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4D nucleome modeling.

Author(s) : Di Stefano M, Paulsen J, Jost D, Marti-Renom M,
Journal : Curr Opin Genet Dev
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 Novel Screen for Expression Regulators of the Telomeric Protein TRF2 Identified Small Molecules That Impair TRF2 Dependent Immunosuppression and Tumor Growth.

Author(s) : El Maï M, Janho Dit Hreich S, Gaggioli C, Roisin A, Wagner N, Ye J, Jalinot P, Cherfils-Vicini J, Gilson E,
Journal : Cancers (Basel)
Telomeric repeat-binding factor 2 (TRF2) is a subunit of the shelterin proteincomplex, which binds to and protects telomeres from unwanted DNA damage response(DDR) activation. TRF2 expression plays a pivotal role in aging and cancer, beingdownregulated during cellular senescence and overexpressed during oncogenesis.Cancers overexpressing TRF2 often exhibit a poor prognosis. In cancer cells, TRF2plays multiple functions, including telomere protection and non-cell autonomousroles, promoting neo-angiogenesis and immunosuppression. We present here anoriginal screening strategy, which enables identification of small molecules thatdecrease or increase TRF2 expression. By screening a small library of Food andDrug Agency (FDA)-approved drugs, we identified two molecules (AR-A014418 andalexidine·2HCl) that impaired tumor growth, neo-angiogenesis andimmunosuppression by downregulating TRF2 expression in a mouse xenograft model.These results support the chemotherapeutic strategy of downregulating TRF2expression to treat aggressive human tumors and validate this cell-based assaycapable of screening for potential anti-cancer and anti-aging molecules bymodulating TRF2 expression levels.

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
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.

Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease.

Author(s) : Girard V, Jollivet F, Knittelfelder O, Celle M, Arsac J, Chatelain G, Van den Brink D, Baron T, Shevchenko A, Kühnlein R, Davoust N, Mollereau B,
Journal : PLoS Genet
Parkinson's disease (PD) is a neurodegenerative disorder characterized byalpha-synuclein (αSyn) aggregation and associated with abnormalities in lipidmetabolism. The accumulation of lipids in cytoplasmic organelles called lipiddroplets (LDs) was observed in cellular models of PD. To investigate thepathophysiological consequences of interactions between αSyn and proteins thatregulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, inwhich human αSyn is specifically expressed in photoreceptor neurons. We first foundthat overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), whichlimit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loadedLDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipidanabolic (diacylglycerol acyltransferase 1/midway, fatty acid transportprotein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating thatalternative mechanisms regulate neuronal LD homeostasis. Interestingly, theaccumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 orKlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, whichlocalized to LDs in both Drosophila photoreceptor neurons and in human neuroblastomacells. Finally, the accumulation of LDs increased the resistance of αSyn toproteolytic digestion, a characteristic of αSyn aggregation in human neurons. Wepropose that αSyn cooperates with LD proteins to inhibit lipolysis and that bindingof αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn inneurons.

Asymmetry is defined during meiosis in the oocyte of the parthenogenetic nematode Diploscapter pachys.

Author(s) : Eweis D, Delattre M, Plastino J,
Journal : Dev Biol
Asymmetric cell division is an essential feature of normal development and certainpathologies. The process and its regulation have been studied extensively in theCaenorhabditis elegans embryo, particularly how symmetry of the actomyosin corticalcytoskeleton is broken by a sperm-derived signal at fertilization, upstream ofpolarity establishment. Diploscapter pachys is the closest parthenogenetic relativeto C. elegans, and D. pachys one-cell embryos also divide asymmetrically. Howeverhow polarity is triggered in the absence of sperm remains unknown. In post-meioticembryos, we find that the nucleus inhabits principally one embryo hemisphere, thefuture posterior pole. When forced to one pole by centrifugation, the nucleusreturns to its preferred pole, although poles appear identical as concerns corticalruffling and actin cytoskeleton. The location of the meiotic spindle also correlateswith the future posterior pole and slight actin enrichment is observed at that polein some early embryos along with microtubule structures emanating from the meioticspindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachysoocytes. All together our results are consistent with the idea that polarity of theD. pachys embryo is attained during meiosis, seemingly based on the location of themeiotic spindle, by a mechanism that may be present but suppressed in C. elegans.

