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2012

[Mice are not Men and yet... how humanized mice inform us about human infectious diseases].

Author(s) : Cachat A, Villaudy J, Rigal D, Gazzolo L, Duc Dodon M,
Journal : Med Sci (Paris)
2012
The study of human pathologies is often limited by the absence of animal models which are robust, cost-effective and reproduce the hallmarks of human infections. While mice have been frequently employed to study human diseases, many of important pathogens display unique human tropism. These last two decades the graft of human progenitor cells or tissues into -immunodeficient mice has allowed the elaboration of so called humanized mice. Humanized mouse technology has maderapid progress, and it is now possible to achieve high levels of human chimerismin various organs and tissues, particularly the immune system and the liver. Thereview briefly summarizes the different models of humanized mice available for in vivo experiments. With a focus on lymphotropic, monocytotropic and hepatotropic viruses, we here discuss the current status and future prospects of these modelsfor studying the pathogenesis of infectious diseases. Furthermore, they provide a powerful tool for the development of innovative therapies.

A mathematical model to study the dynamics of epithelial cellular networks.

Author(s) : Abate A, Vincent S, Dobbe R, Silletti A, Master N, Axelrod J, Tomlin C,
Journal : IEEE/ACM Trans Comput Biol Bioinform
2012
Epithelia are sheets of connected cells that are essential across the animal kingdom. Experimental observations suggest that the dynamical behavior of many single-layered epithelial tissues has strong analogies with that of specific mechanical systems, namely large networks consisting of point masses connected through spring-damper elements and undergoing the influence of active and dissipating forces. Based on this analogy, this work develops a modeling framework to enable the study of the mechanical properties and of the dynamic behavior of large epithelial cellular networks. The model is built first by creating a network topology that is extracted from the actual cellular geometry as obtained from experiments, then by associating a mechanical structure and dynamics to the network via spring-damper elements. This scalable approach enables running simulations of large network dynamics: the derived modeling framework in particular is predisposed to be tailored to study general dynamics (for example, morphogenesis) of various classes of single-layered epithelial cellular networks. In this contribution, we test the model on a case study of the dorsal epithelium of the Drosophila melanogaster embryo during early dorsal closure (and, less conspicuously, germband retraction).

Alternative splicing and cancer.

Author(s) : Auboeuf D, Carmo-Fonseca M, Valcarcel J, Biamonti G,
Journal : J Nucleic Acids
2012

Analysis of co-transcriptional RNA processing by RNA-ChIP assay.

Author(s) : Bittencourt D, Auboeuf D,
Journal : Methods Mol Biol
2012
It was initially assumed that RNA biogenesis and processing were two independentprocesses with transcripts undergoing splicing only after being completely synthesized and released from the DNA template. However, transcription and splicing are tightly linked and increasing evidence shows that nascent transcripts can undergo splicing in the vicinity of chromatin while still attached to the RNA polymerase II (RNAPII) transcriptional machinery. These co-transcriptionally spliced RNA molecules are very labile due to dynamic processing and represent a minor subpopulation among total cellular RNA species.Thus, it is difficult to isolate these RNAs in order to study the dynamics and mechanisms of co-transcriptional RNA splicing. To overcome this problem, the RNA-chromatin immunoprecipitation (ChIP) assay, adapted from classical ChIP, allows to co-purify and isolate nascent RNAs after immunoprecipitation of RNAPII. Thanks to this technique, we have shown that co-transcriptional RNA splicing occurs with distinct efficiencies for different genes and different exons of a given transcript and can represent a rate-limiting step in the biological response of messenger RNA synthesis to extracellular stimuli and drug treatments.

Chance at the heart of the cell.

Author(s) : Gandrillon O, Kolesnik-Antoine D, Kupiec J, Beslon G,
Journal : Prog Biophys Mol Biol
2012

Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation.

Author(s) : Soto-Rifo R, Limousin T, Rubilar P, Ricci E, Decimo D, Moncorge O, Trabaud M, Andre P, Cimarelli A, Ohlmann T,
Journal : Nucleic Acids Res
2012
The 5'-untranslated region (5'-UTR) of the genomic RNA of human immunodeficiencyviruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5'-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5'-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which wasmuch inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens.

Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival.

Author(s) : Dourlen P, Bertin B, Chatelain G, Robin M, Napoletano F, Roux M, Mollereau B,
Journal : PLoS Genet
2012
Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In thisstudy, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatpmutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

Dual role of the ddx5/ddx17 RNA helicases in the control of the pro-migratory NFAT5 transcription factor.

