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Assigning function to natural allelic variation via dynamic modeling of gene network induction.

Author(s) : Richard M, Chuffart F, Duplus-Bottin H, Pouyet F, Spichty M, Fulcrand E, Entrevan M, Barthelaix A, Springer M, Jost D, Yvert G,
Journal : Mol Syst Biol
More and more natural DNA variants are being linked to physiological traits. Yet, understanding what differences they make on molecular regulations remains challenging. Important properties of gene regulatory networks can be captured bycomputational models. If model parameters can be "personalized" according to thegenotype, their variation may then reveal how DNA variants operate in the network. Here, we combined experiments and computations to visualize natural alleles of the yeast GAL3 gene in a space of model parameters describing the galactose response network. Alleles altering the activation of Gal3p by galactose were discriminated from those affecting its activity (production/degradation or efficiency of the activated protein). The approach allowed us to correctly predict that a non-synonymous SNP would change the binding affinity of Gal3p with the Gal80p transcriptional repressor. Our results illustrate how personalizing gene regulatory models can be used for the mechanistic interpretation of geneticvariants.

Condensin controls cellular RNA levels through the accurate segregation of chromosomes instead of directly regulating transcription.

Author(s) : Hocquet C, Robellet X, Modolo L, Sun X, Burny C, Cuylen-Haering S, Toselli E, Clauder-Munster S, Steinmetz L, Haering C, Marguerat S, Bernard P,
Journal : Elife
Condensins are genome organisers that shape chromosomes and promote their accurate transmission. Several studies have also implicated condensins in gene expression, although any mechanisms have remained enigmatic. Here, we report on the role of condensin in gene expression in fission and budding yeasts. In contrast to previous studies, we provide compelling evidence that condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis. We further show that the changes in gene expression in post-mitotic fission yeast cells that result from condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite condensin inactivation. Thus, chromosome instability, rather than a direct role of condensin in the transcription process, changes gene expression. This knowledge challenges the concept of gene regulation by canonical condensin complexes.

DRP-1-mediated apoptosis induces muscle degeneration in dystrophin mutants.

Author(s) : Scholtes C, Bellemin S, Martin E, Carre-Pierrat M, Mollereau B, Gieseler K, Walter L,
Journal : Sci Rep
Mitochondria are double-membrane subcellular organelles with highly conserved metabolic functions including ATP production. Mitochondria shapes change continually through the combined actions of fission and fusion events rendering mitochondrial network very dynamic. Mitochondria are largely implicated in pathologies and mitochondrial dynamics is often disrupted upon muscle degeneration in various models. Currently, the exact roles of mitochondria in the molecular mechanisms that lead to muscle degeneration remain poorly understood. Here we report a role for DRP-1 in regulating apoptosis induced by dystrophin-dependent muscle degeneration. We found that: (i) dystrophin-dependent muscle degeneration was accompanied by a drastic increase in mitochondrial fragmentation that can be rescued by genetic manipulations of mitochondrial dynamics (ii) the loss of function of the fission gene drp-1 or the overexpression of the fusion genes eat-3 and fzo-1 provoked a reduction of muscle degeneration and an improved mobility of dystrophin mutant worms (iii) the functions of DRP-1 in apoptosis and of others apoptosis executors are important for dystrophin-dependent muscle cell death (iv) DRP-1-mediated apoptosis is alsolikely to induce age-dependent loss of muscle cell. Collectively, our findings point toward a mechanism involving mitochondrial dynamics to respond to trigger(s) of muscle degeneration via apoptosis in Caenorhabditis elegans.

Evolution of mitotic spindle behavior during the first asymmetric embryonic division of nematodes.

Author(s) : Valfort A, Launay C, Semon M, Delattre M,
Journal : PLoS Biol
Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded thefirst embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at, constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra-and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject tosystem drift.

Genomics of cellular proliferation in periodic environmental fluctuations.

