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2014

'Particle genetics': treating every cell as unique.

Author(s) : Yvert G,
Journal : Trends Genet
2014
Genotype-phenotype relations are usually inferred from a deterministic point of view. For example, quantitative trait loci (QTL), which describe regions of the genome associated with a particular phenotype, are based on a mean trait difference between genotype categories. However, living systems comprise huge numbers of cells (the 'particles' of biology). Each cell can exhibit substantialphenotypic individuality, which can have dramatic consequences at the organismallevel. Now, with technology capable of interrogating individual cells, it is time to consider how genotypes shape the probability laws of single cell traits. The possibility of mapping single cell probabilistic trait loci (PTL), which link genomic regions to probabilities of cellular traits, is a promising step in thisdirection. This approach requires thinking about phenotypes in probabilistic terms, a concept that statistical physicists have been applying to particles fora century. Here, I describe PTL and discuss their potential to enlarge our understanding of genotype-phenotype relations.

A genetic screen for functional partners of condensin in fission yeast.

Author(s) : Robellet X, Fauque L, Legros P, Mollereau E, Janczarski S, Parrinello H, Desvignes J, Thevenin M, Bernard P,
Journal : G3 (Bethesda)
2014
Mitotic chromosome condensation is a prerequisite for the accurate segregation of chromosomes during cell division, and the conserved condensin complex a central player of this process. However, how condensin binds chromatin and shapes mitotic chromosomes remain poorly understood. Recent genome-wide binding studies showingthat in most species condensin is enriched near highly expressed genes suggest aconserved link between condensin occupancy and high transcription rates. To gaininsight into the mechanisms of condensin binding and mitotic chromosome condensation, we searched for factors that collaborate with condensin through a synthetic lethal genetic screen in the fission yeast Schizosaccharomyces pombe. We isolated novel mutations affecting condensin, as well as mutations in four genes not previously implicated in mitotic chromosome condensation in fission yeast. These mutations cause chromosome segregation defects similar to those provoked by defects in condensation. We also identified a suppressor of the cut3-477 condensin mutation, which largely rescued chromosome segregation duringanaphase. Remarkably, of the five genes identified in this study, four encode transcription co-factors. Our results therefore provide strong additional evidence for a functional connection between chromosome condensation and transcription.

A recently evolved class of alternative 3'-terminal exons involved in cell cycle regulation by topoisomerase inhibitors.

Author(s) : Dutertre M, Chakrama F, Combe E, Desmet F, Mortada H, Polay Espinoza M, Gratadou L, Auboeuf D,
Journal : Nat Commun
2014
Alternative 3'-terminal exons, which use intronic polyadenylation sites, are generally less conserved and expressed at lower levels than the last exon of genes. Here we discover a class of human genes, in which the last exon appeared recently during evolution, and the major gene product uses an alternative 3'-terminal exon corresponding to the ancestral last exon of the gene. This novel class of alternative 3'-terminal exons are downregulated on a large scale by doxorubicin, a cytostatic drug targeting topoisomerase II, and play a role in cell cycle regulation, including centromere-kinetochore assembly. The RNA-binding protein HuR/ELAVL1 is a major regulator of this specific set of alternative 3'-terminal exons. HuR binding to the alternative 3'-terminal exon in the pre-messenger RNA promotes its splicing, and is reduced by topoisomerase inhibitors. These findings provide new insights into the evolution, function andmolecular regulation of alternative 3'-terminal exons.

Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

Author(s) : Cluet D, Stebe P, Riche S, Spichty M, Delattre M,
Journal : PLoS One
2014
The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.

Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

Author(s) : Cluet D, Stebe P, Riche S, Spichty M, Delattre M,
Journal : PLoS One
2014
The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.

Bifurcation in epigenetics: implications in development, proliferation, and diseases.

Author(s) : Jost D,
Journal : Phys Rev E Stat Nonlin Soft Matter Phys
2014
Cells often exhibit different and stable phenotypes from the same DNA sequence. Robustness and plasticity of such cellular states are controlled by diverse transcriptional and epigenetic mechanisms, among them the modification of biochemical marks on chromatin. Here, we develop a stochastic model that describes the dynamics of epigenetic marks along a given DNA region. Through mathematical analysis, we show the emergence of bistable and persistent epigenetic states from the cooperative recruitment of modifying enzymes. We alsofind that the dynamical system exhibits a critical point and displays, in the presence of asymmetries in recruitment, a bifurcation diagram with hysteresis. These results have deep implications for our understanding of epigenetic regulation. In particular, our study allows one to reconcile within the same formalism the robust maintenance of epigenetic identity observed in differentiated cells, the epigenetic plasticity of pluripotent cells during differentiation, and the effects of epigenetic misregulation in diseases. Moreover, it suggests a possible mechanism for developmental transitions where the system is shifted close to the critical point to benefit from high susceptibility to developmental cues.