Auxin confers protection against ER stress in Caenorhabditis elegans.

Author(s) : Bhoi A, Palladino F, Fabrizio P,
Journal : Biol Open
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.

Baboon Envelope Pseudotyped "Nanoblades" Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34(+) Cells and Knock-in of AAV6-Encoded Donor DNA in CD34(+) Cells.

Author(s) : Gutierrez-Guerrero A, Abrey Recalde M, Mangeot P, Costa C, Bernadin O, Périan S, Fusil F, Froment G, Martinez-Turtos A, Krug A, Martin F, Benabdellah K, Ricci E, Giovannozzi S, Gijsbers R, Ayuso E, Cosset F, Verhoeyen E,
Journal : Front Genome Ed
Programmable nucleases have enabled rapid and accessible genome engineering ineukaryotic cells and living organisms. However, their delivery into human bloodcells can be challenging. Here, we have utilized "nanoblades," a new technology thatdelivers a genomic cleaving agent into cells. These are modified murine leukemiavirus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structuralprotein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 proteincomplexed with the guide RNAs. Highly efficient gene editing was obtained in celllines, IPS and primary mouse and human cells. Here, we showed that nanoblades wereremarkably efficient for entry into human T, B, and hematopoietic stem andprogenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelope glycoproteins. A brief incubation of human T and Bcells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletionin the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34(+) cells(HSPCs) treated with the same nanoblades allowed 30-40% exon 1 drop-out in the WASgene locus. Importantly, no toxicity was detected upon nanoblade-mediated geneediting of these blood cells. Finally, we also treated HSPCs with nanoblades incombination with a donor-encoding rAAV6 vector resulting in up to 40% of stableexpression cassette knock-in into the WAS gene locus. Summarizing, this newtechnology is simple to implement, shows high flexibility for different targetsincluding primary immune cells of human and murine origin, is relatively inexpensiveand therefore gives important prospects for basic and clinical translation in thearea of gene therapy.

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

Author(s) : Chédin F, Hartono S, Sanz L, Vanoosthuyse V,
Journal : EMBO J
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.

Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila.

Author(s) : Arsac J, Sedru M, Dartiguelongue M, Vulin J, Davoust N, Baron T, Mollereau B,
Journal : Int J Mol Sci
Parkinson's disease (PD) is characterized by the progressive accumulation ofneuronal intracellular aggregates largely composed of alpha-Synuclein (αSyn)protein. The process of αSyn aggregation is induced during aging and enhanced byenvironmental stresses, such as the exposure to pesticides. Paraquat (PQ) is anherbicide which has been widely used in agriculture and associated with PD. PQ isknown to cause an increased oxidative stress in exposed individuals but theconsequences of such stress on αSyn conformation remains poorly understood. To studyαSyn pathogenic modifications in response to PQ, we exposed Drosophila expressinghuman αSyn to a chronic PQ protocol. We first showed that PQ exposure and αSynexpression synergistically induced fly mortality. The exposure to PQ was alsoassociated with increased levels of total and phosphorylated forms of αSyn in theDrosophila brain. Interestingly, PQ increased the detection of soluble αSyn inhighly denaturating buffer but did not increase αSyn resistance to proteinase Kdigestion. These results suggest that PQ induces the accumulation of toxic solubleand misfolded forms of αSyn but that these toxic forms do not form fibrils oraggregates that are detected by the proteinase K assay. Collectively, our resultsdemonstrate that Drosophila can be used to study the effect of PQ or otherenvironmental neurotoxins on αSyn driven pathology.

