Our research led to the concept that Set1C subunits, in addition to regulating H3K4 methylation, may be directly involved in biological functions by recruiting specific protein partners. Based on a systematic two-hybrid screens performed in collaboration with Bernhard Dichtl using Set1 domains as well as each Set1C subunits as baits, our current research investigates many unsuspected functions of Set1C.

HISTONE STRESS: AN UNEXPLORED SOURCE OF CHROMOSOMAL INSTABILITY IN CANCER

In collaboration with Manuel Mendoza and Sebastian Chavez, we showed in S. cerevisiae that abnormal accumulation of histones in wild-type cells was sufficient to promote aberrant endomitosis and induce whole genome duplications. We showed using live imaging that high levels of histones were able to promote the delocalization of the whole mitotic machinery into the daughter cell before mitosis and lead to aberrant endomitosis in which both nuclei remain trapped in the daughter cell.