Author(s) : Germann S, Gratadou L, Zonta E, Dardenne E, Gaudineau B, Fougere M, Samaan S, Dutertre M, Jauliac S, Auboeuf D,
Journal : Oncogene
2012
Ddx5 and ddx17 are two highly related RNA helicases involved in both transcription and splicing. These proteins coactivate transcription factors involved in cancer such as the estrogen receptor alpha, p53 and beta-catenin. Ddx5 and ddx17 are part of the splicing machinery and can modulate alternative splicing, the main mechanism increasing the proteome diversity. Alternative splicing also has a role in gene expression level regulation when it is coupled to the nonsense-mediated mRNA decay (NMD) pathway. In this work, we report that ddx5 and ddx17 have a dual role in the control of the pro-migratory NFAT5 transcription factor. First, ddx5 and ddx17 act as transcriptional coactivators of NFAT5 and are required for activating NFAT5 target genes involved in tumor cell migration. Second, at the splicing level, ddx5 and ddx17 increase the inclusion of NFAT5 exon 5. As exon 5 contains a pre-mature translation termination codon, its inclusion leads to the regulation of NFAT5 mRNAs by the NMD pathway and to a decrease in NFAT5 protein level. Therefore, we demonstratedfor the first time that a transcriptional coregulator can simultaneously regulate the transcriptional activity and alternative splicing of a transcription factor.This dual regulation, where ddx5 and ddx17 enhance the transcriptional activity of NFAT5 although reducing its protein expression level, suggests a critical role for ddx5 and ddx17 in tumor cell migration through the fine regulation of NFAT5 pathway.

ER stress inhibits neuronal death by promoting autophagy.

Author(s) : Fouillet A, Levet C, Virgone A, Robin M, Dourlen P, Rieusset J, Belaidi E, Ovize M, Touret M, Nataf S, Mollereau B,
Journal : Autophagy
2012
Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro.We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning haspotential therapeutic value for the treatment of neurodegenerative diseases.

Genetic modifiers of chromatin acetylation antagonize the reprogramming of epi-polymorphisms.

Author(s) : Abraham A, Nagarajan M, Veyrieras J, Bottin H, Steinmetz L, Yvert G,
Journal : PLoS Genet
2012
Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or "epi-polymorphisms") are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing "labile" from "persistent" epi-polymorphisms. Hundreds of geneticloci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially withthe genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, "labile" and "persistent" epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA-encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics.

Guidelines for the use and interpretation of assays for monitoring autophagy.

Author(s) : Klionsky D, al,
Journal : Autophagy
2012
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitorthe numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection andinterpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the systembeing used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

HTLV-1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells.

Author(s) : Zane L, Sibon D, Capraro V, Galia P, Karam M, Delfau-Larue M, Gilson E, Gessain A, Gout O, Hermine O, Mortreux F, Wattel E,
Journal : Int J Cancer
2012
Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression.

INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation.

Author(s) : Neusiedler J, Mocquet V, Limousin T, Ohlmann T, Morris C, Jalinot P,
Journal : RNA
2012
The INT6/EIF3E protein has been implicated in mouse and human breast carcinogenesis. This subunit of the eIF3 translation initiation factor that includes a PCI domain exhibits specific features such as presence in the nucleusand ability to interact with other important cellular protein complexes like the26S proteasome and the COP9 signalosome. It has been previously shown that INT6 was not essential for bulk translation, and this protein is considered to regulate expression of specific mRNAs. Based on the results of a two-hybrid screen performed with INT6 as bait, we characterize in this article the MIF4GD/SLIP1 protein as an interactor of this eIF3 subunit. MIF4GD was previously shown to associate with SLBP, which binds the stem-loop located at the 3' end ofthe histone mRNAs, and to be necessary for efficient translation of these cell cycle-regulated mRNAs that lack a poly(A) tail. In line with the interaction of both proteins, we show using the RNA interference approach that INT6 is also essential to S-phase histone mRNA translation. This was observed by analyzing expression of endogenous histones and by testing heterologous constructs placingthe luciferase reporter gene under the control of the stem-loop element of various histone genes. With such a reporter plasmid, silencing and overexpression of INT6 exerted opposite effects. In agreement with these results, INT6 and MIF4GD were observed to colocalize in cytoplasmic foci. We conclude from these data that INT6, by establishing interactions with MIF4GD and SLBP, plays an important role in translation of poly(A) minus histone mRNAs.