Cohesin regulates homology search during recombinational DNA repair

Author(s) : Piazza A, Bordelet H, Dumont A, Thierry A, Savocco J, Girard F, Koszul R,
Journal : Nature Cell Biology
Homologous recombination repairs DNA double-strand breaks (DSB) using an intact dsDNA molecule as a template. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA filament assembled on each DSB end. Whether, how and to what extent a DSB impacts chromatin folding, and how this (re)organization in turns influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in Saccharomyces cerevisiae. Although cohesin folds chromosomes into cohesive arrays of ~20-kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB–dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during recombinational DNA repair.

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
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.

Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs.

Author(s) : Sun Y, Wang R, Du K, Zhu J, Zheng J, Xie L, Pereira A, Zhang C, Ricci E, Li X,
Journal : Nat Commun
PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essentialfor fertility. piRNAs originate from transposable element (TE) RNAs, long non-codingRNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, withthe last being the least characterized of the three piRNA classes. Here, wedemonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and thatpost-termination 80S ribosomes guide piRNA production on 3´UTRs in mice andchickens. At the pachytene stage, when other co-translational RNA surveillancepathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer roundsof translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNAprecursor mRNAs code for distinct proteins in mice and chickens, they all harborembedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a functionof the piRNA pathway in fine-tuning protein production and reveal a conserved piRNAbiogenesis mechanism that recognizes translating RNAs in amniotes.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Author(s) : Zhai L, Rety S, Chen W, Song Z, Auguin D, Sun B, Dou S, Xi X,
Journal : Nucleic Acids Res
Telomerase plays critical roles in cellular aging, in the emergence and/ordevelopment of cancer, and in the capacity for stem-cell renewal, consists of acatalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA(TER). TERs from diverse organisms contain two conserved structural elements: thetemplate-pseudoknot (T-PK) and a helical three-way junction (TWJ).Species-specific features of the structure and function of telomerase makeobtaining a more in-depth understanding of the molecular mechanism of telomeraseparticularly important. Here, we report the first structural studies ofN-terminally truncated TERTs from Candida albicans and Candida tropicalis in apoform and complexed with their respective TWJs in several conformations. We foundthat Candida TERT proteins perform only one round of telomere addition in thepresence or absence of PK/TWJ and display standard reverse transcriptaseactivity. The C-terminal domain adopts at least two extreme conformations andundergoes conformational interconversion, which regulates the catalytic activity.Most importantly, we identified a conserved tertiary structural motif, called theU-motif, which interacts with the reverse transcriptase domain and is crucial forcatalytic activity. Together these results shed new light on the structure andmechanics of fungal TERTs, which show common TERT characteristics, but alsodisplay species-specific features.

Decreased expression of the translation factor eIF3e induces senescence in breast cancer cells via suppression of PARP1 and activation of mTORC1.

Author(s) : Morris C, Durand S, Jalinot P,
Journal : Oncotarget
Altered expression of the translation factor eIF3e is associated with breastcancer occurrence. We have previously shown that eIF3e deficiency leads to animpaired DNA damage response with a marked decrease in DNA repair by homologousrecombination. Here, we explored the possibility to exploit this DNA repairdefect in targeted cancer therapy using PARP inhibitors. Surprisingly,eIF3e-deficient breast cancer cells are resistant to these drugs, in contrast toBRCA1-deficient cells. Studying this, we found that eIF3e-depleted cellssynthesize lowered amounts of PARP1 protein, due to a weakened translation of thecorresponding mRNA, associated with a strong decrease in cellularpoly(ADP-ribosyl)ation. Additionally, we discovered that the mTORC1 signalingpathway is aberrantly activated in response to eIF3e suppression. Together, thesePARP1 and mTORC1 dysfunctions upon eIF3e depletion are causally linked toinduction of cellular senescence associated with a pro-inflammatory secretoryphenotype. This study provides mechanistic insights into how eIF3e protectsagainst breast cancer, with potential novel cancer therapeutic opportunities.While PARP inhibitors appear as inappropriate drugs for eIF3e-deficient breasttumors, our findings suggest that such cancers may benefit from senolytic drugsor mTORC1 inhibitors.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").

