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2013

A long noncoding RNA mediates both activation and repression of immune response genes.

Author(s) : Carpenter S, Aiello D, Atianand M, Ricci E, Gandhi P, Hall L, Byron M, Monks B, Henry-Bezy M, Lawrence J, O'Neill L, Moore M, Caffrey D, Fitzgerald K,
Journal : Science
2013
An inducible program of inflammatory gene expression is central to antimicrobialdefenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.

Absolute requirement of cholesterol binding for Hedgehog gradient formation in Drosophila.

Author(s) : Ducuing A, Mollereau B, Axelrod J, Vincent S,
Journal : Biol Open
2013
How morphogen gradients are shaped is a major question in developmental biology,but remains poorly understood. Hedgehog (Hh) is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to HhN-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N) remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand.

Antiretroviral therapy promotes an inflammatory-like pattern of human T-cell lymphotropic virus type 1 (HTLV-1) replication in human immunodeficiency virus type 1/HTLV-1 co-infected individuals.

Author(s) : Pomier C, Rabaaoui S, Pouliquen J, Couppie P, El Guedj M, Nacher M, Lacoste V, Wattel E, Kazanji M, Mortreux F,
Journal : J Gen Virol
2013
Upon antiretroviral therapy (ART) human immunodeficiency virus (HIV)/human T-cell lymphotropic virus type 1 (HTLV-1) co-infected individuals frequently develop neurological disorders through hitherto unknown mechanisms. Here, we show that effective anti-HIV ART increases HTLV-1 proviral load through a polyclonal integration pattern of HTLV-1 in both CD4(+) and CD8(+) T-cell subsets that is reminiscent of that typically associated with HTLV-1-related inflammatory conditions. These data indicate that preventing ART-triggered clonal expansion of HTLV-1-infected cells in co-infected individuals deserves investigation.

Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster.

Author(s) : Gutierrez L, Zubow K, Nield J, Gambis A, Mollereau B, Lazaro F, Missirlis F,
Journal : Metallomics
2013
Metals have vital functions as prosthetic groups in enzymes, but in labile form they can propagate oxidative stress. The primary function of ferritin is to store bioavailable iron in the form of ferrihydrite. In animals, ferritin is also usedto traffic and recycle iron, and to modulate intestinal iron absorption. However, the effect of ferritin accumulation on cellular iron bioavailability remains poorly understood. Moreover, putative in vivo interactions of ferritin with other metal ions have been proposed, but their physiological relevance remains unclear. Here, heterozygous mutant and overexpression ferritin strains of Drosophila melanogaster were subjected to dietary iron manipulations to study the dynamics of iron partition between ferritin and other proteins. Quantitative magnetic analysis of whole fly samples indicated that iron loading of the ferritin core varied in the different genotypes. Total paramagnetic iron content, a likely correlate of bioavailable iron, was reduced in flies overexpressing ferritin when compared with control white flies. Further, three-dimensional maps of the ferritin protein shell and iron core were obtained from single particle transmission electron microscopy imaging and confirmed the similarity between Drosophila and Trichoplusia ferritin structures. Purified Drosophila ferritin also contained small amounts of zinc and manganese. Flies that overexpressed ferritin accumulated in their bodies half the amount of manganese compared to their respective controls. Our results indicate that ferritin may be involved inthe homeostasis of other divalent metals, besides iron, and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells.

Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

Author(s) : Dibenedetto S, Cluet D, Stebe P, Baumle V, Leault J, Terreux R, Bickle M, Chassey B, Mikaelian I, Colas P, Spichty M, Zoli M, Rudkin B,
Journal : Mol Cell Proteomics
2013
We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two bindingproteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants ofthe variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42,we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.

Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

Author(s) : Dibenedetto S, Cluet D, Stebe P, Baumle V, Leault J, Terreux R, Bickle M, Chassey B, Mikaelian I, Colas P, Spichty M, Zoli M, Rudkin B,
Journal : Mol Cell Proteomics
2013
We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two bindingproteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants ofthe variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42,we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.

