Telomeres and chromatin

Telomeres are chromosome-capping structures that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusions and illegitimate recombination. They are made of repetitive DNA sequences that are folded into a particular chromatin structure organized by specific DNA-protein interactions. Telomeres are known to be natural hard-to-replicate regions of the genome also defined as fragile sites because of the many obstacles that prevent the progression of replication forks at terminal sequences. Replication stress due to replication forks pausing or stalling is a potential source of dysfunctional telomeres and hence genome instability, a recognized hallmark of cancer.

We have shown in the recent years how components of the yeast shelterin complex recruit accessory factors to promote efficient replication of the terminal sequences (Matmati et al., 2018& 2020; Vaurs et al.,2022). Our lab has also been spearheading in establishing how stalled replication forks and eroded telomeres are relocated to the nuclear pore complex (NPC) for promoting accurate replication resumption (Aguilera et al., 2020) or recombination-based alternative lengthening of telomeres in the absence of telomerase (Churikov et al., 2016, Charifi et al., 2021; Aguilera et al., 2022). The lab has been also pioneering in describing the role of RPA in replication, maintenance and recombination of telomeres in yeast models (Maestroni et al. 2020; Corda et al., 2021; Audry et al., 2015)

We have established new approaches/tools to explore the mechanisms that protect and restart the replication forks at telomeres and their spatial and temporal regulation. We have implemented in both yeast models strong artificial telomeric DNA barriers to address in different mutant backgrounds the dynamic of replication and the mechanism

of fork restart. We analyze the replication dynamics at these barriers and identify the

proteome of forks arrested at telomeres, in different genetic conditions that modify their nuclear positioning, the compartmentalization of repair factors or the stress response.

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.

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

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.

We further investigate how Set1C cooperate with chromatin remodelers to promote the remodeling of newly replicated chromatin to facilitate fork progression or restart.