INT6/EIF3E interacts with ATM and is required for proper execution of the DNA damage response in human cells.

Author(s) : Morris C, Tomimatsu N, Richard D, Cluet D, Burma S, Khanna K, Jalinot P,
Journal : Cancer Res
2012
Altered expression of the INT6 gene, encoding the e subunit of the translationalinitiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in theDNA damage response. INT6 was required for cell survival following gamma-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with gamma-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, thisresult could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.

Mathematical model of the primary CD8 T cell immune response: stability analysis of a nonlinear age-structured system.

Author(s) : Terry E, Marvel J, Arpin C, Gandrillon O, Crauste F,
Journal : J Math Biol
2012
The primary CD8 T cell immune response, due to a first encounter with a pathogen, happens in two phases: an expansion phase, with a fast increase of T cell count,followed by a contraction phase. This contraction phase is followed by the generation of memory cells. These latter are specific of the antigen and will allow a faster and stronger response when encountering the antigen for the second time. We propose a nonlinear mathematical model describing the T CD8 immune response to a primary infection, based on three nonlinear ordinary differential equations and one nonlinear age-structured partial differential equation, describing the evolution of CD8 T cell count and pathogen amount. We discuss in particular the roles and relevance of feedback controls that regulate the response. First we reduce our system to a system with a nonlinear differential equation with a distributed delay. We study the existence of two steady states, and we analyze the asymptotic stability of these steady states. Second we study the system with a discrete delay, and analyze global asymptotic stability of steady states. Finally, we show some simulations that we can obtain from the model and confront them to experimental data.

Modeling erythroblastic islands: using a hybrid model to assess the function of central macrophage.

Author(s) : Fischer S, Kurbatova P, Bessonov N, Gandrillon O, Volpert V, Crauste F,
Journal : J Theor Biol
2012
The production and regulation of red blood cells, erythropoiesis, occurs in the bone marrow where erythroid cells proliferate and differentiate within particular structures, called erythroblastic islands. A typical structure of these islands consists of a macrophage (white cell) surrounded by immature erythroid cells (progenitors), with more mature cells on the periphery of the island, ready to leave the bone marrow and enter the bloodstream. A hybrid model, coupling a continuous model (ordinary differential equations) describing intracellular regulation through competition of two key proteins, to a discrete spatial model describing cell-cell interactions, with growth factor diffusion in the medium described by a continuous model (partial differential equations), is proposed toinvestigate the role of the central macrophage in normal erythropoiesis. Intracellular competition of the two proteins leads the erythroid cell to eitherproliferation, differentiation, or death by apoptosis. This approach allows considering spatial aspects of erythropoiesis, involved for instance in the occurrence of cellular interactions or the access to external factors, as well as dynamics of intracellular and extracellular scales of this complex cellular process, accounting for stochasticity in cell cycle durations and orientation ofthe mitotic spindle. The analysis of the model shows a strong effect of the central macrophage on the stability of an erythroblastic island, when assuming the macrophage releases pro-survival cytokines. Even though it is not clear whether or not erythroblastic island stability must be required, investigation of the model concludes that stability improves responsiveness of the model, hence stressing out the potential relevance of the central macrophage in normal erythropoiesis.

Monitoring single-cell bioenergetics via the coarsening of emulsion droplets.

Author(s) : Boitard L, Cottinet D, Kleinschmitt C, Bremond N, Baudry J, Yvert G, Bibette J,
Journal : Proc Natl Acad Sci U S A
2012
Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water throughhydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.

Novel roles of Caenorhabditis elegans heterochromatin protein HP1 and linker histone in the regulation of innate immune gene expression.