Cause-specific telomere factors deregulation in hepatocellular carcinoma.

Author(s) : El Idrissi M, Hervieu V, Merle P, Mortreux F, Wattel E,
Journal : J Exp Clin Cancer Res
2013
BACKGROUND: Among the numerous genetic defects associated with hepatocarcinogenesis, telomere abnormalities appear to play a role both in tumorpromotion and maintenance. Telomeres, the chromosome extremities, are protected by specific proteins, the shelterin complex and by additional factors. Besides telomerase dysregulation, expression changes of these telomere factors have beenobserved in cancers. METHODS: Here, we tested the hypothesis that such dysregulation might occur in hepatocellular carcinoma (HCC) with specific patterns depending on the cause of HCC. We compared telomere length, telomerase activity (TA), hTERT and telomere genes expression using PCR and Western-blot analyses between non-cirrhotic liver, peritumoral cirrhotic tissue (40 samples) and cancerous tissue (40 samples) derived from 40 patients with HBV-, HCV-, or alcohol-related HCC. RESULTS: Alterations in TA, hTERT expression and telomere length between non-cirrhotic, cirrhotic, and tumor samples were not significantly influenced by the cause of HCC. In contrast, the expression pattern of hTR, shelterin, and non-shelterin telomere protective factors clearly distinguished the 3 causes of cirrhosis and HCC. For patients with HBV diseased liver, when compared with non-cirrhotic liver, the cirrhotic tissue underexpressed all shelterin and all but HMRE11A and RAD50 non-shelterin telomere factors. For HCV the expression level of POT1, RAP1, Ku80, and RAD50 was higher in cirrhotic thanin non-cirrhotic liver samples without evidence for significant transcriptional change for the remaining genes. For alcohol-related liver diseases, the expression level of POT1, RAP1, TIN2, hMRE11A, hMRE11B, Ku70, Ku80, RAD50, TANK1, and PINX1 was higher in cirrhotic than in non-cirrhotic liver samples. For the 3causes of HCC, there was no significant change in shelterin and non-shelterin gene expression between cirrhosis and HCC samples. CONCLUSIONS: These results validate our hypotheses and demonstrate that cirrhosis and HCC add-up numerous telomere dysfunctions including numerous cause-specific changes that appear to occur early during the course of the disease.

Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma.

Author(s) : Rosewick N, Momont M, Durkin K, Takeda H, Caiment F, Cleuter Y, Vernin C, Mortreux F, Wattel E, Burny A, Georges M, Van den Broeke A,
Journal : Proc Natl Acad Sci U S A
2013
Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy, however, remains poorly understood, especially as viral mRNA/protein are tightlysilenced in tumors. Here, using deep sequencing of broad windows of small RNA sizes in the bovine leukemia virus ovine model of leukemia/lymphoma, we provide in vivo evidence of the production of noncanonical RNA polymerase III (Pol III)-transcribed viral microRNAs in leukemic B cells in the complete absence of Pol II 5'-LTR-driven transcriptional activity. Processed from a cluster of five independent self-sufficient transcriptional units located in a proviral region dispensable for in vivo infectivity, bovine leukemia virus microRNAs represent approximately 40% of all microRNAs in both experimental and natural malignancy. They are subject to strong purifying selection and associate with Argonautes, consistent with a critical function in silencing of important cellular and/or viral targets. Bovine leukemia virus microRNAs are strongly expressed in preleukemic and malignant cells in which structural and regulatory gene expression is repressed, suggesting a key role in tumor onset and progression. Understanding how Pol III-dependent microRNAs subvert cellular and viral pathways will contribute to deciphering the intricate perturbations that underlie malignant transformation.

Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation.

