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Thierry, A.

Publications and source records attributed to Thierry, A..

At least 19 recordsLinked to original sources

Extracellular DNASE1L3 dysfunction fuels obesity-driven inflammation and metabolic syndrome

Obesity, a global health crisis affecting 16% of the world population, is characterized by chronic inflammation that contributes to health complications such as type 2 diabetes and metabolic dysfunction-associated steatotic liver disease (MASLD). Emerging evidence suggests that self-DNA released from dying cells aberrantly activates inflammatory responses during obesity. However, the role of extracellular deoxyribonucleases (DNASEs), which at steady state regulate abundance of extracellular self-DNA, remains poorly understood in this context. Here, we show that individuals with obesity exhibit elevated levels of circulating cell-free DNA (cfDNA) with a distinctive end-motif signature, anti-DNASE1L3 autoantibodies and a reduction in circulating DNASE activity. These cfDNA alterations correlate with the severity of obesity and can be corrected by therapeutic intervention such as bariatric surgery. Similarly, mice fed a high-fat diet (HFD) displayed increased cfDNA levels and decreased DNASE activity. Genetic deficiency of the extracellular nuclease DNASE1L3 in mice worsened HFD-induced metabolic complications, including glucose intolerance, insulin resistance, MASLD, and metabolic tissue inflammation. Conversely, targeted supplementation of DNASE1L3 in the liver using adeno-associated viral vectors protected obese mice from developing MASLD and liver inflammation. These findings uncover a novel role of DNASE1L3 in controlling obesity-associated inflammation and its potential therapeutic use for preventing metabolic disease.

pathology↗

Bacteriophage PAK_P3 genome structuration and dynamics during infection of Pseudomonas aeruginosa reveal specific interactions patterns

Bacteriophages, or phages, are highly abundant and diverse genomic entities that play an important role in microbial ecology, evolution, and horizontal gene transfer. While the dynamic changes in genome organization in cellular organisms have been well described, the 3D folding of phage genomes during the infection of their host is extremely limited. Understanding how phage genomes fold, invade and rearrange the genome of their host to functionally organize the optimal expression of their genes remains unknown. Here, we explore the spatial dynamics of the virulent double-stranded DNA phage PAK_P3 during infection of its host Pseudomonas aeruginosa and reveal how its genome rapidly decondenses to adopt a specific 3D organization that reflects its transcriptional program. Concomitantly, the host genome decondenses as gene expression wanes and transcription induced domains vanish. We also uncover specific and discrete bridging of the host and the phage genomes, showing how the phage genome exploits the spatial genome architecture of its host to succeed in its infection cycle. Our data highlights an unprecedented level of genome folding and gene expression regulation during a viral infection.

microbiology↗

Z-DNA formation induces the totipotent-like state and primes Zscan4-dependent chromatin compartmentalization

A remarkable transition during murine development is the progression from the 1-cell embryo to the 2-cell stage, accompanied by the activation of a specific set of embryonic genes, epigenome reprogramming, and nuclear architecture reorganization. Some of these characteristics are recapitulated in vitro with the spontaneous emergence of 2-cell-like cells from mouse embryonic stem cells, which exhibit a transcriptomic signature resembling the 2-cell stage, including the expression of genes such as Dux, Zscan4, and the repetitive element MERVL, and a more relaxed chromatin state. Here, we show that inter- and intra-chromosomal interactions, driven by Zscan4 chromatin factors, form during this transition and segregate into a distinct genomic compartment (Z compartment). Mechanistically, the formation of Z-DNA, an alternative DNA conformation regulated by polyamine levels, promotes the emergence of totipotent-like cells and the establishment of the Z compartment. This compartment is characterized by a decrease in active histone marks and a reduced expression of genes associated with differentiation and late developmental processes. Overall, these findings suggest that Z-DNA formation may play a dual role, first in activating ZGA genes and later in guiding genome compartmentalization to safeguard the totipotent-like state by restricting the expression of non-ZGA genes within a permissive chromatin environment.

cell biology↗

HU promotes higher-order chromosome organisation and influences DNA replication rates in Streptococcus pneumoniae

