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2021

4D nucleome modeling.

Author(s) : Di Stefano M, Paulsen J, Jost D, Marti-Renom M,
Journal : Curr Opin Genet Dev
2021
The intrinsic dynamic nature of chromosomes is emerging as a fundamental componentin regulating DNA transcription, replication, and damage-repair among other nuclearfunctions. With this increased awareness, reinforced over the last ten years, manynew experimental techniques, mainly based on microscopy and chromosome conformationcapture, have been introduced to study the genome in space and time. Owing to theincreasing complexity of these cutting-edge techniques, computational approacheshave become of paramount importance to interpret, contextualize, and complement suchexperiments with new insights. Hence, it is becoming crucial for experimentalbiologists to have a clear understanding of the diverse theoretical modelingapproaches available and the biological information each of them can provide.

A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.

Author(s) : Duplus-Bottin H, Spichty M, Triqueneaux G, Place C, Mangeot P, Ohlmann T, Vittoz F, Yvert G,
Journal : Elife
2021
Optogenetics enables genome manipulations with high spatiotemporal resolution,opening exciting possibilities for fundamental and applied biological research.Here, we report the development of LiCre, a novel light-inducible Cre recombinase.LiCre is made of a single flavin-containing protein comprising the AsLOV2photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizingmutations in its N-terminal and C-terminal domains. LiCre can be activated withinminutes of illumination with blue light, without the need of additional chemicals.When compared to existing photoactivatable Cre recombinases based on two splitunits, LiCre displayed faster and stronger activation by light as well as a lowerresidual activity in the dark. LiCre was efficient both in yeast, where it allowedus to control the production of β-carotene with light, and in human cells. Given itssimplicity and performances, LiCre is particularly suited for fundamental andbiomedical research, as well as for controlling industrial bioprocesses.

Auxin confers protection against ER stress in Caenorhabditis elegans.

Author(s) : Bhoi A, Palladino F, Fabrizio P,
Journal : Biol Open
2021
Auxins are plant growth regulators that influence most aspects of plant developmentthrough complex mechanisms. The development of an auxin-inducible degradation (AID)system has enabled rapid, conditional protein depletion in yeast and cultured cells.More recently, the system was successfully adapted to C aenorhabditis elegans toachieve auxin-dependent degradation of targets in all tissues and developmentalstages. Whether auxin treatment alone has an impact on nematode physiology is anopen question. Here we show that indole-3-acetic acid (IAA), the auxin most commonlyused to trigger AID in worms, functions through the conserved IRE-1/XBP-1 branch ofthe Unfolded Protein Response (UPR) to promote resistance to endoplasmic reticulum(ER) stress. Because the UPR not only plays a central role in restoring ERhomeostasis, but also promotes lipid biosynthesis and regulates lifespan, we suggestthat extreme caution should be exercised when using the AID system to study theseand related processes.

Best practices for the visualization, mapping, and manipulation of R-loops.

Author(s) : Chédin F, Hartono S, Sanz L, Vanoosthuyse V,
Journal : EMBO J
2021
R-loops represent an abundant class of large non-B DNA structures in genomes. Eventhough they form transiently and at modest frequencies, interfering with R-loopformation or dissolution has significant impacts on genome stability. Addressing themechanism(s) of R-loop-mediated genome destabilization requires a precisecharacterization of their distribution in genomes. A number of independent methodshave been developed to visualize and map R-loops, but their results are at timesdiscordant, leading to confusion. Here, we review the main existing methodologiesfor R-loop mapping and assess their limitations as well as the robustness ofexisting datasets. We offer a set of best practices to improve the reproducibilityof maps, hoping that such guidelines could be useful for authors and referees alike.Finally, we propose a possible resolution for the apparent contradictions in R-loopmapping outcomes between antibody-based and RNase H1-based mapping approaches.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