Author(s) : Joly-Tonetti N, Vinuelas J, Gandrillon O, Lamartine J,
Journal : BMC Genomics
2013
BACKGROUND: MicroRNAs (miRNAs), a group of short non-coding RNAs that negativelyregulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity isan important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. RESULTS: We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. Wefound that proliferative cells irradiated at 6 Gy display a global fall of miRNAexpression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated bythese responding miRNAs. CONCLUSIONS: Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern stronglyrelated to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.

Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation.

Author(s) : Dichtel-Danjoy M, Ma D, Dourlen P, Chatelain G, Napoletano F, Robin M, Corbet M, Levet C, Hafsi H, Hainaut P, Ryoo H, Bourdon J, Mollereau B,
Journal : Cell Death Differ
2013
Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expressionand growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DDeltaNp53). Historically, DDeltaNp53 wasthe first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DDeltaNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DDeltaNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DDeltaNp53 induced Wingless (Wg)expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DDeltaNp53, Dp53 did not induce Wg expression inthe absence of the endogenous p53 gene. Thus, we propose that DDeltaNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

Endogenous nuclear RNAi mediates behavioral adaptation to odor.

Author(s) : Juang B, Gu C, Starnes L, Palladino F, Goga A, Kennedy S, L'Etoile N,
Journal : Cell
2013
Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show thatthe endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior.This class of siRNA may act broadly as a rheostat allowing prolonged stimulationto dampen gene expression and promote cellular memory formation. PAPERFLICK:

Establishing links between endoplasmic reticulum-mediated hormesis and cancer.

Author(s) : Mollereau B,
Journal : Mol Cell Biol
2013

Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos.

Author(s) : Riche S, Zouak M, Argoul F, Arneodo A, Pecreaux J, Delattre M,
Journal : J Cell Biol
2013
During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Galpha-GPR-LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and(3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.

Genome-wide analysis of thyroid hormone receptors shared and specific functions in neural cells.

Author(s) : Chatonnet F, Guyot R, Benoit G, Flamant F,
Journal : Proc Natl Acad Sci U S A
2013
TRalpha1 and TRbeta1, the two main thyroid hormone receptors in mammals, are transcription factors that share similar properties. However, their respective functions are very different. This functional divergence might be explained in two ways: it can reflect different expression patterns or result from different intrinsic properties of the receptors. We tested this second hypothesis by comparing the repertoires of 3,3',5-triiodo-L-thyronine (T3)-responsive genes oftwo neural cell lines, expressing either TRalpha1 or TRbeta1. Using transcriptome analysis, we found that a substantial fraction of the T3 target genes display a marked preference for one of the two receptors. So when placed alone in identical situations, the two receptors have different repertoires of target genes. Chromatin occupancy analysis, performed at a genome-wide scale, revealed that TRalpha1 and TRbeta1 cistromes were also different. However, receptor-selective regulation of T3 target genes did not result from receptor-selective chromatin occupancy of their promoter regions. We conclude that modification of TRalpha1 and TRbeta1 intrinsic properties contributes in a large part to the divergent evolution of the receptors' function, at least during neurodevelopment.

hCAF1/CNOT7 regulates interferon signalling by targeting STAT1.

Author(s) : Chapat C, Kolytcheff C, Le Romancer M, Auboeuf D, De La Grange P, Chettab K, Sentis S, Corbo L,
Journal : EMBO J
2013
Stringent regulation of the interferon (IFN) signalling pathway is essential formaintaining the immune response to pathogens and tumours. The transcription factor STAT1 is a crucial mediator of this response. Here, we show that hCAF1/CNOT7 regulates class I and II IFN pathways at different crucial steps. Inresting cells, hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistently, hCAF1 silencing enhances STAT1 basal promoter occupancy associatedwith increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradationof some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activationis associated with immune disorders and cancer, hCAF1 could play a major role ininnate immunity and oncogenesis, contributing to tumour escape.

HTLV-1 bZIP factor impedes the menin tumor suppressor and upregulates JunD-mediated transcription of the hTERT gene.