C. elegans epigenetic regulation in development and aging.

Author(s) : Gonzalez-Aguilera C, Palladino F, Askjaer P,
Journal : Brief Funct Genomics
2014
The precise developmental map of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation-resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity.

Chromatin redistribution of the DEK oncoprotein represses hTERT transcription in leukemias.

Author(s) : Karam M, Thenoz M, Capraro V, Robin J, Pinatel C, Lancon A, Galia P, Sibon D, Thomas X, Ducastelle-Lepretre S, Nicolini F, El-Hamri M, Chelghoun Y, Wattel E, Mortreux F,
Journal : Neoplasia
2014
Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this contextof chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis.

CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation.

Author(s) : Vanoosthuyse V, Legros P, van der Sar S, Yvert G, Toda K, Le Bihan T, Watanabe Y, Hardwick K, Bernard P,
Journal : PLoS Genet
2014
Functional links connecting gene transcription and condensin-mediated chromosomecondensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3' end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does notaffect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1(Dis2) with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologueof vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatasesPP1 and Ssu72.

Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

Author(s) : Sharma R, Jost D, Kind J, Gomez-Saldivar G, van Steensel B, Askjaer P, Vaillant C, Meister P,
Journal : Genes Dev
2014
The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-widegene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while inhermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domainsin males, while the DCC prevents this in hermaphrodites.

Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases.

Author(s) : Hetz C, Mollereau B,
Journal : Nat Rev Neurosci
2014
The unfolded protein response (UPR) is a homeostatic mechanism by which cells regulate levels of misfolded proteins in the endoplasmic reticulum (ER). Although it is well characterized in non-neuronal cells, a proliferation of papers over the past few years has revealed a key role for the UPR in normal neuronal function and as an important driver of neurodegenerative diseases. A complex scenario is emerging in which distinct UPR signalling modules have specific and even opposite effects on neurodegeneration depending on the disease context. Here, we provide an overview of the most recent findings addressing the biological relevance of ER stress in the nervous system.

Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin.

Author(s) : Mallinjoud P, Villemin J, Mortada H, Polay Espinoza M, Desmet F, Samaan S, Chautard E, Tranchevent L, Auboeuf D,
Journal : Genome Res
2014
Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type-specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution.

Getting the better of ER stress.

Author(s) : Mollereau B, Manie S, Napoletano F,
Journal : J Cell Commun Signal
2014
Research over the past few years has highlighted the ability of the unfolded protein response (UPR) to minimize the deleterious effects of accumulated misfolded proteins under both physiological and pathological conditions. The endoplasmic reticulum (ER) adapts to endogenous and exogenous stressors by expanding its protein-folding capacity and by stimulating protective processes such as autophagy and antioxidant responses. Although it is clear that severe ERstress can elicit cell death, several recent studies have shown that low levels of ER stress may actually be beneficial to cells by eliciting an adaptive UPR that 'preconditions' the cell to a subsequent lethal insult; this process is called ER hormesis. The findings have important implications for the treatment of a wide variety of diseases associated with defective proteostasis, including neurodegenerative diseases, diabetes, and cancer. Here, we review the physiological and pathological functions of the ER, with a particular focus on the molecular mechanisms that lead to ER hormesis and cellular protection, and discuss the implications for disease treatment.

HIV-2 genomic RNA accumulates in stress granules in the absence of active translation.

Author(s) : Soto-Rifo R, Valiente-Echeverria F, Rubilar P, Garcia-de-Gracia F, Ricci E, Limousin T, Decimo D, Mouland A, Ohlmann T,
Journal : Nucleic Acids Res
2014
During the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells.This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding proteinand stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observedthat increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a 'packageable' gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein.

How does evolution tune biological noise?

Author(s) : Richard M, Yvert G,
Journal : Front Genet
2014
Part of molecular and phenotypic differences between individual cells, between body parts, or between individuals can result from biological noise. This sourceof variation is becoming more and more apparent thanks to the recent advances indynamic imaging and single-cell analysis. Some of these studies showed that the link between genotype and phenotype is not strictly deterministic. Mutations canchange various statistical properties of a biochemical reaction, and thereby theprobability of a trait outcome. The fact that they can modulate phenotypic noisebrings up an intriguing question: how may selection act on these mutations? In this review, we approach this question by first covering the evidence that biological noise is under genetic control and therefore a substrate for evolution. We then sequentially inspect the possibilities of negative, neutral, and positive selection for mutations increasing biological noise. Finally, we hypothesize on the specific case of H2A.Z, which was shown to both buffer phenotypic noise and modulate transcriptional efficiency.

How mRNA is misspliced in acute myelogenous leukemia (AML)?

Author(s) : Mohamed A, Thenoz M, Solly F, Balsat M, Mortreux F, Wattel E,
Journal : Oncotarget
2014
Approximately one-third of expressed genes are misspliced in AML, opening the possibility that additional factors than splicing factor mutations might cause RNA missplicing in these diseases. AML cells harbor a constellation of epigenetic modifications and regularly express large amounts of WT1 transcripts. Histone acetylation/methylation and DNA CpG methylation favor either exon skipping or inclusion, mainly through interfering with RNA Pol II-mediated elongation. This can result either from the binding of various factors on Pol II or alternativelyfrom the recruitment of DNA binding factors that create roadblocks to Pol II-induced elongation. WT1 exhibits pleiotropic effects on mRNA splicing, which mainly result from the binding properties of WT1 via its zinc fingers domains toDNA, RNA, and proteins. Through the repression of the kinase SRPK1, WT1 modifiesthe splicing of VEGF, which plays important roles in hematopoiesis and angiogenesis. At the protein level, WT1 interacts with the splicing factors U2AF2, WTAP, and RPM4. Therefore, AML cells appear to have acquired numerous properties known to interfere with mRNA splicing. The challenge is now to elucidate these links in order to trigger mRNA splicing at the therapeutic level.

HTLV-1 bZIP factor HBZ promotes cell proliferation and genetic instability by activating OncomiRs.

Author(s) : Vernin C, Thenoz M, Pinatel C, Gessain A, Gout O, Delfau-Larue M, Nazaret N, Legras-Lachuer C, Wattel E, Mortreux F,
Journal : Cancer Res
2014
Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4(+) and CD8(+) T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4(+) phenotype. Theviral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved insustaining cell proliferation and silencing virus expression. Here, we show thatan HBZ-miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4(+) cells, but not CD8(+) T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1-infected CD4(+) T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLLcells.

HTLV-1-infected CD4+ T-cells display alternative exon usages that culminate in adult T-cell leukemia.

Author(s) : Thenoz M, Vernin C, Mortada H, Karam M, Pinatel C, Gessain A, Webb T, Auboeuf D, Wattel E, Mortreux F,
Journal : Retrovirology
2014
BACKGROUND: Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression. RESULTS: Exon expression analysis shows that patients' untransformed and malignant HTLV-1(+) CD4(+) T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involvedin cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4(+) T-cells. Furthermore, untransformedinfected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL. CONCLUSIONS: Taken together, thesedata reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.

Human T-cell leukemia virus type 3 (HTLV-3) and HTLV-4 antisense-transcript-encoded proteins interact and transactivate Jun family-dependent transcription via their atypical bZIP motif.

Author(s) : Larocque E, Andre-Arpin C, Borowiak M, Lemay G, Switzer W, Duc Dodon M, Mesnard J, Barbeau B,
Journal : J Virol
2014
Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque E Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the roleof HBZ in HTLV-1-associated diseases. IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins fromother HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteinsare limited to our previously reported study (M. Larocque E Halin, S. Landry, S.J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.

Immunological alterations and associated diseases in mandrills (Mandrillus sphinx) naturally co-infected with SIV and STLV.

Author(s) : Souquiere S, Makuwa M, Salle B, Lepelletier Y, Mortreux F, Hermine O, Kazanji M,
Journal : Virology
2014
Mandrills are naturally infected with simian T-cell leukaemia virus type 1 (STLV-1) and simian immunodeficiency virus (SIV)mnd. In humans, dual infection with human immunodeficiency virus (HIV) and human T-cell lymphotropic virus type1 (HTLV-1) may worsen their clinical outcome. We evaluated the effect of co-infection in mandrills on viral burden, changes in T-cell subsets and clinical outcome. The SIV viral load was higher in SIV-infected mandrills than in co-infected animals, whereas the STLV-1 proviral load was higher in co-infected than in mono-infected groups. Dually infected mandrills had a statistically significantly lower CD4+ T-cell count, a lower proportion of naive CD8+ T cells and a higher proportion of central memory cells. CD4(+) and CD8(+) T cells from SIV-infected animals had a lower percentage of Ki67 than those from the other groups. Co-infected monkeys had higher percentages of activated CD4(+) and CD8(+) T cells. Two co-infected mandrills with high immune activation and clonal integration of STLV provirus showed pathological manifestations (infective dermatitis and generalised scabies) rarely encountered in nonhuman primates.