Author(s) : Studencka M, Konzer A, Moneron G, Wenzel D, Opitz L, Salinas-Riester G, Bedet C, Kruger M, Hell S, Wisniewski J, Schmidt H, Palladino F, Schulze E, Jedrusik-Bode M,
Journal : Mol Cell Biol
2012
Linker histone (H1) and heterochromatin protein 1 (HP1) are essential componentsof heterochromatin which contribute to the transcriptional repression of genes. It has been shown that the methylation mark of vertebrate histone H1 is specifically recognized by the chromodomain of HP1. However, the exact biological role of linker histone binding to HP1 has not been determined. Here, we investigate the function of the Caenorhabditis elegans H1 variant HIS-24 and theHP1-like proteins HPL-1 and HPL-2 in the cooperative transcriptional regulation of immune-relevant genes. We provide the first evidence that HPL-1 interacts with HIS-24 monomethylated at lysine 14 (HIS-24K14me1) and associates in vivo with promoters of genes involved in antimicrobial response. We also report an increase in overall cellular levels and alterations in the distribution of HIS-24K14me1 after infection with pathogenic bacteria. HIS-24K14me1 localization changes frombeing mostly nuclear to both nuclear and cytoplasmic in the intestinal cells of infected animals. Our results highlight an antimicrobial role of HIS-24K14me1 and suggest a functional link between epigenetic regulation by an HP1/H1 complex andthe innate immune system in C. elegans.

Programmed genome rearrangements: in lampreys, all cells are not equal.

Author(s) : Semon M, Schubert M, Laudet V,
Journal : Curr Biol
2012
How can organisms silence deleterious gene loci? A recent study has shed light on a very brute mechanism in a jawless vertebrate: the irreversible deletion of massive chunks of genomic DNA.

Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

Author(s) : Raverdeau M, Gely-Pernot A, Feret B, Dennefeld C, Benoit G, Davidson I, Chambon P, Mark M, Ghyselinck N,
Journal : Proc Natl Acad Sci U S A
2012
Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiatedifferentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates theeffects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

Author(s) : Moutier E, Ye T, Choukrallah M, Urban S, Osz J, Chatagnon A, Delacroix L, Langer D, Rochel N, Moras D, Benoit G, Davidson I,
Journal : J Biol Chem
2012
Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separatedby 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodiesor F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

Splicing programs and cancer.

Author(s) : Germann S, Gratadou L, Dutertre M, Auboeuf D,
Journal : J Nucleic Acids
2012
Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets.

Splicing switch of an epigenetic regulator by RNA helicases promotes tumor-cell invasiveness.

Author(s) : Dardenne E, Pierredon S, Driouch K, Gratadou L, Lacroix-Triki M, Espinoza M, Zonta E, Germann S, Mortada H, Villemin J, Dutertre M, Lidereau R, Vagner S, Auboeuf D,
Journal : Nat Struct Mol Biol
2012
Both epigenetic and splicing regulation contribute to tumor progression, but thepotential links between these two levels of gene-expression regulation in pathogenesis are not well understood. Here, we report that the mouse and human RNA helicases Ddx17 and Ddx5 contribute to tumor-cell invasiveness by regulatingalternative splicing of several DNA- and chromatin-binding factors, including the macroH2A1 histone. We show that macroH2A1 splicing isoforms differentially regulate the transcription of a set of genes involved in redox metabolism. In particular, the SOD3 gene that encodes the extracellular superoxide dismutase and plays a part in cell migration is regulated in an opposite manner by macroH2A1 splicing isoforms. These findings reveal a new regulatory pathway in which splicing factors control the expression of histone variant isoforms that in turndrive a transcription program to switch tumor cells to an invasive phenotype.

Tax protein-induced expression of antiapoptotic Bfl-1 protein contributes to survival of human T-cell leukemia virus type 1 (HTLV-1)-infected T-cells.

Author(s) : Macaire H, Riquet A, Moncollin V, Biemont-Trescol M, Duc Dodon M, Hermine O, Debaud A, Mahieux R, Mesnard J, Pierre M, Gazzolo L, Bonnefoy N, Valentin H,
Journal : J Biol Chem
2012
Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor(HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4(+) T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restrictedto HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-kappaB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation,whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-kappaB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-x(L), and Bcl-2. Indeed, both Bfl-1 and Bcl-x(L) knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-x(L) in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-x(L) represent potential therapeutic targets for ATLL treatment.

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

Author(s) : Vera-Otarola J, Solis L, Soto-Rifo R, Ricci E, Pino K, Tischler N, Ohlmann T, Darlix J, Lopez-Lastra M,
Journal : J Virol
2012
The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein.In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggestthat translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The human T-lymphotropic virus type 1 tax protein inhibits nonsense-mediated mRNA decay by interacting with INT6/EIF3E and UPF1.

Author(s) : Mocquet V, Neusiedler J, Rende F, Cluet D, Robin J, Terme J, Duc Dodon M, Wittmann J, Morris C, Le Hir H, Ciminale V, Jalinot P,
Journal : J Virol
2012
In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibitedin C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required forefficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation.