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2012

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

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.

Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant.

Author(s) : Schneiderman J, Orsi G, Hughes K, Loppin B, Ahmad K,
Journal : Proc Natl Acad Sci U S A
2012
Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions ofthe genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin.Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose thatXnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.

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.

Repeated evolution of testis-specific new genes: the case of telomere-capping genes in Drosophila.

Author(s) : Dubruille R, Marais G, Loppin B,
Journal : Int J Evol Biol
2012
Comparative genome analysis has allowed the identification of various mechanismsinvolved in gene birth. However, understanding the evolutionary forces driving new gene origination still represents a major challenge. In particular, an intriguing and not yet fully understood trend has emerged from the study of new genes: many of them show a testis-specific expression pattern, which has remained poorly understood. Here we review the case of such a new gene, which involves a telomere-capping gene family in Drosophila. hiphop and its testis-specific paralog K81 are critical for the protection of chromosome ends in somatic cells and male gametes, respectively. Two independent functional studies recently proposed that these genes evolved under a reproductive-subfunctionalization regime. The 2011 release of new Drosophila genome sequences from the melanogaster group of species allowed us to deepen our phylogenetic analysis of the hiphop/K81 family. This work reveals an unsuspected dynamic of gene birth and death within the group, with recurrent duplication events through retroposition mechanisms. Finally, we discuss the plausibility of different evolutionary scenarios that could explain the diversification of this gene family.

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

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

Stimulation of gross chromosomal rearrangements by the human CEB1 and CEB25 minisatellites in Saccharomyces cerevisiae depends on G-quadruplexes or Cdc13

Author(s) : Piazza A, Serero A, Boule J, Legoix-Ne P, Lopes J, Nicolas A,
Journal : PLoS genetics
2012
Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC–rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC3 G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites‚ distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.

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.

Thermodynamics and kinetics of large-time-step molecular dynamics.

Author(s) : Rao F, Spichty M,
Journal : J Comput Chem
2012
Molecular dynamics (MD) simulations provide essential information about the thermodynamics and kinetics of proteins. Technological advances in both hardwareand algorithms have seen this method accessing timescales that used to be unreachable only few years ago. The quest to simulate slow, biologically relevant macromolecular conformational changes, is still open. Here, we present an approximate approach to increase the speed of MD simulations by a factor of approximately 4.5. This is achieved by using a large integration time step of 7 fs, in combination with frozen covalent bonds and look-up tables for nonbonded interactions of the solvent. Extensive atomistic MD simulations for a flexible peptide in water show that the approach reproduces the peptide's equilibrium conformational changes, preserving the essential properties of both thermodynamics and kinetics. Comparison of this approximate method with state-of-the-art implicit solvation simulations indicates that the former provides a better description of the underlying free-energy surface. Finally, simulations of a 33-residue peptide show that these fast MD settings are readilyapplicable to investigate biologically relevant systems.

Towards experimental manipulation of stochasticity in gene expression.

Author(s) : Vinuelas J, Kaneko G, Coulon A, Beslon G, Gandrillon O,
Journal : Prog Biophys Mol Biol
2012
For decades, most of molecular biology was driven by the "central dogma" in which the phenotype is defined by the genotype following a fully deterministic point of view. However, during the last 10 years, a wealth of studies has demonstrated that a given genotype can generate multiple phenotypes in identical environmental conditions, mainly because of the inherently probabilistic nature of the transcription process. It has also been shown that cells can tune this variability at the molecular level. Although previously described as a useless "noise", stochastic gene expression has now been shown by many authors to be an essential part of diverse biological processes. Chromatin dynamics having a central role in higher eukaryotes, we decided to investigate its involvement in the generation and control of stochasticity in gene expression (SGE). Our experiments reveal that the chromatin environment of a gene plays an important role in regulating SGE. Indeed, we find that histone acetylation and DNA methylation significantly affect SGE, suggesting that cells are able to adjust the variability of the expression of their genes through modification of chromatin marks. Given that the alteration of chromatin marks is itself subject to the expression of chromatin modifiers, our results shed light on a complex circular causality with on the one hand, the effect of gene expression on chromatin and on the other hand, the influence of the local chromatin environment of a gene on the dynamics of its expression.

Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm.

Author(s) : Raychaudhuri N, Dubruille R, Orsi G, Bagheri H, Loppin B, Lehner C,
Journal : PLoS Biol
2012
In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles duringmeiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamineexchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid ispresent in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion,paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount ofCid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.
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2011

Epigenetic maintenance of telomere identity in Drosophila: buckle up for the sperm ride.

Author(s) : Dubruille R, Loppin B,
Journal : Cell Cycle
2011
A critical function of telomeres is to prevent the ligation of chromosome ends by DNA repair enzymes. In most eukaryotes, telomeric DNA consists in large arrays of G-rich tandem repeats that are recognized by DNA binding capping proteins. Drosophila telomeres are unusual as they lack short tandem repeats. However, Drosophila capping proteins can bind chromosome extremities in a DNA sequence-independent manner. This epigenetic protection of fly telomeres has been essentially studied in somatic cells where capping proteins such as HOAP or HP1 are essential in preventing chromosome end-to-end fusions. HipHop and K81 are two recently identified paralogous capping proteins with complementary expression patterns. While HipHop is involved in telomere capping in somatic cells, K81 hasspecialized in the protection of telomeres in post-meiotic male germ cells. Remarkably, K81 is required for the stabilization of HOAP and HP1 at telomeres during the massive paternal chromatin remodeling that occurs during spermiogenesis and at fertilization. We thus propose that the maintenance of capping proteins at Drosophila sperm telomeres is crucial for the transmission of telomere identity to the diploid zygote. :

Epigenetics in C. elegans: facts and challenges.

Author(s) : Wenzel D, Palladino F, Jedrusik-Bode M,
Journal : Genesis
2011
Epigenetics is defined as the study of heritable changes in gene expression thatare not accompanied by changes in the DNA sequence. Epigenetic mechanisms include histone post-translational modifications, histone variant incorporation, non-coding RNAs, and nucleosome remodeling and exchange. In addition, the functional compartmentalization of the nucleus also contributes to epigenetic regulation of gene expression. Studies on the molecular mechanisms underlying epigenetic phenomena and their biological function have relied on various model systems, including yeast, plants, flies, and cultured mammalian cells. Here we will expose the reader to the current understanding of epigenetic regulation in the roundworm C. elegans. We will review recent models of nuclear organization and its impact on gene expression, the biological role of enzymes modifying corehistones, and the function of chromatin-associated factors, with special emphasis on Polycomb (PcG) and Trithorax (Trx-G) group proteins. We will discuss how the C. elegans model has provided novel insight into mechanisms of epigenetic regulation as well as suggest directions for future research.

Finding modulators of stochasticity levels by quantitative genetics.

Author(s) : Fehrmann S, Yvert G,
Journal : Methods Mol Biol
2011
Although bakers and wine makers constantly select, compare, and hunt for new wild strains of Saccharomyces cerevisiae, yeast geneticists have long focused on a few "standard" strains to ensure reproducibility and easiness of experimentation. And so far, the wonderful natural resource of wild genetic variation has been poorlyexploited in most academic laboratories. We describe here how one can use this resource to investigate the molecular sources of stochasticity in a gene regulatory network. The approach is general enough to be applied to any network of interest, as long as the experimental read-out offers robust statistics. For a given network, a typical study first identifies two backgrounds A and B displaying different levels of stochasticity and then study the network in A x Bprogeny. Taking advantage of microarrays or resequencing technologies, genotyping of appropriate segregants can then lead to the genomic regions housing modulators of stochasticity. The powerful toolbox available to manipulate the yeast genome offers several ways to narrow these regions further and to unambiguously demonstrate the regulatory consequences of DNA polymorphisms.

G-quadruplex-induced instability during leading-strand replication

Author(s) : Lopes J, Piazza A, Bermejo R, Kriegsman B, Colosio A, Teulade-Fichou M, Foiani M, Nicolas A,
Journal : The EMBO journal
2011
G‐quadruplexes are four‐stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G‐quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G‐quadruplex‐prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild‐type cells but is very frequently destabilized upon treatment with the potent Phen‐DC3 G‐quadruplex ligand, or in the absence of the G‐quadruplex‐unwinding Pif1 helicase, only when the G‐rich strand is the template of leading‐strand replication. The orientation‐dependent instability is associated with the formation of Rad51–Rad52‐dependent X‐shaped intermediates during replication detected by two‐dimensional (2D) gels, and relies on the presence of intact G‐quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G‐quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G‐rich telomeres.

Genome-wide in silico identification of new conserved and functional retinoic acid receptor response elements (direct repeats separated by 5 bp).