Nucleoid-associated proteins (NAPs) are crucial for maintaining chromosomal compaction and architecture and are actively involved in DNA replication, recombination, repair, and gene regulation. In the opportunistic pathogen Streptococcus pneumoniae, HU is the only identified NAP, and its role in chromosome conformation and other essential processes has not yet been investigated. Here, we use a multi-scale approach to explore the role of HU in chromosome conformation and segregation dynamics. By combining superresolution microscopy and whole-genome binding analysis, we describe the nucleoid as a dynamic structure where HU binds transiently across the entire nucleoid, with a preference for the origin of replication over the terminus. Reducing cellular HU levels impacts nucleoid maintenance and disrupts robust nucleoid scaling with cell size. This effect is similar to the distortion caused by fluoroquinolone-antibiotics, supporting earlier observations that HU is essential for maintaining DNA supercoiling. Furthermore, in cells lacking HU, the replication machinery is misplaced, and cells are unable to initiate and proceed with on-going replication. Chromosome conformation capture (Hi-C) experiments revealed that HU is required to maintain cohesion between the two chromosomal arms, in a similar way to the structural maintenance of the chromosome complex SMC. Together, we show that by promoting long-range chromosome interactions and supporting the architecture of the domain encompassing the origin, HU is fundamental for chromosome integrity and the intimately related processes of chromosome replication and segregation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/615122v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@10cfa9eorg.highwire.dtl.DTLVardef@119ae12org.highwire.dtl.DTLVardef@f35cd4org.highwire.dtl.DTLVardef@1537e7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

Chromosome-level genome assembly of the European Green woodpecker Picus viridis

The European Green Woodpecker, Picus viridis, is a widely distributed species found in the Western Palearctic region. Here we assembled a highly contiguous genome assembly for this species using a combination of short and long reads sequencing and scaffolded with chromatin conformation capture (Hi-C). The final genome assembly was 1.28 Gb and features a scaffold N50 of 37Mb and a scaffold L50 of 39.165 Mb. The assembly incorporates 89.4% of the genes identified in birds in OrthoDB. Gene and repetitive content annotation on the assembly detected 15,805 genes and a [~]30.1% occurrence of repetitive elements, respectively. Analysis of synteny demonstrates the fragmented nature of the Picus viridis genome when compared to the chicken (Gallus gallus). The assembly and annotations produced in this study will certainly help for further research into the genomics of P. viridis and the comparative evolution of woodpeckers.

genomics↗

Mechanism of homology search expansion during recombinational DNA break repair

Homology search catalyzed by a RecA/Rad51 nucleoprotein filament (NPF) is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short NPFs is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection by Exo1 drives this transition from local to genome-wide search by providing ssDNA substrates for assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by NPFs formed on the two DSB ends. Finally, an autonomous genetic element on chromosome III engages the NPF and stimulates homology search in its vicinity. This work reveals the mechanism of the progressive and uneven expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization. Highlights- Cohesin-mediated chromatin loops constrain homology search in cis for NPF regions close to the resection front - Stiffening of ssDNA by Rad51 enables genome-wide homology search by DSB-proximal sites - Exo1-mediated long-range resection promotes genome-wide homology search - DSB end-tethering promotes coordinated homology search by NPFs formed on both DSB ends - The recombination enhancer focuses homology search in its vicinity

genetics↗

Anchoring of parasitic plasmids to inactive regions of eukaryotic chromosomes through nucleosome signal

Natural plasmids are common in prokaryotes but few have been documented in eukaryotes. The natural 2{micro} plasmid present in budding yeast Saccharomyces cerevisiae is one of the most well characterized. This highly stable genetic element coexists with its host for millions of years, efficiently segregating at each cell division through a mechanism that remains poorly understood. Using proximity ligation (Hi-C, Micro-C) to map the contacts between the 2{micro} and yeast chromosomes under dozens of different biological conditions, we found that the plasmid tether preferentially on regions with low transcriptional activity, often corresponding to long inactive genes. Common players in chromosome structure such as members of the structural maintenance of chromosome complexes (SMC) are not involved in these contacts which depend instead on a nucleosomal signal associated with a depletion of RNA Pol II. These contacts are stable throughout the cell cycle, and can be established within minutes. This strategy may involve other types of DNA molecules and species other than S. cerevisiae, as suggested by the binding pattern of the natural plasmid along the silent regions of the chromosomes of Dictyostelium discoideum.