Author(s) : Spanier B, Laurençon A, Weiser A, Pujol N, Omi S, Barsch A, Korf A, Meyer S, Ewbank J, Paladino F, Garvis S, Aguilaniu H, Witting M,
Journal : Metabolomics
2021
INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample tobe detected and quantified. Modern lipidomics techniques are ultra-sensitive assaysthat enable the discovery of novel biomarkers in a variety of fields and provide newinsight in mechanistic investigations. Despite much progress in lipidomics, thereremains, as for all high throughput "omics" strategies, the need to developstrategies to standardize and integrate quality control into studies in order toenhance robustness, reproducibility, and usability of studies within specific fieldsand beyond. OBJECTIVES: We aimed to understand how much results from lipid profilingin the model organism Caenorhabditis elegans are influenced by different cultureconditions in different laboratories. METHODS: In this work we have undertaken aninter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans anddaf-2(e1370) mutants lacking a functional insulin receptor. Sample were collectedfrom worms grown in four separate laboratories under standardized growth conditions.We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MSanalysis. RESULTS: We found common qualitative changes in several marker lipids insamples from the individual laboratories. On the other hand, even in this controlledexperimental system, the exact fold-changes for each marker varied betweenlaboratories. CONCLUSION: Our results thus reveal a serious limitation to thereproducibility of current lipid profiling experiments and reveal challenges to theintegration of such data from different laboratories.

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system.

Author(s) : Herbette M, Wei X, Chang C, Larracuente A, Loppin B, Dubruille R,
Journal : PLoS Genet
2021
Segregation Distorter (SD) is a male meiotic drive system in Drosophilamelanogaster. Males heterozygous for a selfish SD chromosome rarely transmit thehomologous SD+ chromosome. It is well established that distortion results from aninteraction between Sd, the primary distorting locus on the SD chromosome and itstarget, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecularand cellular mechanisms leading to post-meiotic SD+ sperm elimination remainunclear. Here we show that SD/SD+ males of different genotypes but with similarlystrong degrees of distortion have distinct spermiogenic phenotypes. In somegenotypes, SD+ spermatids fail to fully incorporate protamines after the removal ofhistones, and degenerate during the individualization stage of spermiogenesis. Incontrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed,yet spermatid nuclei are abnormally compacted, and mature sperm nuclei areeventually released in the seminal vesicle. Our analyses of different SD+chromosomes suggest that the severity of the spermiogenic defects associates withthe copy number of the Rsp satellite. We propose that when Rsp copy number is veryhigh (> 2000), spermatid nuclear compaction defects reach a threshold that triggersa checkpoint controlling sperm chromatin quality to eliminate abnormal spermatidsduring individualization.

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae.

Author(s) : Duveau F, Vande Zande P, Metzger B, Diaz C, Walker E, Tryban S, Siddiq M, Yang B, Wittkopp P,
Journal : Elife
2021
Heritable variation in a gene's expression arises from mutations impacting cis- andtrans-acting components of its regulatory network. Here, we investigate howtrans-regulatory mutations are distributed within the genome and within a generegulatory network by identifying and characterizing 69 mutations withtrans-regulatory effects on expression of the same focal gene in Saccharomycescerevisiae. Relative to 1766 mutations without effects on expression of this focalgene, we found that these trans-regulatory mutations were enriched in codingsequences of transcription factors previously predicted to regulate expression ofthe focal gene. However, over 90% of the trans-regulatory mutations identifiedmapped to other types of genes involved in diverse biological processes includingchromatin state, metabolism, and signal transduction. These data show how geneticchanges in diverse types of genes can impact a gene's expression in trans, revealingproperties of trans-regulatory mutations that provide the raw material fortrans-regulatory variation segregating within natural populations.

Neuronal perception of the social environment generates an inherited memory that controls the development and generation time of C. elegans

Author(s) : Perez M, Shamalnasab M, Mata-Cabana A, Della Valle S, Olmedo M, Francesconi M, Lehner B,
Journal : Current Biology
2021
An old and controversial question in biology is whether information perceived by the nervous system of an animal can “cross the Weismann barrier” to alter the phenotypes and fitness of their progeny. Here, we show that such intergenerational transmission of sensory information occurs in the model organism, C. elegans, with a major effect on fitness. Specifically, that perception of social pheromones by chemosensory neurons controls the post-embryonic timing of the development of one tissue, the germline, relative to others in the progeny of an animal. Neuronal perception of the social environment thus intergenerationally controls the generation time of this animal.

Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae

Author(s) : Piazza A, Rajput P, Heyer W,
Journal : Methods in Molecular Biology
2021
DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIR in various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defects for genomic instability by MIR.

Polymer modelling unveils the roles of heterochromatin and nucleolar organizing regions in shaping 3D genome organization in Arabidopsis thaliana.

Author(s) : Di Stefano M, Nützmann H, Marti-Renom M, Jost D,
Journal : Nucleic Acids Res
2021
The 3D genome is characterized by a complex organization made of genomic andepigenomic layers with profound implications on gene regulation and cell function.However, the understanding of the fundamental mechanisms driving the crosstalkbetween nuclear architecture and (epi)genomic information is still lacking. Theplant Arabidopsis thaliana is a powerful model organism to address these questionsowing to its compact genome for which we have a rich collection of microscopy,chromosome conformation capture (Hi-C) and ChIP-seq experiments. Using polymermodelling, we investigate the roles of nucleolus formation and epigenomics-driveninteractions in shaping the 3D genome of A. thaliana. By validation of severalpredictions with published data, we demonstrate that self-attracting nucleolarorganizing regions and repulsive constitutive heterochromatin are major mechanismsto regulate the organization of chromosomes. Simulations also suggest thatinterphase chromosomes maintain a partial structural memory of the V-shapes, typicalof (sub)metacentric chromosomes in anaphase. Additionally, self-attraction betweenfacultative heterochromatin regions facilitates the formation of Polycomb bodieshosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin arehighly-conserved in eukaryotic cells, our findings pave the way for a comprehensivecharacterization of the generic principles that are likely to shape and regulate the3D genome in many species.

Recombination-mediated genome rearrangements

Author(s) : Savocco J, Piazza A,
Journal : Current Opinion in Genetics and Development
2021
Homologous recombination (HR) is a universal DNA double-strand break (DSB) repair pathway that uses an intact DNA molecule as a template. Signature HR reactions are homology search and DNA strand invasion catalyzed by the prototypical RecA-ssDNA filament (Rad51 and Dmc1 in eukaryotes), which produces heteroduplex DNA-containing joint molecules (JMs). These reactions uniquely infringe on the DNA strands association established at replication, on the basis of substantial sequence similarity. For that reason, and despite the high fidelity of its templated nature, DSB repair by HR authorizes the alteration of genome structure, guided by repetitive DNA elements. The resulting structural variations (SVs) can involve vast genomic regions, potentially affecting multiple coding sequences and regulatory elements at once, with possible pathological consequences. Here, we discuss recent advances in our understanding of genetic and molecular vulnerabilities of HR leading to SVs, and of the various fidelity-enforcing factors acting across scales on the balancing act of this complex pathway. An emphasis is put on extra-chomosomal DNAs, both product of, and substrate for HR-mediated chromosomal rearrangements.

RNA polymerase backtracking results in the accumulation of fission yeast condensin at active genes.

Author(s) : Rivosecchi J, Jost D, Vachez L, Gautier F, Bernard P, Vanoosthuyse V,
Journal : Life Sci Alliance
2021
The mechanisms leading to the accumulation of the SMC complexes condensins aroundspecific transcription units remain unclear. Observations made in bacteria suggestedthat RNA polymerases (RNAPs) constitute an obstacle to SMC translocation,particularly when RNAP and SMC travel in opposite directions. Here we show infission yeast that gene termini harbour intrinsic condensin-accumulating featureswhatever the orientation of transcription, which we attribute to the frequentbacktracking of RNAP at gene ends. Consistent with this, to relocate backtrackedRNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation ofcondensin at gene ends and to redistribute it evenly within transcription units,indicating that RNAP backtracking may play a key role in positioning condensin.Formalization of this hypothesis in a mathematical model suggests that the inclusionof a sub-population of RNAP with longer dwell-times is essential to fullyrecapitulate the distribution profiles of condensin around active genes. Takentogether, our data strengthen the idea that dense arrays of proteins tightly boundto DNA alter the distribution of condensin on chromosomes.