Biology subjects

Koszul, R.

Publications and source records attributed to Koszul, R..

46 records · Page 3Linked to original sources

bioRxiv2020

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↗

bioRxiv2020

Chromosight: A computer vision program for pattern detection in chromosome contact maps

Chromosomes of all species studied so far display a variety of higher order organizational features such as domains or loops often associated to biological functions and visible on Hi-C contact maps. We developed Chromosight, an algorithm inspired from computer vision that can detect patterns in Hi-C maps. Chromosight has greater sensitivity than existing methods, while being faster and applicable to any type of genomes, including bacteria, viruses, yeasts and mammals. Code and documentation: https://github.com/koszullab/chromosight

bioinformatics↗

bioRxiv2019

Chromosome-level quality scaffolding of brown algal genomes using InstaGRAAL, a proximity ligation-based scaffolder

Hi-C has become a popular technique in recent genome assembly projects. Hi-C exploits contact frequencies between pairs of loci to bridge and order contigs in draft genomes, resulting in chromosome-level assemblies. However, application of this approach is currently hampered by a lack of robust programs that are capable of effectively treating this type of data, particularly open source programs. We developed instaGRAAL, a complete overhaul of the GRAAL program, which has adapted the latter to allow efficient assembly of large genomes. Both GRAAL, and instaGRAAL use a Markov Chain Monte Carlo algorithm to perform Hi-C scaffolding, but instaGRAAL features a number of improvements including a modular polishing approach that optionally integrates independent data. To validate the program, we used it to generate chromosome-level assemblies for two brown algae, Desmarestia herbacea and the model Ectocarpus sp., and quantified improvements compared to the initial draft for the latter. Overall, instaGRAAL is a program able to generate, using default parameters with minimal human intervention, near-complete assemblies.

genomics↗

bioRxiv2019

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↗

bioRxiv2019

Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

Higher-order chromosome folding and segregation is tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remains poorly characterized, especially in non-model species. Here, we investigate the role of DNA partitioning protein ParB and condensin complexes, two key players in bacterial chromosome structuring, in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for a reliable chromosome segregation. Using a combination of chromatin immunoprecipitation and photoactivated single molecule localization microscopy evidences the formation of distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further identified and functionally characterized two condensin paralogs. Whereas SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while showing that some condensin complexes evolved functions uncoupled from chromosome folding.

microbiology↗

bioRxiv2019

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↗

bioRxiv2018

Dynamic Processing of Displacement Loops During Recombinational DNA Repair

Displacement-loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the network of D-loop formation and reversal pathways. Nascent D-loops are detected within 2 hrs of DSB formation and extended over the next 2 hrs in a system allowing break-induced replication. The majority of D-loops are disrupted in wild type cells by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a novel layer of HR control in cells relying on nascent D-loop dynamics, revealing unsuspected complexities, and identifying a surprising role for a conserved Rad54 paralog.

molecular biology↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2017

Redesigning chromosomes to optimize conformation capture (Hi-C) assays

In all chromosome conformation capture based experiments the accuracy with which contacts are detected varies considerably because of the uneven distribution of restriction sites along genomes. In addition, repeated sequences as well as homologous, large identical regions remain invisible to the assay because of the ambiguities they introduce during the alignment of the sequencing reads along the genome. As a result, the investigation of homologs during meiosis prophase through 3C studies has been limited. Here, we redesigned and reassembled in yeast a 145kb region with regularly spaced restriction sites for various enzymes. Thanks to this Syn-3C design, we enhanced the signal to noise ratio and improved the visibility of the entire region. We also improved our understanding of Hi-C data and definition of resolution. The redesigned sequence is now distinguishable from its native homologous counterpart in an isogenic diploid strain. As a proof of principle, we track the establishment of homolog pairing during meiotic prophase in a synchronized population. This provides new insights on the individualization and pairing of homologs, as well as on their internal restructuration into arrays of loops during meiosis prophase. Overall, we show the interest of redesigned genomic regions to explore complex biological questions otherwise difficult to address.

synthetic biology↗

bioRxiv2016

Choreography of budding yeast chromosomes during the cell cycle

To ensure the proper transmission of the genetic information, DNA molecules must be faithfully duplicated and segregated. These processes involve dynamic modifications of chromosomes internal structure to promote their individualization, as well as their global repositioning into daughter cells (Guacci et al., 1994; Kleckner et al., 2014; Mizuguchi et al., 2014). In eukaryotes, these events are regulated by conserved architectural proteins, such as structural maintenance of chromosomes (SMC i.e. cohesin and condensin) complexes (Aragon et al., 2013a; Uhlmann, 2016). Although the roles of these factors have been actively investigated, the genome-wide chromosomal architecture and dynamics both at small and large-scales during cell division remains elusive. Here we report a comprehensive Hi-C (Dekker et al., 2002; Lieberman-Aiden et al., 2009) analysis of the dynamic changes of chromosomes structure over the Saccharomyces cerevisiae cell cycle. We uncover specific SMC-dependent structural transitions between the different phases of the mitotic cycle. During replication, cohesion establishment promotes the increase of long-range intra-chromosomal contacts. This process correlates with the individualization of chromosomes, which culminates at metaphase. Mitotic chromosomes are then abruptly reorganized in anaphase by the mechanical forces exerted by the mitotic spindle on the centromere cluster. The formation of a condensin-dependent loop, that bridges centromere cluster with the cenproximal flanking region of the rDNA, suggests that these forces may directly facilitate nucleolus segregation. This work provides a comprehensive overview of chromosome dynamics during the cell cycle of a unicellular eukaryote that recapitulates and unveils new features of highly conserved stages of the cell division.

genetics↗