genomics↗

Absence of chromosome axis proteins recruitment prevents meiotic recombination chromosome-wide in the budding yeast Lachancea kluyveri

Meiotic recombination shows broad variations across species and along chromosomes, and is often suppressed at and around genomic regions determining sexual compatibility such as mating type loci in fungi. Here we show that the absence of Spo11-DSBs and meiotic recombination on Lakl0C-left, the chromosome arm containing the sex locus of the Lachancea kluyveri budding yeast, results from the absence of recruitment of the two chromosome axis proteins Red1 and Hop1, essential for proper Spo11-DSBs formation. Furthermore, cytological observation of spread pachytene meiotic chromosomes reveals that Lakl0C-left does not undergo synapsis. However, we show that the behavior of Lakl0C-left is independent of its particularly early replication timing and is not accompanied by any peculiar chromosome structure as detectable by Hi-C in this yet poorly studied yeast. Finally, we observed an accumulation of heterozygous mutations on Lakl0C-left and a sexual dimorphism of the haploid meiotic offspring, supporting a direct effect of this absence of meiotic recombination on L. kluyveri genome evolution and fitness. Because suppression of meiotic recombination on sex chromosomes is widely observed across eukaryotes, the novel mechanism for recombination suppression described here may apply to other species, with the potential to impact sex chromosome evolution.

genetics↗

Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants

SUMMARYAnosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals developed a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS- CoV-2 variants were found to be neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validated the olfactory pathway as a major entry point into the brain in vivo and demonstrated in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=151 HEIGHT=200 SRC="FIGDIR/small/505985v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@8225ceorg.highwire.dtl.DTLVardef@80342aorg.highwire.dtl.DTLVardef@e0dfd1org.highwire.dtl.DTLVardef@1861e13_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin re-organization

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain, and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila. Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization, suggesting that changes in chromosomal folding are associated with host cell biology during infection.

genomics↗

Plasma derived cell-free mitochondrial DNA originates mainly from circulating cell-free mitochondria

Circulating mitochondrial DNA (cir-mtDNA) could have a potential comparable to circulating nuclear DNA (cir-nDNA), with numerous applications. However, research and development in this area have fallen behind, particularly considering its origin and structural features. To tackle this, we initially combined Q-PCR and low-pass whole genome sequencing in the same analytical strategy previously and successfully used for cir-nDNA. This revealed unexplained structural patterns and led us to correlate these data with observations made during physical examinations such as filtration, and differential centrifugation in various plasma preparations. Both the integrity index and number of reads revealed a very minor proportion of low size-ranged fragments (<1000 bp) in plasma obtained with a standard preparation (0.06%). Filtration and high speed second step centrifugation revealed that 98.7 and 99.4% corresponded to extracellular mitochondria either free or in large extracellular vesicles. When avoiding platelet activation during plasma preparation, the proportion of both types of entities was still preponderant (76-80%), but the amount of detected mitochondrial DNA decreased 67-fold. In correlation with our previous study on the presence of circulating cell-free mitochondria in blood, our differential centrifugation procedure suggested that cir-mtDNA is also associated with approximately 18% small extracellular vesicles, 1.7% exosomes and 4% protein complexes.

genomics↗

Cohesin is essential for the timely activation of condensin and anaphase segregation

Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role in determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids are maintained during anaphase in the absence of cohesin. Here we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, TEV-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wildtype cells and show that they play a role in the organisation of centromeric regions. Our data demonstrate that in yeast cells, cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating. One sentence summaryCohesin complexes on yeast chromosomes provide organisation to segregating chromatids.

cell biology↗

Nucleosome positioning on large tandem DNA repeats of the '601' sequence engineered in Saccharomyces cerevisiae