Author(s) : Salignon J, Richard M, Fulcrand E, Duplus-Bottin H, Yvert G,
Journal : Mol Syst Biol
Living systems control cell growth dynamically by processing information from their environment. Although responses to a single environmental change have beenintensively studied, little is known about how cells react to fluctuating conditions. Here, we address this question at the genomic scale by measuring therelative proliferation rate (fitness) of 3,568 yeast gene deletion mutants in out-of-equilibrium conditions: periodic oscillations between two environmental conditions. In periodic salt stress, fitness and its genetic variance largely depended on the oscillating period. Surprisingly, dozens of mutants displayed pronounced hyperproliferation under short stress periods, revealing unexpected controllers of growth under fast dynamics. We validated the implication of the high-affinity cAMP phosphodiesterase and of a regulator of protein translocationto mitochondria in this group. Periodic oscillations of extracellular methionine, a factor unrelated to salinity, also altered fitness but to a lesser extent and for different genes. The results illustrate how natural selection acts on mutations in a dynamic environment, highlighting unsuspected genetic vulnerabilities to periodic stress in molecular processes that are conserved across all eukaryotes.

HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.

Author(s) : Fiorini F, Robin J, Kanaan J, Borowiak M, Croquette V, Le Hir H, Jalinot P, Mocquet V,
Journal : Nat Commun
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

Author(s) : Peres E, Blin J, Ricci E, Artesi M, Hahaut V, Van den Broeke A, Corbin A, Gazzolo L, Ratner L, Jalinot P, Duc Dodon M,
Journal : PLoS Pathog
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (DeltaPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WTthan in DeltaPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from DeltaPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actinfilopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively,our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina.

Author(s) : Van Den Brink D, Cubizolle A, Chatelain G, Davoust N, Girard V, Johansen S, Napoletano F, Dourlen P, Guillou L, Angebault-Prouteau C, Bernoud-Hubac N, Guichardant M, Brabet P, Mollereau B,
Journal : PLoS Genet
Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types inresponse to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient forexpansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic andpromoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.

Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing.

Author(s) : Boland B, Yu W, Corti O, Mollereau B, Henriques A, Bezard E, Pastores G, Rubinsztein D, Nixon R, Duchen M, Mallucci G, Kroemer G, Levine B, Eskelinen E, Mochel F, Spedding M, Louis C, Martin O, Millan M,
Journal : Nat Rev Drug Discov
Neurodegenerative disorders of ageing (NDAs) such as Alzheimer disease, Parkinson disease, frontotemporal dementia, Huntington disease and amyotrophic lateral sclerosis represent a major socio-economic challenge in view of their high prevalence yet poor treatment. They are often called 'proteinopathies' owing to the presence of misfolded and aggregated proteins that lose their physiological roles and acquire neurotoxic properties. One reason underlying the accumulation and spread of oligomeric forms of neurotoxic proteins is insufficient clearance by the autophagic-lysosomal network. Several other clearance pathways are also compromised in NDAs: chaperone-mediated autophagy, the ubiquitin-proteasome system, extracellular clearance by proteases and extrusion into the circulation via the blood-brain barrier and glymphatic system. This article focuses on emerging mechanisms for promoting the clearance of neurotoxic proteins, a strategy that may curtail the onset and slow the progression of NDAs.

Single cell expression analysis uncouples transdifferentiation and reprogramming

Author(s) : Francesconi M, Di Stefano B, Berenguer C, de Andres M, Mendez Lago M, Guillaumet-Adkins A, Rodriguez-Esteban G, Gut M, Gut I, Heyn H, Lehner B, Graf T,
Journal : bioRxiv
Many somatic cell types are plastic, having the capacity to convert into other specialized cells (transdifferentiation)(1) or into induced pluripotent stem cells (iPSCs, reprogramming)(2) in response to transcription factor over-expression. To explore what makes a cell plastic and whether these different cell conversion processes are coupled, we exposed bone marrow derived pre-B cells to two different transcription factor overexpression protocols that efficiently convert them either into macrophages or iPSCs and monitored the two processes over time using single cell gene expression analysis. We found that even in these highly efficient cell fate conversion systems, cells differ in both their speed and path of transdifferentiation and reprogramming. This heterogeneity originates in two starting pre-B cell subpopulations, large pre-BII and the small pre-BII cells they normally differentiate into. The large cells transdifferentiate slowly but exhibit a high efficiency of iPSC reprogramming. In contrast, the small cells transdifferentiate rapidly but are highly resistant to reprogramming. Moreover, the large B cells induce a stronger transient granulocyte/macrophage progenitor (GMP)-like state, while the small B cells undergo a more direct conversion to the macrophage fate. The large cells are cycling and exhibit high Myc activity whereas the small cells are Myc low and mostly quiescent. The observed heterogeneity of the two cell conversion processes can therefore be traced to two closely related cell types in the starting population that exhibit different types of plasticity. These data show that a somatic cell’s propensity for either transdifferentiation and reprogramming can be uncoupled.