Modeling epigenome folding: formation and dynamics of topologically associated chromatin domains.

Author(s) : Jost D, Carrivain P, Cavalli G, Vaillant C,
Journal : Nucleic Acids Res
2014
Genomes of eukaryotes are partitioned into domains of functionally distinct chromatin states. These domains are stably inherited across many cell generations and can be remodeled in response to developmental and external cues, hence contributing to the robustness and plasticity of expression patterns and cell phenotypes. Remarkably, recent studies indicate that these 1D epigenomic domainstend to fold into 3D topologically associated domains forming specialized nuclear chromatin compartments. However, the general mechanisms behind such compartmentalization including the contribution of epigenetic regulation remain unclear. Here, we address the question of the coupling between chromatin foldingand epigenome. Using polymer physics, we analyze the properties of a block copolymer model that accounts for local epigenomic information. Considering copolymers build from the epigenomic landscape of Drosophila, we observe a very good agreement with the folding patterns observed in chromosome conformation capture experiments. Moreover, this model provides a physical basis for the existence of multistability in epigenome folding at sub-chromosomal scale. We show how experiments are fully consistent with multistable conformations where topologically associated domains of the same epigenomic state interact dynamically with each other. Our approach provides a general framework to improve our understanding of chromatin folding during cell cycle and differentiation andits relation to epigenetics.

MyLabStocks: a web-application to manage molecular biology materials.

Author(s) : Chuffart F, Yvert G,
Journal : Yeast
2014
Laboratory stocks are the hardware of research. They must be stored and managed with mimimum loss of material and information. Plasmids, oligonucleotides and strains are regularly exchanged between collaborators within and between laboratories. Managing and sharing information about every item is crucial for retrieval of reagents, for planning experiments and for reproducing past experimental results. We have developed a web-based application to manage stockscommonly used in a molecular biology laboratory. Its functionalities include user-defined privileges, visualization of plasmid maps directly from their sequence and the capacity to search items from fields of annotation or directly from a query sequence using BLAST. It is designed to handle records of plasmids,oligonucleotides, yeast strains, antibodies, pipettes and notebooks. Based on PHP/MySQL, it can easily be extended to handle other types of stocks and it can be installed on any server architecture. MyLabStocks is freely available from: https://forge.cbp.ens-lyon.fr/redmine/projects/mylabstocks under an open source licence.

Post-transcriptional regulation of gene expression in innate immunity.

Author(s) : Carpenter S, Ricci E, Mercier B, Moore M, Fitzgerald K,
Journal : Nat Rev Immunol
2014
Innate immune responses combat infectious microorganisms by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. Multiple genes within each of these functional categories are coordinately and temporally regulated inresponse to distinct external stimuli. The substantial potential of these responses to drive pathological inflammation and tissue damage highlights the need for rigorous control of these responses. Although transcriptional control of inflammatory gene expression has been studied extensively, the importance of post-transcriptional regulation of these processes is less well defined. In thisReview, we discuss the regulatory mechanisms that occur at the level of mRNA splicing, mRNA polyadenylation, mRNA stability and protein translation, and thathave instrumental roles in controlling both the magnitude and duration of the inflammatory response.

RIPiT-Seq: a high-throughput approach for footprinting RNA:protein complexes.

Author(s) : Singh G, Ricci E, Moore M,
Journal : Methods
2014
Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacityto differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds.

RNA helicases DDX5 and DDX17 dynamically orchestrate transcription, miRNA, and splicing programs in cell differentiation.

Author(s) : Dardenne E, Polay Espinoza M, Fattet L, Germann S, Lambert M, Neil H, Zonta E, Mortada H, Gratadou L, Deygas M, Chakrama F, Samaan S, Desmet F, Tranchevent L, Dutertre M, Rimokh R, Bourgeois C, Auboeuf D,
Journal : Cell Rep
2014
The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 alsofunction as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation thatdynamically orchestrate several layers of gene expression.