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

synthetic biology↗

Cohesin regulates homology search during recombinational DNA repair

Homologous recombination (HR) is a ubiquitous DNA double-strand break (DSB) repair mechanism that promotes cell survival. It entails a potentially genome-wide homology search step, carried out along a conserved RecA/Rad51-ssDNA nucleoprotein filament (NPF) assembled on each DSB ends1-3. This search is subdued to NPF-dsDNA collision probability, dictated in part by chromatin conformation2,4. In contrast to the extensive knowledge about chromatin composition and mobility changes elicited by the DNA damage checkpoint (DDC)5-7, whether, how, and to which extent a DSB impacts spatial chromatin organization, and whether this organization in turns influences the homology search process, remains ill-defined8,9. Here we characterize two layers of spatial chromatin reorganization following DSB formation in S. cerevisiae. While cohesin folds chromosomes into cohesive arrays of 10-20 kb long chromatin loops as cells arrest in G2/M10,11, the DSB-flanking regions locally interact in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin and HR proteins. This local structure blocks cohesin progression, constraining the extending NPF at loop base. Functionally this organization promotes side-specific cis DSB-dsDNA interactions that scales with loop expansion span, and provides a kinetic advantage for identification of intra- over inter-chromosomal homologies. We propose that cohesins regulate homology search by promoting cis dsDNA over-sampling, both upon loop expansion-coupled unidimensional dsDNA scanning, NPF trapping, and chromosome individualization, largely independent of their role in sister chromatid cohesion.

genetics↗

Phages - bacteria interactions network of the healthy human gut

With an estimated 1031 particles on earth, bacteriophages are the most abundant genomic entities across all habitats and important drivers of microbial communities. Growing evidence suggest that they play roles in intestinal human microbiota homeostasis, and recent metagenomics studies on the viral fraction of this ecosystem have provided crucial information about their diversity and specificity. However, the bacterial hosts of this viral fraction, a necessary information to characterize further the balance of these ecosystems, remain poorly characterized. Here we unveil, using an enhanced metagenomic Hi-C approach, a large network of 6,651 host-phage relationships in the healthy human gut allowing to study in situ phage-host ratio. We notably found that half of these contigs appear to be sleeping prophages whereas [1/4] exhibit a higher coverage than their associated MAG representing potentially active phages impacting the ecosystem. We also detect different candidate members of the crAss-like phage family as well as their bacterial hosts showing that these elusive phages infect different genus of Bacteroidetes. This work opens the door to single sample analysis and concomitant study of phages and bacteria in complex communities.

microbiology↗

FACT mediates cohesin function on chromatin

Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion 1,2 and the organisation of higher-order structures during interphase 3 and mitosis 4,5. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes 6-8. Here we show that cohesin role in chromosome organisation requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin-dependent compartmentalization of chromosomes into loop domains.

molecular biology↗

A major role for Eco1 in regulating cohesin-mediated mitotic chromosome folding

Understanding how chromatin organizes spatially into chromatid and how sister chromatids are maintained together during mitosis is of fundamental importance in chromosome biology. Cohesin, a member of the Structural Maintenance of Chromosomes (SMC) complex family, holds sister chromatids together 1-3 and promotes long-range intra-chromatid DNA looping 4,5. These cohesin-mediated DNA loops are important for both higher-order mitotic chromatin compaction6,7 and, in some organisms, compartmentalization of chromosomes during interphase into topologically associating domains (TADs) 8,9. Our understanding of the mechanism(s) by which cohesin generates large DNA loops remains incomplete. It involves a combination of molecular partners and active expansion/extrusion of DNA loops. Here we dissect the roles on loop formation of three partners of the cohesin complex: Pds5 10, Wpl1 11 and Eco1 acetylase 12, during yeast mitosis. We identify a new function for Eco1 in negatively regulating cohesin translocase activity, which powers loop extrusion. In the absence of negative regulation, the main barrier to DNA loop expansion appears to be the centromere. Those results provide new insights on the mechanisms regulating cohesin dependent DNA looping.

genetics↗