Author(s) : Mangeot P, Guiguettaz L, Sohier T, Ricci E,
Journal : J Vis Exp
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systemhas democratized genome-editing in eukaryotic cells and led to the development ofnumerous innovative applications. However, delivery of the Cas9 protein andsingle-guide RNA (sgRNA) into target cells can be technically challenge. Classicalviral vectors, such as those derived from lentiviruses (LVs) or adeno-associatedviruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9protein and its associated sgRNA in many primary cells and in vivo. Nevertheless,these vectors can suffer from drawbacks such as integration of the transgene in thetarget cell genome, a limited cargo capacity, and long-term expression of the Cas9protein and guide RNA in target cells. To overcome some of these problems, adelivery vector based on the murine Leukemia virus (MLV) was developed to packagethe Cas9 protein and its associated guide RNA in the absence of any codingtransgene. By fusing the Cas9 protein to the C-terminus of the structural proteinGag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA(named "Nanoblades") were formed. Nanoblades can be collected from the culturemedium of producer cells, purified, quantified, and used to transduce target cellsand deliver the active Cas9/sgRNA complex. Nanoblades deliver theirribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary andimmortalized cells and can be programmed for other applications, such as transienttranscriptional activation of targeted genes, using modified Cas9 proteins.Nanoblades are capable of in vivo genome-editing in the liver of injected adult miceand in oocytes to generate transgenic animals. Finally, they can be complexed withdonor DNA for "transfection-free" homology-directed repair. Nanoblade preparation issimple, relatively low-cost, and can be easily carried out in any cell biologylaboratory.

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
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.

Endogenous Bos taurus RECQL is predominantly monomeric and more active than oligomers.

Author(s) : Liu N, Song Z, Guo H, Yin H, Chen W, Dai Y, Xin B, Ai X, Ji L, Wang Q, Hou X, Dou S, Rety S, Xi X,
Journal : Cell Rep
There is broad consensus that RecQ family helicase is a high-order oligomer thatdissociates into a dimer upon ATP binding. This conclusion is based mainly onstudies of highly purified recombinant proteins, and the oligomeric states ofRecQ helicases in living cells remain unknown. We show here that, in contrast tocurrent models, monomeric RECQL helicase is more abundant than oligomer/dimerforms in living cells. Further characterization of endogenous BtRECQL andisolated monomeric BtRECQL using various approaches demonstrates that bothendogenous and recombinant monomeric BtRECQL effectively function as monomers,displaying higher helicase and ATPase activities than dimers and oligomers.Furthermore, monomeric BtRECQL unfolds intramolecular G-quadruplex DNA asefficiently as human RECQL and BLM helicases. These discoveries have implicationsfor understanding endogenous RECQL oligomeric structures and their regulation. Itis worth revisiting oligomeric states of the other members of the RecQ familyhelicases in living cells.

Integrative Cell Type-Specific Multi-Omics Approaches Reveal Impaired Programs of Glial Cell Differentiation in Mouse Culture Models of DM1

Author(s) : González-Barriga A, Lallemant L, Dincã D, Braz S, Polvèche H, Magneron P, Pionneau C, Huguet-Lachon A, Claude J, Chhuon C, Guerrera I, Bourgeois C, Auboeuf D, Gourdon G, Gomes-Pereira M,
Journal : Front Cell Neurosci
gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila.

Author(s) : Child M, Bateman J, Jahangiri A, Reimer A, Lammers N, Sabouni N, Villamarin D, McKenzie-Smith G, Johnson J, Jost D, Garcia H,
Journal : Elife
Three-dimensional eukaryotic genome organization provides the structural basis forgene regulation. In Drosophila melanogaster, genome folding is characterized bysomatic homolog pairing, where homologous chromosomes are intimately paired from endto end; however, how homologs identify one another and pair has remained mysterious.Recently, this process has been proposed to be driven by specifically interacting'buttons' encoded along chromosomes. Here, we turned this hypothesis into aquantitative biophysical model to demonstrate that a button-based mechanism can leadto chromosome-wide pairing. We tested our model using live-imaging measurements ofchromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. We showsolid agreement between model predictions and experiments in the pairing dynamics ofindividual homologous loci. Our results strongly support a button-based mechanism ofsomatic homolog pairing in Drosophila and provide a theoretical framework forrevealing the molecular identity and regulation of buttons.