Author(s) : Borowiak M, Kuhlmann A, Girard S, Gazzolo L, Mesnard J, Jalinot P, Dodon M,
Journal : Carcinogenesis
2013
Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD toenhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression inATL cells confirms that these proteins are differentially involved in telomeraseregulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.

HTLV-1-associated inflammatory myopathies: low proviral load and moderate inflammation in 13 patients from West Indies and West Africa.

Author(s) : Desdouits M, Cassar O, Maisonobe T, Desrames A, Aouba A, Hermine O, Mikol J, Polivka M, Penisson-Besnier I, Marcorelles P, Zagnoli F, Papo T, Lacour A, Amoura Z, Haroche J, Cherin P, Teixeira A, Benveniste O, Herson S, Morin A, Mortreux F, Wattel E, Huerre M, Cumont M, Martin-Latil S, Butler-Browne G, Gout O, Taylor G, Gessain A, Ozden S, Ceccaldi P,
Journal : J Clin Virol
2013
BACKGROUND: The Human T-cell Leukemia Virus type 1 (HTLV-1) is the causative agent of several inflammatory diseases, including HTLV-1-associated inflammatorymyopathies (HAIM). Little is known about the virological and immunological characteristics of this viral disease. OBJECTIVES: To characterize the histological and virological features of HAIM patients, in order to better understand the pathogenetic mechanisms and unravel new biological markers of this disease. STUDY DESIGN: We conducted a retrospective study on 13 patients with HAIM, based on blood and muscle samples. We included blood samples from HTLV-1-infected individuals without myopathy as controls. Muscle biopsies were used for a broad immunohistological evaluation of tissue damage and inflammation, as well as identification of infected cells through in situ hybridization. DNA extracted from patients' PBMC was used to identify the virus genotype by sequencing and to assess the proviral load by quantitative PCR. Anti-viral antibodies in plasma samples were titrated by indirect immunofluorescence. RESULTS: Patients originate from HTLV-1 endemic areas, the West Indies and West Africa. Histological alterations and inflammation in patients muscles were mostly moderate, with classical features of idiopathic myositis and rare HTLV-1-infected infiltrating cells. In all patients, HTLV-1 belonged to the A subtype, transcontinental subgroup. Anti-HTLV-1 antibodies titers were high, but the proviral load was not elevated compared to asymptomatic HTLV-1 carriers. CONCLUSION: We show here that muscle inflammation is moderate in HAIM, and accompanied by a low HTLV-1 proviral load, suggesting that the pathogenetic events do not exactly mirror those of other HTLV-1-associated inflammatory diseases.

miRNA repression of translation in vitro takes place during 43S ribosomal scanning.

Author(s) : Ricci E, Limousin T, Soto-Rifo R, Rubilar P, Decimo D, Ohlmann T,
Journal : Nucleic Acids Res
2013
microRNAs (miRNAs) regulate gene expression at multiple levels by repressing translation, stimulating deadenylation and inducing the premature decay of target messenger RNAs (mRNAs). Although the mechanism by which miRNAs repress translation has been widely studied, the precise step targeted and the molecularinsights of such repression are still evasive. Here, we have used our newly designed in vitro system, which allows to study miRNA effect on translation independently of deadenylation. By using specific inhibitors of various stages of protein synthesis, we first show that miRNAs target exclusively the early steps of translation with no effect on 60S ribosomal subunit joining, elongation or termination. Then, by using viral proteases and IRES-driven mRNA constructs, we found that translational inhibition takes place during 43S ribosomal scanning and requires both the poly(A) binding protein and eIF4G independently from their physical interaction.

Mosaicism of HTLV-1 5' LTR CpG methylation in the absence of malignancy.

Author(s) : Sibon D, Zane L, Idrissi M, Delfau-Larue M, Gessain A, Gout O, Mortreux F, Wattel E,
Journal : Virus Res
2013
Viral expression varies widely between untransformed HTLV-1 positive clones derived from infected individuals without malignancy. Here we show that, in the absence of malignancy, 68% of HTLV-1 positive clones carry deleted (10%) or methylated (58%) 5' LTR. These changes were found to contribute to the fluctuation of viral expression between clones. 5' LTR deletions strongly impaired tax expression and thereby clonal expansion, telomere homeostasis, and genetic instability. The effects of CpG methylation on viral transcription were qualitatively and quantitatively different from those of LTR deletions and preserved the preleukemic features of HTLV-1(+)CD4(+) clones. 5' LTR methylationvaried not only between clones but also between cells belonging to the same clones, between distinct integrated sequences within the same cells, and betweenCpG dyads along the 5' LTR. Such distributions suggest that a dynamic methylation spectrum might help sustain persistent infection.

Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability.

Author(s) : Fehrmann S, Bottin-Duplus H, Leonidou A, Mollereau E, Barthelaix A, Wei W, Steinmetz L, Yvert G,
Journal : Mol Syst Biol
2013
Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P(met17)-GFPreporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P(met17)-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transportergenes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts.

Author(s) : Vinuelas J, Kaneko G, Coulon A, Vallin E, Morin V, Mejia-Pous C, Kupiec J, Beslon G, Gandrillon O,
Journal : BMC Biol
2013
BACKGROUND: A number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells. RESULTS: For this purpose, we generated isogenic chicken-cell populations expressing a fluorescent reporterintegrated in one copy per clone. Although the clones differed only in the genetic locus at which the reporter was inserted, they showed markedly differentfluorescence distributions, revealing different levels of stochastic gene expression. Use of chromatin-modifying agents showed that direct manipulation ofchromatin dynamics had a marked effect on the extent of stochastic gene expression. To better understand the molecular mechanism involved in these phenomena, we fitted these data to a two-state model describing the opening/closing process of the chromatin. We found that the differences between clones seemed to be due mainly to the duration of the closed state, and that theagents we used mainly seem to act on the opening probability. CONCLUSIONS: In this study, we report biological experiments combined with computational modeling, highlighting the importance of chromatin dynamics in stochastic gene expression. This work sheds a new light on the mechanisms of gene expression in higher eukaryotic cells, and argues in favor of relatively slow dynamics with long (hours to days) periods of quiet state.

Single-cell phenomics reveals intra-species variation of phenotypic noise in yeast.

Author(s) : Yvert G, Ohnuki S, Nogami S, Imanaga Y, Fehrmann S, Schacherer J, Ohya Y,
Journal : BMC Syst Biol
2013
BACKGROUND: Most quantitative measures of phenotypic traits represent macroscopic contributions of large numbers of cells. Yet, cells of a tissue do not behave similarly, and molecular studies on several organisms have shown that regulations can be highly stochastic, sometimes generating diversified cellular phenotypes within tissues. Phenotypic noise, defined here as trait variability among isogenic cells of the same type and sharing a common environment, has therefore received a lot of attention. Given the potential fitness advantage provided by phenotypic noise in fluctuating environments, the possibility that it is directly subjected to evolutionary selection is being considered. For selection to act, phenotypic noise must differ between contemporary genotypes. Whether this is thecase or not remains, however, unclear because phenotypic noise has very rarely been quantified in natural populations. RESULTS: Using automated image analysis,we describe here the phenotypic diversity of S. cerevisiae morphology at single-cell resolution. We profiled hundreds of quantitative traits in more than1,000 cells of 37 natural strains, which represent various geographical and ecological origins of the species. We observed abundant trait variation between strains, with no correlation with their ecological origin or population history.Phenotypic noise strongly depended on the strain background. Noise variation waslargely trait-specific (specific strains showing elevated noise for subset of traits) but also global (a few strains displaying elevated noise for many unrelated traits). CONCLUSIONS: Our results demonstrate that phenotypic noise does differ quantitatively between natural populations. This supports the possibility that, if noise is adaptive, microevolution may tune it in the wild. This tuning may happen on specific traits or by varying the degree of global phenotypic buffering.

TAF15 is important for cellular proliferation and regulates the expression of a subset of cell cycle genes through miRNAs.

Author(s) : Ballarino M, Jobert L, Dembele D, de la Grange P, Auboeuf D, Tora L,
Journal : Oncogene
2013
TAF15 (formerly TAFII68) is a member of the FET (FUS, EWS, TAF15) family of RNA-and DNA-binding proteins whose genes are frequently translocated in sarcomas. Byperforming global gene expression profiling, we found that TAF15 knockdown affects the expression of a large subset of genes, of which a significant percentage is involved in cell cycle and cell death. In agreement, TAF15 depletion had a growth-inhibitory effect and resulted in increased apoptosis. Among the TAF15-regulated genes, targets of microRNAs (miRNAs) generated from the onco-miR-17 locus were overrepresented, with CDKN1A/p21 being the top miRNAs-targeted gene. Interestingly, the levels of onco-miR-17 locus coded miRNAs (miR-17-5p and miR-20a) were decreased upon TAF15 depletion and shown to affect the post-transcriptional regulation of TAF15-dependent genes, such as CDKN1A/p21. Thus, our results demonstrate that TAF15 is required to regulate gene expressionof cell cycle regulatory genes post-transcriptionally through a pathway involving miRNAs. The findings that high TAF15 levels are needed for rapid cellular proliferation and that endogenous TAF15 levels decrease during differentiation strongly suggest that TAF15 is a key regulator of maintaining a highly proliferative rate of cellular homeostasis.

The RNA helicase DDX5/p68 is a key factor promoting c-fos expression at different levels from transcription to mRNA export.

Author(s) : Zonta E, Bittencourt D, Samaan S, Germann S, Dutertre M, Auboeuf D,
Journal : Nucleic Acids Res
2013
It is widely accepted that pre-mRNA maturation, including splicing, is tightly coupled to both transcription and mRNA export, but factors linking the three processes are less understood. By analysing the estrogen-regulated expression ofthe c-fos mRNA that is processed during transcription, we show that the ddx5 RNAhelicase, is required throughout the major nuclear steps of the expression of the c-fos gene, from transcription to mRNA export. Indeed, ddx5, whose recruitment on the c-fos gene was increased upon estrogen treatment, was required for the full transcriptional activation of the c-fos gene. In addition, ddx5 was required forc-fos co-transcriptional RNA splicing. When splicing occurred post-transcriptionally in the absence of ddx5, the c-fos mRNA was poorly exported into the cytosol because of inefficient recruitment of the TAP mRNA export receptor. Finally, ddx5 was present in the c-fos messenger ribonucleoprotein together with mRNA export factors, which further supports that ddx5 is a key operator in the c-fos 'mRNA factory'.

The Tomato/GFP-FLP/FRT method for live imaging of mosaic adult Drosophila photoreceptor cells.

Author(s) : Dourlen P, Levet C, Mejat A, Gambis A, Mollereau B,
Journal : J Vis Exp
2013
The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying thefactors regulating PR survival and function. It can be used for kinetic analysesof PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly.This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

Twist-DNA: computing base-pair and bubble opening probabilities in genomic superhelical DNA.

Author(s) : Jost D,
Journal : Bioinformatics
2013
SUMMARY: Local opening of the DNA double helix is required in many fundamental biological processes and is, in part, controlled by the degree of superhelicity imposed in vivo by the protein machinery. In particular, positions of superhelically destabilized regions correlate with regulatory sites along the genome. Based on a self-consistent linearization of a thermodynamic model of superhelical DNA introduced by Benham, we have developed a program that predictsthe locations of these regions by efficiently computing base-pair and bubble opening probabilities in genomic DNA. The program allows visualization of results in standard genome browsers to compare DNA opening properties with other available datasets. AVAILABILITY AND IMPLEMENTATION: Source codes freely available for download at http://www.cbp.ens-lyon.fr/doku.php?id=developpement:productions:logiciels:twistd na, implemented in Fortran90 and supported on any Unix-based operating system (Linux, Mac OS X).