Strengths and Weaknesses of the Current Strategies to Map and Characterize R-Loops.

Author(s) : Vanoosthuyse V,
Journal : Noncoding RNA
R-loops are evolutionarily conserved three-stranded structures that result from the formation of stable DNA:RNA hybrids in the genome. R-loops have attracted increasing interest in recent years as potent regulators of gene expression and genome stability. In particular, their strong association with severe replication stress makes them potential oncogenic structures. Despite their importance, the rules that govern their formation and their dynamics are still controversial andan in-depth description of their direct impact on chromatin organization and DNAtransactions is still lacking. To better understand the diversity of R-loop functions, reliable, accurate, and quantitative mapping techniques, as well as functional assays are required. Here, I review the different approaches that arecurrently used to do so and to highlight their individual strengths and weaknesses. In particular, I review the advantages and disadvantages of using the S9.6 antibody to map R-loops in vivo in an attempt to propose guidelines for best practices.

The Ectodysplasin receptor EDAR acts as a tumor suppressor in melanoma by conditionally inducing cell death.

Author(s) : Vial J, Royet A, Cassier P, Tortereau A, Dinvaut S, Maillet D, Gratadou-Hupon L, Creveaux M, Sadier A, Tondeur G, Leon S, Depaepe L, Pantalacci S, de la Fouchardiere A, Micheau O, Dalle S, Laudet V, Mehlen P, Castets M,
Journal : Cell Death Differ
Ectodysplasin receptor EDAR is seen as a typical Tumor Necrosis Factor receptor (TNFR) family member known to interact with its ligand Eda-A1, and signaling mainly through the nuclear factor-kappaB (NF-kappaB) and c-jun N-terminal kinases pathways. Mutations in genes that encode proteins involved in EDAR transduction cascade cause anhidrotic ectodermal dysplasia. Here, we report an unexpected pro-apoptotic activity of EDAR when unbound to its ligand Eda-A1, which is independent of NF-kappaB pathway. Contrarily to other death receptors, EDAR doesrecruit caspase-8 to trigger apoptosis but solely upon ligand withdrawal, thereby behaving as the so-called dependence receptors. We propose that pro-apoptotic activity of unbound EDAR confers it a tumor suppressive activity. Along this line, we identified loss-of-pro-apoptotic function mutations in EDAR gene in human melanoma. Moreover, we show that the invalidation of EDAR in mice promotesmelanoma progression in a B-Raf mutant background. Together, these data support the view that EDAR constrains melanoma progression by acting as a dependence receptor.

The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain.

Author(s) : Issa A, Sun J, Petitgas C, Mesquita A, Dulac A, Robin M, Mollereau B, Jenny A, Cherif-Zahar B, Birman S,
Journal : Autophagy
The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerativeconditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (lysosomal-associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. Here we report that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA(A30P)). Using KFERQ-tagged fluorescent biosensors, we observed that LAMP2Aexpression stimulated selective autophagy in the adult brain and not in the larval fat body, but did not increase this process under starvation conditions. Noteworthy, we found that neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formationof autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of theautophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can potentiate autophagic flux in theDrosophila brain, leading to enhanced stress resistance and neuroprotection. ABBREVIATIONS: Act5C: actin 5C; a.E.: after eclosion; Atg5: autophagy-related 5;Atg8a/LC3: autophagy-related 8a; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; elav: embryonic lethal abnormal vision; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; GABARAP: GABA typeA receptor-associated protein; Hsc70-4: heat shock protein cognate 4; HSPA8/Hsc70: heat shock protein family A (Hsp70) member 8; LAMP2: lysosomal associated membrane protein 2; MDA: malondialdehyde; PA-mCherry: photoactivable mCherry; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PD: Parkinson disease; Ref(2)P/p62: refractory to sigma P; ROS: reactive oxygen species; RpL32/rp49: ribosomal protein L32; RT-PCR: reverse transcription polymerase chain reaction; SING: startle-induced negative geotaxis; SNCA/alpha-synuclein: synuclein alpha; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; UAS: upstream activating sequence.

The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

Author(s) : Lambert M, Terrone S, Giraud G, Benoit-Pilven C, Cluet D, Combaret V, Mortreux F, Auboeuf D, Bourgeois C,
Journal : Nucleic Acids Res
The Repressor Element 1-silencing transcription factor (REST) represses a numberof neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulatorsof DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.