Loop extrusion as a mechanism for formation of DNA damage repair foci.

Author(s) : Arnould C, Rocher V, Finoux A, Clouaire T, Li K, Zhou F, Caron P, Mangeot P, Ricci E, Mourad R, Haber J, Noordermeer D, Legube G,
Journal : Nature
The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genomeintegrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers theestablishment of megabase-sized, chromatin domains decorated with phosphorylatedhistone H2AX (γH2AX), which act as seeds for the formation of DNA-damage responsefoci(1). It is unclear how these foci are rapidly assembled to establish a'repair-prone' environment within the nucleus. Topologically associating domains area key feature of 3D genome organization that compartmentalize transcription andreplication, but little is known about their contribution to DNA repairprocesses(2,3). Here we show that topologically associating domains are functionalunits of the DNA damage response, and are instrumental for the correct establishmentof γH2AX-53BP1 chromatin domains in a manner that involves one-sidedcohesin-mediated loop extrusion on both sides of the DSB. We propose a model inwhich H2AX-containing nucleosomes are rapidly phosphorylated as they actively passby DSB-anchored cohesin. Our work highlights the importance of chromosomeconformation in the maintenance of genome integrity and demonstrates theestablishment of a chromatin modification by loop extrusion.

Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): a novel function for the viral EBNA-LP protein

Author(s) : Manet E, Polvèche H, Mure F, Mrozek-Gorska P, Roisné-Hamelin F, Hammerschmidt W, Auboeuf D, Gruffat H,
Journal : Nucleic Acids Res
Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides-besides transcription-an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP-together with the RBM4 splicing factor-in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X.

Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease.

Author(s) : Eshraghi M, Karunadharma P, Blin J, Shahani N, Ricci E, Michel A, Urban N, Galli N, Sharma M, Ramírez-Jarquín U, Florescu K, Hernandez J, Subramaniam S,
Journal : Nat Commun
The polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) andneurodegeneration, but the mechanisms remain unclear. Here, we found that mHttpromotes ribosome stalling and suppresses protein synthesis in mouse HD striatalneuronal cells. Depletion of mHtt enhances protein synthesis and increases the speedof ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro.Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but itsdepletion has no discernible effect on protein synthesis or ribosome stalling in HDcells. We found interactions of ribosomal proteins and translating ribosomes withmHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seqindicates a widespread shift in ribosome occupancy toward the 5' and 3' end andunique single-codon pauses on selected mRNA targets in HD cells, compared tocontrols. Thus, mHtt impedes ribosomal translocation during translation elongation,a mechanistic defect that can be exploited for HD therapeutics.

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
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
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.

Organization of DNA Replication Origin Firing in Xenopus Egg Extracts: The Role of Intra-S Checkpoint.

Author(s) : Ciardo D, Haccard O, Narassimprakash H, Arbona J, Hyrien O, Audit B, Marheineke K, Goldar A,
Journal : Genes (Basel)
During cell division, the duplication of the genome starts at multiple positionscalled replication origins. Origin firing requires the interaction of rate-limitingfactors with potential origins during the S(ynthesis)-phase of the cell cycle.Origins fire as synchronous clusters which is proposed to be regulated by theintra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and thecheckpoint protein Chk1 over-expressed replication pattern of single DNA moleculesfrom Xenopus sperm chromatin replicated in egg extracts, we demonstrate that thequantitative modelling of data requires: (1) a segmentation of the genome intoregions of low and high probability of origin firing; (2) that regions with highprobability of origin firing escape intra-S checkpoint regulation and (3) thevariability of the rate of DNA synthesis close to replication forks is a necessaryingredient that should be taken in to account in order to describe the dynamic ofreplication origin firing. This model implies that the observed origin clusteringemerges from the apparent synchrony of origin firing in regions with highprobability of origin firing and challenge the assumption that the intra-Scheckpoint is the main regulator of origin clustering.

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
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
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
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
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.