Author(s) : Lalevee S, Anno Y, Chatagnon A, Samarut E, Poch O, Laudet V, Benoit G, Lecompte O, Rochette-Egly C,
Journal : J Biol Chem
2011
The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzedfor their localization and conservation in vertebrates. We selected 138 elementslocated +/-10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provideda global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development

Author(s) : Grellscheid S, Dalgliesh C, Storbeck M, Best A, Liu Y, Jakubik M, Mende Y, Ehrmann I, Curk T, Rossbach K, Bourgeois C, St?venin J, Grellscheid D, Jackson M, Wirth B, Elliott D,
Journal : PLoS Genet
2011

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2beta in development.

Author(s) : Grellscheid S, Dalgliesh C, Storbeck M, Best A, Liu Y, Jakubik M, Mende Y, Ehrmann I, Curk T, Rossbach K, Bourgeois C, Stevenin J, Grellscheid D, Jackson M, Wirth B, Elliott D,
Journal : PLoS Genet
2011
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2beta (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2beta is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2beta binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specificSfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defectsin brain development and allowed correlation of genuine physiologically Tra2betaregulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2beta binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2beta protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2beta. Versions of Tra2beta lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2beta protein.

Identification of human, rat and chicken ribosomal proteins by a combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Author(s) : Nguyen-Lefebvre A, Gonin-Giraud S, Scherl A, Arboit P, Granger L, Sanchez J, Diaz J, Gandrillon O, Madjar J,
Journal : J Proteomics
2011
To identify the exact spot position of human, rat and chicken ribosomal proteins(RP) separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), a 2-DE system was designed to separate RP with a pI>8.6 according to their charge in the first dimension and to their molecular mass in the second dimension. Individual proteins were excised from the gels and identified by mass spectrometry after digestion by trypsin. In addition, a mixture of purified RP from these three species was also analyzed by tandem mass tag spectrometry. By combining those two methods 74 RP from human, 76 from rat and 67 from chicken were identified according to the nomenclature initially defined for rat liver RPand by using the Swiss-Prot/trEMBL databases. Whereas human and rat RP were welldescribed, most of RP from chicken were not characterized in databases, since 35out of 67 chicken RP identified in this study were not listed yet. We propose here the first comprehensive description of chicken RP and their comparison to those from human and rat.

Integrated genome-scale prediction of detrimental mutations in transcription networks.

Author(s) : Francesconi M, Jelier R, Lehner B,
Journal : PLoS Genet
2011
A central challenge in genetics is to understand when and why mutations alter the phenotype of an organism. The consequences of gene inhibition have been systematically studied and can be predicted reasonably well across a genome. However, many sequence variants important for disease and evolution may alter gene regulation rather than gene function. The consequences of altering a regulatory interaction (or "edge") rather than a gene (or "node") in a network have not been as extensively studied. Here we use an integrative analysis and evolutionary conservation to identify features that predict when the loss of a regulatory interaction is detrimental in the extensively mapped transcription network of budding yeast. Properties such as the strength of an interaction, location and context in a promoter, regulator and target gene importance, and the potential for compensation (redundancy) associate to some extent with interaction importance. Combined, however, these features predict quite well whether the loss of a regulatory interaction is detrimental across many promoters and for many different transcription factors. Thus, despite the potential for regulatory diversity, common principles can be used to understand and predict when changes in regulation are most harmful to an organism.

microRNA complements in deuterostomes: origin and evolution of microRNAs.

Author(s) : Campo-Paysaa F, Semon M, Cameron R, Peterson K, Schubert M,
Journal : Evol Dev
2011
Although numerous studies have emphasized the role of microRNAs (miRNAs) in the control of many different cellular processes, they might also exert a profound effect on the macroevolution of animal body plans. It has been hypothesized that, because miRNAs increase genic precision and are continuously being added to metazoan genomes through geologic time, miRNAs might be instrumental for canalization of development and morphological evolution. Nonetheless, an outstanding question remains: how are new miRNAs constantly evolving? To addressthis question, we assessed the miRNA complements of four deuterostome species, chosen because of their sequenced genomes and well-resolved phylogeny. Our comparative analysis shows that each of these four species is characterized by aunique repertoire of miRNAs, with few instances of miRNA loss. Moreover, we findthat almost half of the miRNAs identified in this study are located in intronic regions of protein coding genes, suggesting that new miRNAs might arise from intronic regions in a process we term intronic exaptation. We also show that miRNAs often occur within cotranscribed clusters, and describe the biological function of one of these conserved clusters, the miR-1/miR-133 cluster. Taken together, our work shows that miRNAs can easily emerge within already transcribed regions of DNA, whether it be introns or preexisting clusters of miRNAs and/or miRNAs and protein coding genes, and because of their regulatory roles, these novel players change the structure of gene regulatory networks, with potential macroevolutionary results.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Author(s) : Fugier C, Klein A, Hammer C, Vassilopoulos S, Ivarsson Y, Toussaint A, Tosch V, Vignaud A, Ferry A, Messaddeq N, Kokunai Y, Tsuburaya R, de la Grange P, Dembele D, Francois V, Precigout G, Boulade-Ladame C, Hummel M, Lopez de Munain A, Sergeant N, Laquerri?re A, Thibault C, Deryckere F, Auboeuf D, Garcia L, Zimmermann P, Udd B, Schoser B, Takahashi M, Nishino I, Bassez G, Laporte J, Furling D, Charlet-Berguerand N,
Journal : Nat Med
2011

Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy

Author(s) : Rau F, Freyermuth F, Fugier C, Villemin J, Fischer M, Jost B, Dembele D, Gourdon G, Nicole A, Duboc D, Wahbi K, Day J, Fujimura H, Takahashi M, Auboeuf D, Dreumont N, Furling D, Charlet-Berguerand N,
Journal : Nat Struct Mol Biol
2011

Molecular design of a splicing switch responsive to the RNA binding protein Tra2beta.

Author(s) : Grellscheid S, Dalgliesh C, Rozanska A, Grellscheid D, Bourgeois C, Stevenin J, Elliott D,
Journal : Nucleic Acids Res
2011
Tra2beta regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2beta most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2beta were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2beta AGAA-rich binding site. Surprisingly disruption ofeach single ESE prevented Tra2beta-mediated activation, although single mutated exons could still bind Tra2beta protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2beta-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangementof multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2beta, and co-ordinately regulates HIPK3-T exon splicing inclusion.

Molecular design of a splicing switch responsive to the RNA binding protein Tra2β

Author(s) : Grellscheid S, Dalgliesh C, Rozanska A, Grellscheid D, Bourgeois C, St?venin J, Elliott D,
Journal : Nucleic Acids Res
2011

Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.

Author(s) : Royer C, Briolay J, Garel A, Brouilly P, Sasanuma S, Sasanuma M, Shimomura M, Keime C, Gandrillon O, Huang Y, Chavancy G, Mita K, Couble P,
Journal : Insect Biochem Mol Biol
2011
Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3' side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-specific and highly abundant silk proteins. Strikingly,in addition to tags from silk protein mRNAs, 19 abundant tags were found (>/= 0.1%), in the median cell library, which were absent in the posterior cell tag collection. With the exception of tags from SP1 mRNA, no PSG specific tags were found in this subset class. The analysis of some of the MSG-specific transcripts, suggested that middle silk gland cells have diversified functions, in addition to their well characterized role in silk sericins synthesis and secretion.

Outcome of children treated with haematopoietic-stem cell transplantations from donors expressing the rare C77G variant of the PTPRC (CD45) gene

Author(s) : Samaan S, Gu?rin-El Khourouj V, Auboeuf D, Peltier L, P?dron B, Ouach?e-Chardin M, Gourgouillon N, Baruchel A, Dalle J, Sterkers G,
Journal : Br J Haematol
2011

Pharmacological inhibition of Frizzled-7 displays anti-tumor properties in hepatocellular carcinoma.

Author(s) : Nambotin S, Lefrancois L, Sainsily X, Berthillon P, Kim M, Wands J, Chevallier M, Jalinot P, Scoazec J, Trepo C, Zoulim F, Merle P,
Journal : J Hepatol
2011
BACKGROUND & AIMS: We previously reported the frequent overexpression of the FZD7 membrane receptor in hepatocellular carcinoma (HCC) and its role for controllingcancer phenotype. Herein, this study aimed at assessing the anticancer properties of compounds inhibiting FZD7 activity by disrupting its binding with the cytosolic Dishevelled (DVL) adaptator. METHODS: We have designed small interfering peptides (RHPDs) that are able to enter within cells and to competitively antagonize the binding of FZD7 to the PDZ domain of DVL. Their anti-neoplastic properties were assessed in vitro on a panel of human HCC cell lines and in vivo on the SV40-TAg transgenic mouse model of HCC. RESULTS: We have shown that RHPDs decrease cell viability via apoptosis depending on their affinity for PDZ, with a therapeutic index between cancerous and non-cancerous cells. RHPD properties were linked to beta-catenin degradation and PKCdelta activation. In transgenic mice, intra-tumor injection of RHPDs inhibited HCC progression. CONCLUSIONS: We have completed a proof-of-concept showing that in vitro and in vivo the pharmacological inhibition of FZD7 displays anti-cancerousproperties against HCC. The mechanisms can involve beta-catenin and PKCdelta modulations. Further studies are warranted to design protocols showing the compatibility with systemic in vivo applications.

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

Author(s) : Schmidt U, Basyuk E, Robert M, Yoshida M, Villemin J, Auboeuf D, Aitken S, Bertrand E,
Journal : J Cell Biol
2011

Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias

Author(s) : Capraro V, Zane L, Poncet D, Perol D, Galia P, Preudhomme C, Bonnefoy-Berard N, Gilson E, Thomas X, El-Hamri M, Chelghoun Y, Michallet M, Wattel E, Mortreux F, Sibon D,
Journal : Exp Hematol
2011

The emerging role of pre-messenger RNA splicing in stress responses: sending alternative messages and silent messengers

Author(s) : Dutertre M, Sanchez G, Barbier J, Corcos L, Auboeuf D,
Journal : RNA Biol
2011

Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila.

Author(s) : Gambis A, Dourlen P, Steller H, Mollereau B,
Journal : Dev Biol
2011
We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined acollection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression ofp35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The "Tomato/GFP-FLP/FRT" method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.
[1] 2

2010

Complex patterns of gene expression in human T cells during in vivo aging.

Author(s) : Remondini D, Salvioli S, Francesconi M, Pierini M, Mazzatti D, Powell J, Zironi I, Bersani F, Castellani G, Franceschi C,
Journal : Mol Biosyst
2010
Human aging is associated with complex alterations that contribute to remodelling of physiological processes and ultimately manifests in loss of tissue/organ function. Peripheral blood T cells do not escape this phenomenon and undergo profound remodelling with aging. Thus, investigating the effects of aging on T cells transcriptomics and identifying the underlying regulatory mechanisms can be of extreme importance to understand the aging process in the Immune System (IS).To this aim, we performed an analysis of gene expression data of T cells collected from peripheral blood of 25 healthy human donors of different age from25 to more than 95 years, in order to characterize changes that occur throughoutthe entire adult lifespan. By means of microarray analysis, we observed large groups of genes exhibiting non-monotonic expression patterns over time: such behaviour, that could not be observed in typical "two-group" experiments (e.g. young vs. old people) highlights similarities in gene expression profiles of young and "successfully aged" individuals. Genes whose expression profiles change during lifespan were grouped into three main patterns (eigenmodes) to which different biological functions were significantly associated. The analysis of KEGG pathways to which these genes belong indicated that the biological processes altered in T cell aging are not only those typically associated with immune cells (Jak-STAT signalling, T cell receptor signalling, cytokine-cytokine receptor interactions, etc.) but also some not specific of immune cells, such as long-term depression, PPAR and mTOR signalling, glucose and glutathione metabolism, suggesting that T cell aging may be representative of a more generalised aging phenomenon. Thus, the T cell may represent a useful cellular model to study organismal aging. We further searched for over-represented transcription factor binding sites (TFBSs) in the promoter regions of genes clustered by similarity of their age-related patterns to evidence possible co-regulation. A comparison between over-representation of TFBSs and the time course of the corresponding transcription factor (TF) expression levels revealed that a restricted group of TFs may play a central role in driving aging-specific changes in gene expressionof T cells.

Conformational free-energy difference of a miniprotein from nonequilibrium simulations

Author(s) : Spichty M, Cecchini M, Karplus M,
Journal : The Journal of Physical Chemistry Letters
2010
Conformational free-energy differences are essential thermodynamic quantities for understanding the function of many biomolecules. They are accessible from computer simulations, but their accurate calculation is a challenging task. Here nonequilibrium computer simulations and the differential fluctuation theorem are used to evaluate the free-energy difference between two conformational states of a structured miniprotein, the β-hairpin of protein G, with an implicit treatment of the solvent. A molecular dynamics-based protocol is employed for the simulation of rapid switches between the conformational states in both the forward and the reverse direction. From the work performed on the system in the individual switches, the conformational free-energy difference is determined by use of the differential fluctuation theorem. The results are in excellent agreement with reference calculations from a long molecular dynamics simulation and from the confinement method. The nonequilibrium approach is a computationally efficient method for the calculation of conformational free-energy differences for biological systems.

Cotranscriptional exon skipping in the genotoxic stress response

Author(s) : Dutertre M, Sanchez G, De Cian M, Barbier J, Dardenne E, Gratadou L, Dujardin G, Le Jossic-Corcos C, Corcos L, Auboeuf D,
Journal : Nat Struct Mol Biol
2010

Drosophila I-R hybrid dysgenesis is associated with catastrophic meiosis and abnormal zygote formation.

Author(s) : Orsi G, Joyce E, Couble P, McKim K, Loppin B,
Journal : J Cell Sci
2010
The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail toproduce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

Estrogen regulation and physiopathologic significance of alternative promoters in breast cancer

Author(s) : Dutertre M, Gratadou L, Dardenne E, Germann S, Samaan S, Lidereau R, Driouch K, de la Grange P, Auboeuf D,
Journal : Cancer Res
2010

Evolutionary trends of the pharyngeal dentition in Cypriniformes (Actinopterygii: Ostariophysi).

Author(s) : Pasco-Viel E, Charles C, Chevret P, Semon M, Tafforeau P, Viriot L, Laudet V,
Journal : PLoS One
2010
BACKGROUND: The fish order Cypriniformes is one of the most diverse ray-finned fish groups in the world with more than 3000 recognized species. Cypriniformes are characterized by a striking distribution of their dentition: namely the absence of oral teeth and presence of pharyngeal teeth on the last gill arch (fifth ceratobranchial). Despite this limited localisation, the diversity of tooth patterns in Cypriniformes is astonishing. Here we provide a further description of this diversity using X-ray microtomography and we map the resulting dental characters on a phylogenetic tree to explore evolutionary trends. RESULTS: We performed a pilot survey of dental formulae and individual tooth shapes in 34 adult species of Cypriniformes by X-ray microtomography (using either conventional X-ray machine, or synchrotron microtomography when necessary) or by dissecting. By mapping morphological results in a phylogenetic tree, it emerges that the two super-families Cobitoidea and Cyprinoidea have followed twodistinct evolutionary pathways. Furthermore, our analysis supports the hypothesis of a three-row dentition as ancestral for Cyprinoidea and a general trend in tooth row reduction in most derived lineages. Yet, this general scheme must be considered with caution as several events of tooth row gain and loss have occurred during evolutionary history of Cyprinoidea. SIGNIFICANCE: Dentition diversity in Cypriniformes constitutes an excellent model to study the evolutionof complex morphological structures. This morphological survey clearly advocatesfor extending the use of X-ray microtomography to study tooth morphology in Cypriniformes. Yet, our survey also underlines that improved knowledge of Cypriniformes life traits, such as feeding habits, is required as current knowledge is not sufficient to conclude on the link between diet and dental morphology.

Exon-based clustering of murine breast tumor transcriptomes reveals alternative exons whose expression is associated with metastasis

Author(s) : Dutertre M, Lacroix-Triki M, Driouch K, de la Grange P, Gratadou L, Beck S, Millevoi S, Tazi J, Lidereau R, Vagner S, Auboeuf D,
Journal : Cancer Res
2010

Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae

Author(s) : Piazza A, Boule J, Lopes J, Mingo K, Largy E, Teulade-Fichou M, Nicolas A,
Journal : Nucleic acids research
2010
G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.

Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes.

Author(s) : Gallois A, Lachuer J, Yvert G, Wierinckx A, Brunet F, Rabourdin-Combe C, Delprat C, Jurdic P, Mazzorana M,
Journal : J Bone Miner Res
2010
Dendritic cells (DCs), mononuclear cells that initiate immune responses, and osteoclasts (OCs), multinucleated bone-resorbing cells, are hematopoietic cells derived from monocytic precursor cells. Using in vitro generated dendritic cells, we previously showed that human and murine DCs could transdifferentiate into resorbing osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). In this study we globally compared by transcriptomic profiling this new osteoclast differentiation pathway from DCs with the canonical differentiation pathway from monocytes. DNA chip data revealed that starting from two very distinct cell types, treatment with M-CSF and RANKL generated two highly similar types of osteoclast. In particular, DC-derived osteoclasts expressed all the characteristic marker genesof monocyte-derived osteoclasts. Two major molecular events could be observed during osteoclastogenesis: downregulation of a large set of monocyte or DC specific markers, together with upregulation of characteristic osteoclast markergenes. Most interestingly, our transcriptomic data showed a closer molecular profile between DCs and OCs than between monocytes and OCs. Our data establish DCs as a new osteoclast precursor able to generate OCs more efficiently than monocytes.

Highly active antiretroviral treatment against STLV-1 infection combining reverse transcriptase and HDAC inhibitors

Author(s) : Afonso P, Mekaouche M, Mortreux F, Toulza F, Moriceau A, Wattel E, Gessain A, Bangham C, Dubreuil G, Plumelle Y, Hermine O, Estaquier J, Mahieux R,
Journal : Blood
2010

Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-{beta}.

Author(s) : Dreumont N, Bourgeois C, Lejeune F, Liu Y, Ehrmann I, Elliott D, Stevenin J,
Journal : J Cell Sci
2010
RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog beta (Tra2-beta), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-beta. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-beta repressed this exon. We propose that hRBMY associates with regions of the nucleus enrichedin nascent RNA and participates in the regulation of specific splicing events inthe germline by modulating the activity of constitutively expressed splicing factors.

Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-beta

Author(s) : Dreumont N, Bourgeois C, Lejeune F, Liu Y, Ehrmann I, Elliott D, St?venin J,
Journal : J Cell Sci
2010

Localized hypermutation and associated gene losses in legume chloroplast genomes.

Author(s) : Magee A, Aspinall S, Rice D, Cusack B, Semon M, Perry A, Stefanovic S, Milbourne D, Barth S, Palmer J, Gray J, Kavanagh T, Wolfe K,
Journal : Genome Res
2010
Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume "inverted repeat loss" clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Mathematical study of feedback control roles and relevance in stress erythropoiesis.

Author(s) : Crauste F, Demin I, Gandrillon O, Volpert V,
Journal : J Theor Biol
2010
This work is devoted to mathematical modelling of erythropoiesis. We propose a new multi-scale model, in which we bring together erythroid progenitor dynamics and intracellular regulatory network that determines erythroid cell fate. All erythroid progenitors are divided into several sub-populations according to their maturity. Two intracellular proteins, Erk and Fas, are supposed to be determinant for regulation of self-renewal, differentiation and apoptosis. We consider two growth factors, erythropoietin and glucocorticoids, and describe their dynamics.Several feedback controls are introduced in the model. We carry out computer simulations of anaemia and compare the obtained results with available experimental data on induced anaemia in mice. The main objective of this work isto evaluate the roles of the feedback controls in order to provide more insightsinto the regulation of erythropoiesis. Feedback by Epo on apoptosis is shown to be determinant in the early stages of the response to anaemia, whereas regulation through intracellular regulatory network, based on Erk and Fas, appears to operate on a long-term scale.

Natural single-nucleosome epi-polymorphisms in yeast.

Author(s) : Nagarajan M, Veyrieras J, de Dieuleveult M, Bottin H, Fehrmann S, Abraham A, Croze S, Steinmetz L, Gidrol X, Yvert G,
Journal : PLoS Genet
2010
Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did notimply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions.

NFAT3 transcription factor inhibits breast cancer cell motility by targeting the Lipocalin 2 gene

Author(s) : Foug?re M, Gaudineau B, Barbier J, Guaddachi F, Feugeas J, Auboeuf D, Jauliac S,
Journal : Oncogene
2010

On the spontaneous stochastic dynamics of a single gene: complexity of the molecular interplay at the promoter.

Author(s) : Coulon A, Gandrillon O, Beslon G,
Journal : BMC Syst Biol
2010
BACKGROUND: Gene promoters can be in various epigenetic states and undergo interactions with many molecules in a highly transient, probabilistic and combinatorial way, resulting in a complex global dynamics as observed experimentally. However, models of stochastic gene expression commonly consider promoter activity as a two-state on/off system. We consider here a model of single-gene stochastic expression that can represent arbitrary prokaryotic or eukaryotic promoters, based on the combinatorial interplay between molecules andepigenetic factors, including energy-dependent remodeling and enzymatic activities. RESULTS: We show that, considering the mere molecular interplay at the promoter, a single-gene can demonstrate an elaborate spontaneous stochastic activity (eg. multi-periodic multi-relaxation dynamics), similar to what is known to occur at the gene-network level. Characterizing this generic model with indicators of dynamic and steady-state properties (including power spectra and distributions), we reveal the potential activity of any promoter and its influence on gene expression. In particular, we can reproduce, based on biologically relevant mechanisms, the strongly periodic patterns of promoter occupancy by transcription factors (TF) and chromatin remodeling as observed experimentally on eukaryotic promoters. Moreover, we link several of its characteristics to properties of the underlying biochemical system. The model can also be used to identify behaviors of interest (eg. stochasticity induced by high TF concentration) on minimal systems and to test their relevance in larger and more realistic systems. We finally show that TF concentrations can regulate manyaspects of the stochastic activity with a considerable flexibility and complexity. CONCLUSIONS: This tight promoter-mediated control of stochasticity may constitute a powerful asset for the cell. Remarkably, a strongly periodic activity that demonstrates a complex TF concentration-dependent control is obtained when molecular interactions have typical characteristics observed on eukaryotic promoters (high mobility, functional redundancy, many alternate states/pathways). We also show that this regime results in a direct and indirectenergetic cost. Finally, this model can constitute a framework for unifying various experimental approaches. Collectively, our results show that a gene - the basic building block of complex regulatory networks - can itself demonstrate a significantly complex behavior.

Patterning by heritage in mouse molar row development.

Author(s) : Prochazka J, Pantalacci S, Churava S, Rothova M, Lambert A, Lesot H, Klein O, Peterka M, Laudet V, Peterkova R,
Journal : Proc Natl Acad Sci U S A
2010
It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mousemolar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions,and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the "primary enamel knot" (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK.We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transientsignaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which mayexplain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

Perinuclear distribution of heterochromatin in developing C. elegans embryos.

Author(s) : Grant J, Verrill C, Coustham V, Arneodo A, Palladino F, Monier K, Khalil A,
Journal : Chromosome Res
2010
Specific nuclear domains are nonrandomly positioned within the nuclear space, and this preferential positioning has been shown to play an important role in genomeactivity and stability. Well-known examples include the organization of repetitive DNA in telomere clusters or in the chromocenter of Drosophila and mammalian cells, which may provide a means to control the availability of general repressors, such as the heterochromatin protein 1 (HP1). We have specifically characterized the intranuclear positioning of in vivo fluorescence of the Caenorhabditis elegans HP1 homologue HPL-2 as a marker for heterochromatin domains in developing embryos. For this purpose, the wavelet transform modulus maxima (WTMM) segmentation method was generalized and adapted to segment the small embryonic cell nuclei in three dimensions. The implementation of a radial distribution algorithm revealed a preferential perinuclear positioning of HPL-2 fluorescence in wild-type embryos compared with the diffuse and homogeneous nuclear fluorescence observed in the lin-13 mutants. For all other genotypes analyzed, the quantitative analysis highlighted various degrees of preferential HPL-2 positioning at the nuclear periphery, which directly correlates with the number of HPL-2 foci previously counted on 2D projections. Using a probabilistic3D cell nuclear model, we found that any two nuclei having the same number of foci, but with a different 3D probabilistic positioning scheme, can have significantly different counts in the 2D maximum projection, thus showing the deceptive limitations of using techniques of 2D maximum projection foci counts. By this approach, a strong perinuclear positioning of HPL-2 foci was brought into light upon inactivation of conserved chromatin-associated proteins, including the HAT cofactor TRAPP.

Regulation of gene expression in hepatic cells by the mammalian Target of Rapamycin (mTOR).

Author(s) : Jimenez R, Lee J, Francesconi M, Castellani G, Neretti N, Sanders J, Sedivy J, Gruppuso P,
Journal : PLoS One
2010
BACKGROUND: We investigated mTOR regulation of gene expression by studying rapamycin effect in two hepatic cell lines, the non-tumorigenic WB-F344 cells and the tumorigenic WB311 cells. The latter are resistant to the growth inhibitory effects of rapamycin, thus providing us with an opportunity to study the gene expression effects of rapamycin without confounding effects on cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: The hepatic cells were exposed torapamycin for 24 hr. Microarray analysis on total RNA preparations identified genes that were affected by rapamycin in both cell lines and, therefore, modulated independent of growth arrest. Further studies showed that the promoterregions of these genes included E-box-containing transcription factor binding sites at higher than expected rates. Based on this, we tested the hypothesis that c-Myc is involved in regulation of gene expression by mTOR by comparing genes altered by rapamycin in the hepatic cells and by c-Myc induction in fibroblasts engineered to express c-myc in an inducible manner. Results showed enrichment for c-Myc targets among rapamycin sensitive genes in both hepatic cell lines. However, microarray analyses on wild type and c-myc null fibroblasts showed similar rapamycin effect, with the set of rapamycin-sensitive genes being enriched for c-Myc targets in both cases. CONCLUSIONS/SIGNIFICANCE: There is considerable overlap in the regulation of gene expression by mTOR and c-Myc. However, regulation of gene expression through mTOR is c-Myc-independent and cannot be attributed to the involvement of specific transcription factors regulated by the rapamycin-sensitive mTOR Complex 1.

Retinoic acid signaling targets Hox genes during the amphioxus gastrula stage: insights into early anterior-posterior patterning of the chordate body plan.

Author(s) : Koop D, Holland N, Semon M, Alvarez S, de Lera A, Laudet V, Holland L, Schubert M,
Journal : Dev Biol
2010
Previous studies of vertebrate development have shown that retinoic acid (RA) signaling at the gastrula stage strongly influences anterior-posterior (A-P) patterning of the neurula and later stages. However, much less is known about the more immediate effects of RA signaling on gene transcription and developmental patterning at the gastrula stage. To investigate the targets of RA signaling during the gastrula stage, we used the basal chordate amphioxus, in which gastrulation involves very minimal tissue movements. First, we determined the effect of altered RA signaling on expression of 42 genes (encoding transcriptionfactors and components of major signaling cascades) known to be expressed in restricted domains along the A-P axis during the gastrula and early neurula stage. Of these 42 genes, the expression domains during gastrulation of only four (Hox1, Hox3, HNF3-1 and Wnt3) were spatially altered by exposure of the embryos to excess RA or to the RA antagonist BMS009. Moreover, blocking protein synthesis with puromycin before adding RA or BMS009 showed that only three of these genes (Hox1, Hox3 and HNF3-1) are direct RA targets at the gastrula stage. From these results we conclude that in the amphioxus gastrula RA signaling primarily acts via regulation of Hox transcription to establish positional identities along theA-P axis and that Hox1, Hox3, HNF3-1 and Wnt3 constitute a basal module of RA action during chordate gastrulation.

Specialization of a Drosophila capping protein essential for the protection of sperm telomeres.

Author(s) : Dubruille R, Orsi G, Delabaere L, Cortier E, Couble P, Marais G, Loppin B,
Journal : Curr Biol
2010
BACKGROUND: A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, HipHop. We previously showed that the hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. RESULTS: Here we show that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and isretained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, we demonstrate by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. CONCLUSIONS: Our data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. We propose that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes.

Splicing factor and exon profiling across human tissues

Author(s) : de la Grange P, Gratadou L, Delord M, Dutertre M, Auboeuf D,
Journal : Nucleic Acids Res
2010

Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast.

Author(s) : Bernard P, Drogat J, Dheur S, Genier S, Javerzat J,
Journal : Mol Cell Biol
2010
Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptionalprocessing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether italso relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize atcentromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhapswith other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways.

Synthesis, spectroscopic and DNA alkylating properties of malondialdehyde (MDA) bis-imine fluorescent adducts.

Author(s) : Meguellati K, Spichty M, Ladame S,
Journal : Mol Biosyst
2010
The synthesis of a series of malondialdehyde (MDA) fluorescent adducts that mimic the well-known pentamethine cyanine dyes is reported. This new subclass of bis-imino dyes shares some common spectroscopic properties with their polymethine analogues although absorbing at significantly shorter wavelengths. A small library of trimethine and pentamethine cyanine dye bis-imino analogues have beensynthesised and characterised that cover a spectral range from blue to orange. Of particular interest is their capacity to act as mono- and bis-alkylating agents of nucleosides in general and of cytidine (and 2'-deoxycytidine) in particular.

Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo.

Author(s) : Zane L, Sibon D, Jeannin L, Zandecki M, Delfau-Larue M, Gessain A, Gout O, Pinatel C, Lancon A, Mortreux F, Wattel E,
Journal : Retrovirology
2010
BACKGROUND: Adult T cell leukemia results from the malignant transformation of aCD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to the expression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection. RESULTS: Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month) experimental infections performed in vitro and thoseobserved in cloned T cells from patients infected for >6-26 years, we found thatin chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells. CONCLUSIONS: Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.

Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo

Author(s) : Zane L, Sibon D, Jeannin L, Zandecki M, Delfau-Larue M, Gessain A, Gout O, Pinatel C, Lan?on A, Mortreux F, Wattel E,
Journal : Retrovirology
2010

The 3' untranslated region of the Andes hantavirus small mRNA functionally replaces the poly(A) tail and stimulates cap-dependent translation initiation from the viral mRNA.

Author(s) : Vera-Otarola J, Soto-Rifo R, Ricci E, Ohlmann T, Darlix J, Lopez-Lastra M,
Journal : J Virol
2010
In the process of translation of eukaryotic mRNAs, the 5' cap and the 3' poly(A)tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of theBunyaviridae, lacks a 3' poly(A) tail. Here we report that the 3' untranslated region (3'UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of translation by the 3'UTR of the ANDV SmRNA was found to be independent of viral proteins and of host poly(A)-binding protein.

2009

Epigenetic and replacement roles of histone variant H3.3 in reproduction and development.

Author(s) : Orsi G, Couble P, Loppin B,
Journal : Int J Dev Biol
2009
The nucleosomal organization of eukaryotic chromatin is generally established during DNA replication by the deposition of canonical histones synthesized in S phase. However, cells also use a Replication Independent (RI) nucleosome assembly pathway that allows the incorporation of non-canonical histone variants in the chromatin. H3.3 is a conserved histone variant that is structurally very close to its canonical counterpart but nevertheless possesses specific properties. In this review, we discuss the dual role of H3.3 which functions as a neutral replacement histone, but also participates in the epigenetic transmission of active chromatin states. These properties of H3.3 are also explored in the light of recent studies that implicate this histone and its associated chromatin assembly factors in large scale, replication-independent chromatin remodeling events. In particular,H3.3 appears as a critical player in the transmission of the paternal genome, from sperm to zygote.

ER stress protects from retinal degeneration.

Author(s) : Mendes C, Levet C, Chatelain G, Dourlen P, Fouillet A, Dichtel-Danjoy M, Gambis A, Ryoo H, Steller H, Mollereau B,
Journal : EMBO J
2009
The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivationnor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissuesfrom harmful exogenous stresses.

Extracting signature motifs from promoter sets of differentially expressed genes.

Author(s) : Mitasiunaite I, Rigotti C, Schicklin S, Meyniel L, Boulicaut J, Gandrillon O,
Journal : In Silico Biol
2009
There is a critical need for new and efficient computational methods aimed at discovering putative transcription factor binding sites (TFBSs) in promoter sequences. Among the existing methods, two families can be distinguished: statistical or stochastic approaches, and combinatorial approaches. Here we focus on a complete approach incorporating a combinatorial exhaustive motif extraction, together with a statistical Twilight Zone Indicator (TZI), in two datasets: a positive set and a negative one, which represents the result of a classical differential expression experiment. Our approach relies on the existence of prior biological information in the form of two sets of promoters of differentially expressed genes. We describe the complete procedure used for extracting either exact or degenerated motifs, ranking these motifs, and finding their known related TFBSs. We exemplify this approach using two different sets of promoters.The first set consists in promoters of genes either repressed or not by the transforming form of the v-erbA oncogene. The second set consists in genes the expression of which varies between self-renewing and differentiating progenitors. The biological meaning of the found TFBSs is discussed and, for one TF, its biological involvement is demonstrated. This study therefore illustrates the power of using relevant biological information, in the form of a set of differentially expressed genes that is a classical outcome in most of transcriptomics studies. This allows to severely reduce the search space and to design an adapted statistical indicator. Taken together, this allows the biologist to concentrate on a small number of putatively interesting TFs.

Genomic imprinting in Singapore. Workshop on Genomic Imprinting.

Author(s) : Loppin B, Oakey R,
Journal : EMBO Rep
2009

Heterochronic shifts explain variations in a sequentially developing repeated pattern: palatal ridges of muroid rodents.

Author(s) : Pantalacci S, Semon M, Martin A, Chevret P, Laudet V,
Journal : Evol Dev
2009
Metazoans are largely made of repeated parts, and metazoan evolution is marked by changes in the number of these parts, called meristic evolution. Understanding the mechanisms associated with meristic changes is thus a critical issue to evolutionary developmental biology. Palatal rugae are sensory ridges regularly arranged on the hard palate of mammals. They develop sequentially following mesio-distal growth of the palate, and activation-inhibition mechanisms very likely control spacing and timing of this sequential addition. In this study, wecharacterized trends in rugae number evolution among muroid rodents, showing that most species display 8+/-1 rugae, changes by one being very frequent in the phylogeny. We then compared development of three muroid species: mouse (nine rugae), rat (eight), and golden hamster (seven). We showed that palatal growth rate, spacing, and addition rate in mouse/rat were remarkably similar (with respect to the embryo size difference), and that increase to nine rugae in mouseis achieved by postponing the end of the addition process (hypermorphosis). Sucha heterochronic shift may be typical of +/-1 variations observed among muroid rodents. In contrast, decrease to seven rugae in golden hamster is attributed toearly growth termination (progenesis) of the palate, which correlates with the severe shortening of gestation in this species. Our results provide an experimental support to the intuitive view that heterochronies are especially relevant to meristic evolution of traits that rely on a sequential addition process. We also interpret our results in the light of developmental constraintsspecifically linked to this kind of process.

HPL-2/HP1 prevents inappropriate vulval induction in Caenorhabditis elegans by acting in both HYP7 and vulval precursor cells.

Author(s) : Schott S, Ramos F, Coustham V, Palladino F,
Journal : Genetics
2009
A current model for Caenorhabditis elegans vulval cell fate specification is that SynMuv genes act redundantly in the hyp7 hypodermal syncytium to repress the LIN-3/EGF inducer and prevent ectopic vulval induction of vulva precursor cells (VPCs). Here we show that the SynMuv gene hpl-2/HP1 has an additional function in VPCs, where it may act through target genes including LIN-39/Hox.

Inhibition of the hTERT promoter by the proto-oncogenic protein TAL1.

Author(s) : Terme J, Mocquet V, Kuhlmann A, Zane L, Mortreux F, Wattel E, Duc Dodon M, Jalinot P,
Journal : Leukemia
2009
Telomerase activity, which has fundamental roles in development and carcinogenesis, strongly depends on the expression of human telomerase reverse transcriptase (hTERT), its catalytic subunit. In this report, we show that the basic helix-loop-helix factor, TAL1 (T-cell acute lymphoblastic leukemia 1), is a negative regulator of the hTERT promoter. Indeed, TAL1 overexpression leads to adecrease in hTERT mRNA abundance and hence to reduced telomerase activity. Conversely, suppression of TAL1 by RNA interference in Jurkat cells increases hTERT expression. Analysis by chromatin immunoprecipitation assays showed that TAL1 binds to the hTERT proximal promoter and recruits HDAC1. Considering the relationship recently established between TAL1 and the human T-cell leukemia virus type 1 (HTLV-1) Tax protein, which was confirmed in T lymphocyte clones derived from adult T-cell leukemia patients, we analyzed the effect of TAL1 withrespect to the earlier characterized effects of Tax and HBZ (HTLV-1 basic leucine zipper) on hTERT expression. TAL1 was observed to reinforce the negative effect of Tax, whereas hTERT transactivation by the HBZ-JunD complex was repressed by TAL1 overexpression. Moreover, HBZ was found to induce proteasome-mediated degradation of TAL1. These observations support a model in which Tax and TAL1 byrepressing hTERT would initially favor genomic instability, whereas expression of factors such as HBZ allows at a later stage an increase in hTERT production and consequently in telomerase activity.

MicroRNAs resolve an apparent conflict between annelid systematics and their fossil record.

Author(s) : Sperling E, Vinther J, Moy V, Wheeler B, Semon M, Briggs D, Peterson K,
Journal : Proc Biol Sci
2009
Both the monophyly and inter-relationships of the major annelid groups have remained uncertain, despite intensive research on both morphology and molecular sequences. Morphological cladistic analyses indicate that Annelida is monophyletic and consists of two monophyletic groups, the clitellates and polychaetes, whereas molecular phylogenetic analyses suggest that polychaetes are paraphyletic and that sipunculans are crown-group annelids. Both the monophyly of polychaetes and the placement of sipunculans within annelids are in conflict with the annelid fossil record--the former because Cambrian stem taxa are similar to modern polychaetes in possessing biramous parapodia, suggesting that clitellatesare derived from polychaetes; the latter because although fossil sipunculans areknown from the Early Cambrian, crown-group annelids do not appear until the latest Cambrian. Here we apply a different data source, the presence versus absence of specific microRNAs--genes that encode approximately 22 nucleotide non-coding regulatory RNAs--to the problem of annelid phylogenetics. We show that annelids are monophyletic with respect to sipunculans, and polychaetes are paraphyletic with respect to the clitellate Lumbricus, conclusions that are consistent with the fossil record. Further, sipunculans resolve as the sister group of the annelids, rooting the annelid tree, and revealing the polarity of the morphological change within this diverse lineage of animals.

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates.

Author(s) : Bohne A, Schultheis C, Galiana-Arnoux D, Froschauer A, Zhou Q, Schmidt C, Selz Y, Ozouf-Costaz C, Dettai A, Segurens B, Couloux A, Bernard-Samain S, Barbe V, Chilmonczyk S, Brunet F, Darras A, Tomaszkiewicz M, Semon M, Schartl M, Volff J,
Journal : Integr Zool
2009
In contrast to mammals and birds, fish display an amazing diversity of genetic sex determination systems, with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex-determining genes. To better understand the molecular and evolutionary mechanisms driving this diversity, several fish models are studied in parallel. Besides the medaka (Oryzias latipes Temminck and Schlegel, 1846) for which the master sex-determination gene has been identified, one of the most advanced models for studying sex determination is the Southern platyfish (Xiphophorus maculatus, Gunther 1966). Xiphophorus maculatus belongs to the Poeciliids, a family of live-bearing freshwater fish, including platyfish, swordtails and guppies that perfectly illustrates the diversity of genetic sex-determination mechanisms observed in teleosts. For X. maculatus, bacterial artificial chromosome contigs covering the sex-determination region of the X and Y sex chromosomes have been constructed. Initial molecular analysis demonstrated that the sex-determination region is very unstable and frequently undergoes duplications, deletions, inversions and other rearrangements. Eleven gene candidates linked to the mastersex-determining gene have been identified, some of them corresponding to pseudogenes. All putative genes are present on both the X and the Y chromosomes,suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes. When compared with other fish and tetrapod genomes, syntenies were detected only with autosomes. This observation supports an independent origin of sex chromosomes, not only in different vertebrate lineagesbut also between different fish species.

Monoallelic expression and tissue specificity are associated with high crossover rates.

Author(s) : Necsulea A, Semon M, Duret L, Hurst L,
Journal : Trends Genet
2009
What determines the recombination rate of a gene? Following the observation that, in humans, imprinted genes have unusually high recombination levels, we ask whether increased recombination is seen for other monoallelically expressed genes and, more generally, how transcriptional properties relate to recombination. We find that monoallelically expressed genes do have high crossover rates and discover a striking negative correlation between within-gene crossover rate and expression breadth. We hypothesise that these findings are possibly symptomatic of a more general, adverse relationship between recombination and transcription in the human genome.

Overcoming resistance to conventional drugs in Ewing sarcoma and identification of molecular predictors of outcome.

Author(s) : Scotlandi K, Remondini D, Castellani G, Manara M, Nardi F, Cantiani L, Francesconi M, Mercuri M, Caccuri A, Serra M, Knuutila S, Picci P,
Journal : J Clin Oncol
2009
PURPOSE: The improvement of Ewing sarcoma (EWS) therapy is currently linked to the discovery of strategies to select patients with poor and good prognosis and of modified treatment regimens. In this study, we analyzed the molecular factorsgoverning EWS response to chemotherapy to identify genetic signatures to be usedfor risk-adapted therapy. PATIENTS AND METHODS: Microarray technology was used for profiling 30 primary tumors and seven metastases of patients who were classified according to event-free survival. For selected genes, real-time polymerase chain reaction was applied in 42 EWS primary tumors as validation assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test was used to evaluate in vitro drug sensitivity. RESULTS: We identified molecular signatures that reflect tumor resistance to chemotherapy. Annotation analysis was applied to reveal the biologic functions that critically influenced clinical outcome. The prognostic relevance of glutathione metabolism pathway was validated. The expression of MGST1, the microsomal glutathione S-transferase (GST), was found to clearly predict EWS prognosis. MGST1 expression was associated with doxorubicin chemosensitivity. This prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX),a new anticancer agent that efficiently inhibits GST enzymes. Six cell lines were found to be sensitive to this new drug. CONCLUSION: Classification of EWS patients into high- and low-risk groups is feasible with restricted molecular signatures that may have practical value at diagnosis for selecting patients with EWS who are unresponsive to current treatments. Glutathione metabolism pathway emerged as one of the most significantly altered prognosis-associated pathway. NBDHEX is proposed as a new potential therapeutic possibility.

Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies.

Author(s) : Rajan P, Dalgliesh C, Bourgeois C, Heiner M, Emami K, Clark E, Bindereif A, Stevenin J, Robson C, Leung H, Elliott D,
Journal : BMC Cell Biol
2009
BACKGROUND: Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids. RESULTS: We used proteomicsto search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. Theinteraction between Sam68 and hnRNP L proteins was observed in a cell line whichexhibits low frequency of SNBs suggesting that this association also takes placeoutside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons. CONCLUSION: Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs.

Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies

Author(s) : Rajan P, Dalgliesh C, Bourgeois C, Heiner M, Emami K, Clark E, Bindereif A, Stevenin J, Robson C, Leung H, Elliott D,
Journal : BMC Cell Biol
2009

Rapamycin response in tumorigenic and non-tumorigenic hepatic cell lines.

Author(s) : Jimenez R, Boylan J, Lee J, Francesconi M, Castellani G, Sanders J, Gruppuso P,
Journal : PLoS One
2009
BACKGROUND: The mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to itsgrowth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug. METHODOLOGY/PRINCIPAL FINDINGS: We profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E-dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E-dependent kinase activity was related to incorporation of p27(Kip1) into cyclin E-containing complexes in some but not all cell lines. Similarly, sensitivity ofglobal protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylationof two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicatingthat the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied. CONCLUSIONS/SIGNIFICANCE: We conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous,thus predicting that maintaining or promoting sensitivity will be highly challenging.

Rev-erbalpha2 mRNA encodes a stable protein with a potential role in circadian clock regulation.

Author(s) : Rambaud J, Triqueneaux G, Masse I, Staels B, Laudet V, Benoit G,
Journal : Mol Endocrinol
2009
Circadian rhythms are observed in nearly all aspects of physiology and behavior.In mammals, such biological rhythms are supported by a complex network of self-sustained transcriptional loops and posttranslational modifications, which regulate timely controlled production and degradation of critical factors on a 24-h basis. Among these factors, the orphan nuclear receptor rev-erbalpha plays an essential role by linking together positive and negative regulatory loops. Asan essential part of the circadian core clock mechanism, REV-ERBalpha expressionshows a precisely scheduled oscillation reflecting the tight control of its production and degradation. In previous studies, we identified two alternative transcripts encoding two protein variants referred to as REV-ERBalpha1 and -alpha2. Interestingly, recent work identified structural elements present only in REV-ERBalpha1 that controls its turnover and thereby influences circadian oscillations. In the present work, we comparatively analyze the two variants andshow that REV-ERBalpha2 exhibits a half-life incompatible with a circadian function, suggesting that this variant exerts different biological functions. However, our comparative study clearly indicates undistinguishable DNA-binding properties and transcriptional repression activity as well as a similar regulation mechanism. The only consistent difference appears to be the relative expression level of the two transcripts, rev-erbalpha1 being one to 100 times more expressed than alpha2 depending on tissue and circadian time. Taking this finding into consideration, we reassessed REV-ERBalpha2 turnover and were able to show that this variant exhibits a reduced half-life when coexpressed with REV-ERBalpha1. We propose that the relative expression levels of the two REV-ERBalpha variants fine-tune the circadian period length by regulating REV-ERBalpha half-life.

Reversible synthesis and characterization of dynamic imino analogues of trimethine and pentamethine cyanine dyes.

Author(s) : Meguellati K, Spichty M, Ladame S,
Journal : Org Lett
2009
A new family of unsymmetrical imine-based trimethine and pentamethine cyanine dye analogues is reported that can form under reversible and thermodynamically controlled conditions from non- or weakly emissive amine and aldehyde building blocks. These dynamic fluorophores show spectroscopic properties comparable to those of their parent cyanine dyes and are responsive to external effectors.

Role of RNA structure and protein factors in the control of HIV-1 splicing

Author(s) : Saliou J, Bourgeois C, Ayadi-Ben Mena L, Ropers D, Jacquenet S, Marchand V, Stevenin J, Branlant C,
Journal : Front Biosci (Landmark Ed)
2009

Structural and functional diversity of viral IRESes.

Author(s) : Balvay L, Soto Rifo R, Ricci E, Decimo D, Ohlmann T,
Journal : Biochim Biophys Acta
2009
Some 20 years ago, the study of picornaviral RNA translation led to the characterization of an alternative mechanism of initiation by direct ribosome binding to the 5' UTR. By using a bicistronic vector, it was shown that the 5' UTR of the poliovirus (PV) or the Encephalomyelitis virus (EMCV) had the abilityto bind the 43S preinitiation complex in a 5' and cap-independent manner. This is rendered possible by an RNA domain called IRES for Internal Ribosome Entry Site which enables efficient translation of an mRNA lacking a 5' cap structure. IRES elements have now been found in many different viral families where they often confer a selective advantage to allow ribosome recruitment under conditions where cap-dependent protein synthesis is severely repressed. In this review, we compare and contrast the structure and function of IRESes that are found within 4 distinct family of RNA positive stranded viruses which are the (i) Picornaviruses; (ii) Flaviviruses; (iii) Dicistroviruses; and (iv) Lentiviruses.

T-cell tropism of simian T-cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills (Mandrillus sphinx).

Author(s) : Souquiere S, Mouinga-Ondeme A, Makuwa M, Beggio P, Radaelli A, De Giuli Morghen C, Mortreux F, Kazanji M,
Journal : J Med Primatol
2009
BACKGROUND: Although a wide variety of non-human primates are susceptible to simian T-cell leukaemia virus type 1 (STLV-1), little is known about the virological or molecular determinants of natural STLV-1 infection. METHODS: We determined STLV-1 virus tropism in vivo and its relation to the immune response by evaluating cytokine production and T-cell subsets in naturally infected and uninfected mandrills. RESULTS: With real-time PCR methods, we found that STLV-1 in mandrills infects both CD4(+) and CD8(+) T cells; however, proviral loads were significantly higher (P = 0.01) in CD4(+) than in CD8(+) cells (mean STLV-1 copies number per 100 cells (+/- SD) was 7.8 +/- 8 in CD4(+) T cells and 3.9 +/-4.5 in CD8(+) T cells). After culture, STLV-1 provirus was detected in enriched CD4(+) but not in enriched CD8(+) T cells. After 6 months of culture, STLV-1-transformed cell lines expressing CD3(+), CD4(+) and HLADR(+) were established, and STLV-1 proteins and tax/rex mRNA were detected. In STLV-1 infected monkeys, there was a correlation between high proviral load and elevated levels of interleukin (IL)-2, IL-6, IL-10, interferon-gamma and tumour necrosis factor-alpha. The two monkeys with the highest STLV-1 proviral load had activated CD4(+)HLADR(+) and CD8(+)HLADR(+) T-cell subsets and a high percentage of CD25(+) in CD4(+) and CD8(+) T cells. CONCLUSIONS: Our study provides the first cellular, immunological and virological characterization of natural STLV-1 infection in mandrills and shows that they are an appropriate animal model for further physiopathological studies of the natural history of human T-cell leukaemia viruses.

T-cell tropism of simian T-cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills (Mandrillus sphinx)

Author(s) : Souqui?re S, Mouinga-Ondeme A, Makuwa M, Beggio P, Radaelli A, De Giuli Morghen C, Mortreux F, Kazanji M,
Journal : J Med Primatol
2009

TGF-beta induces degradation of TAL1/SCL by the ubiquitin-proteasome pathway through AKT-mediated phosphorylation.

Author(s) : Terme J, Lhermitte L, Asnafi V, Jalinot P,
Journal : Blood
2009
T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays important roles in differentiation of hematopoietic and endothelialcells and is deregulated in a high percentage of T-cell acute lymphoblastic leukemia (T-ALL). In this report we show that the intracellular concentration ofTAL1 is regulated by transforming growth factor beta (TGF-beta), which triggers its polyubiquitylation and degradation by the proteasome. This effect is mediated by AKT1, which phosphorylates TAL1 at threonine 90. Immunoprecipitation experiments showed that this event increases association of TAL1 with the E3 ubiquitin ligase CHIP. The E47 heterodimerization partner of TAL1 hinders this association. Our observations indicate that activation of the TGF-beta and phosphatidylinositol 3-kinase/AKT pathways might reverse overexpression of TAL1 in leukemic cells by inducing proteolysis of this important oncogene.

The evolutionary context of robust and redundant cell biological mechanisms.

Author(s) : Delattre M, Felix M,
Journal : Bioessays
2009
The robustness of biological processes to perturbations has so far been mainly explored in unicellular organisms; multicellular organisms have been studied fordevelopmental processes or in the special case of redundancy between gene duplicates. Here we explore the robustness of cell biological mechanisms of multicellular organisms in an evolutionary context. We propose that the reuse ofsimilar cell biological mechanisms in different cell types of the same organism has evolutionary implications: (1) the maintenance of apparently redundant mechanisms over evolutionary time may in part be explained by their differentialrequirement in various cell types; (2) the relative requirement for two alternative mechanisms may evolve among homologous cells in different organisms.We present examples of cell biological processes, such as centrosome separation in prophase, spindle formation or cleavage furrow positioning, that support the first proposition. We propose experimental tests of these hypotheses.

The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon

Author(s) : Liu Y, Bourgeois C, Pang S, Kudla M, Dreumont N, Kister L, Sun Y, Stevenin J, Elliott D,
Journal : PLoS Genet
2009

The HSP90 binding mode of a radicicol-like E-oxime determined by docking, binding free energy estimations, and NMR 15N chemical shifts.

Author(s) : Spichty M, Taly A, Hagn F, Kessler H, Barluenga S, Winssinger N, Karplus M,
Journal : Biophys Chem
2009
We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We samplethe macrocyclic scaffold of the unbound ligand by parallel tempering simulationsand dock the most populated conformations to yeast HSP90. Docking poses are thenevaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of backbone (15)N provides an additional evaluation criteria. As a final test we check the binding modes against available structure-activity-relationships. We find that the most likelybinding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex.

The leukemogenic activity of TaxHTLV-1 during human alphabeta T cell development.

Author(s) : Wencker M, Gazzolo L, Duc Dodon M,
Journal : Front Biosci (Schol Ed)
2009
The regulatory Tax protein of HTLV-1 (Human T-cell Leukaemia Virus type 1) is critically involved in the initiation of ATL (adult T-cell leukaemia). Indeed, Tax provides infected T-cells with a growth advantage and with the potential to get transformed through the deregulation of cell-cycle progression and the acquisition of genetic alterations. Considering that leukemias are induced by disturbances in hematopoietic cells development, we hypothesize that the expression of Tax in human immature thymocytes is a prerequisite to the emergence of ATL cells. Studies of alph abeta T-cell development in the thymus have shown that beta-selection, an early important checkpoint, is regulated by transcription factors that are decisive in the control of cell proliferation, differentiation and survival. Interestingly, Tax is endowed with the ability to interfere with the activity of these transcription factors. We therefore propose that the HTLV-1 infection of these specific target thymocytes leads to a transcriptional deregulation of early alphabeta T cell development, thus inducing a pre-leukemogenic event that favours the subsequent proliferation of ATL cells.

The phytoestrogen genistein affects zebrafish development through two different pathways.

Author(s) : Sassi-Messai S, Gibert Y, Bernard L, Nishio S, Ferri Lagneau K, Molina J, Andersson-Lendahl M, Benoit G, Balaguer P, Laudet V,
Journal : PLoS One
2009
BACKGROUND: Endocrine disrupting chemicals are widely distributed in the environment and derive from many different human activities or can also be natural products synthesized by plants or microorganisms. The phytoestrogen, genistein (4', 5, 7-trihydroxy-isoflavone), is a naturally occurring compound found in soy products. Genistein has been the subject of numerous studies because of its known estrogenic activity. METHODOLOGY/PRINCIPAL FINDINGS: We report thatgenistein exposure of zebrafish embryos induces apoptosis, mainly in the hindbrain and the anterior spinal cord. Timing experiments demonstrate that apoptosis is induced during a precise developmental window. Since adding ICI 182,780, an ER antagonist, does not rescue the genistein-induced apoptosis and since there is no synergistic effect between genistein and estradiol, we conclude that this apoptotic effect elicited by genistein is estrogen-receptors independent. However, we show in vitro, that genistein binds and activates the three zebrafish estrogen receptors ERalpha, ERbeta-A and ERbeta-B. Furthermore using transgenic ERE-Luciferase fish we show that genistein is able to activate the estrogen pathway in vivo during larval stages. Finally we show that genistein is able to induce ectopic expression of the aromatase-B gene in an ER-dependent manner in the anterior brain in pattern highly similar to the one resulting fromestrogen treatment at low concentration. CONCLUSION/SIGNIFICANCE: TAKEN TOGETHERTHESE RESULTS INDICATE THAT GENISTEIN ACTS THROUGH AT LEAST TWO DIFFERENT PATHWAYS IN ZEBRAFISH EMBRYOS: (i) it induces apoptosis in an ER-independent manner and (ii) it regulates aromatase-B expression in the brain in an ER-dependent manner. Our results thus highlight the multiplicity of possible actions of phytoestrogens, such as genistein. This suggests that the use of standardized endpoints to study the effect of a given compound, even when this compound has well known targets, may carry the risk of overlooking interesting effects of this compound.

The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo

Author(s) : Ribeyre C, Lopes J, Boulé J, Piazza A, Guédin A, Zakian V, Mergny J, Nicolas A,
Journal : PLoS genetics
2009
In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences

Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2.

Author(s) : Ricci E, Mure F, Gruffat H, Decimo D, Medina-Palazon C, Ohlmann T, Manet E,
Journal : Nucleic Acids Res
2009
The Epstein-Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.

2008

Constraint-based knowledge discovery from SAGE data.

Author(s) : Klemal J, Blachon S, Soulet A, Cremilleux B, Gandrillon O,
Journal : In Silico Biol
2008
Current analyses of co-expressed genes are often based on global approaches suchas clustering or bi-clustering. An alternative way is to employ local methods and search for patterns--sets of genes displaying specific expression properties in a set of situations. The main bottleneck of this type of analysis is twofold--computational costs and an overwhelming number of candidate patterns which can hardly be further exploited. A timely application of background knowledge available in literature databases, biological ontologies and other sources can help to focus on the most plausible patterns only. The paper proposes, implements and tests a flexible constraint-based framework that enables the effective mining and representation of meaningful over-expression patterns representing intrinsic associations among genes and biological situations. The framework can be simultaneously applied to a wide spectrum of genomic data and we demonstrate that it allows to generate new biological hypotheses with clinical implications.

Cotranscriptional splicing potentiates the mRNA production from a subset of estradiol-stimulated genes

Author(s) : Bittencourt D, Dutertre M, Sanchez G, Barbier J, Gratadou L, Auboeuf D,
Journal : Mol Cell Biol
2008

Coupled alteration of transcription and splicing by a single oncogene: boosting the effect on cyclin D1 activity

Author(s) : Sanchez G, Delattre O, Auboeuf D, Dutertre M,
Journal : Cell Cycle
2008

Cross talk between expression of the human T-cell leukemia virus type 1 Tax transactivator and the oncogenic bHLH transcription factor TAL1.

Author(s) : Terme J, Wencker M, Favre-Bonvin A, Bex F, Gazzolo L, Duc Dodon M, Jalinot P,
Journal : J Virol
2008
The human T-cell leukemia virus type 1 (HTLV-1) Tax transactivator is known to induce or repress various cellular genes, several of them encoding transcriptionfactors. As Tax is known to deregulate various basic bHLH factors, we looked more specifically at its effect on TAL1 (T-cell acute lymphoblastic leukemia 1), alsoknown as SCL (stem cell leukemia). Indeed, TAL1 is deregulated in a high percentage of T-cell acute lymphoblastic leukemia cells, and its oncogenic properties are well-established. Here we show that Tax induces transcription of this proto-oncogene by stimulating the activity of the TAL1 gene promoter 1b, through both the CREB and NF-kappaB pathways. It was also observed that TAL1 upregulates HTLV-1 promoter activity, in either the presence or the absence of Tax. The viral promoter is inhibited in trans by expression of the E2A protein E47, and TAL1 is able to abrogate this inhibition. These data show the existenceof a positive feedback loop between Tax and TAL1 expression and support the notion that this proto-oncogene participates in generation of adult T-cell leukemia/lymphoma by increasing the amount of the Tax oncoprotein but also possibly by its own transforming activities.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Author(s) : Solmi R, Lauriola M, Francesconi M, Martini D, Voltattorni M, Ceccarelli C, Ugolini G, Rosati G, Zanotti S, Montroni I, Mattei G, Taffurelli M, Santini D, Pezzetti F, Ruggeri A, Castellani G, Guidotti L, Coppola D, Strippoli P,
Journal : BMC cancer
2008

Early and transient reverse transcription during primary deltaretroviral infection of sheep

Author(s) : Pomier C, Alcaraz M, Debacq C, Lan?on A, Kerkhofs P, Willems L, Wattel E, Mortreux F,
Journal : Retrovirology
2008

Expression levels of estrogen receptor beta are modulated by components of the molecular clock.

Author(s) : Cai W, Rambaud J, Teboul M, Masse I, Benoit G, Gustafsson J, Delaunay F, Laudet V, Pongratz I,
Journal : Mol Cell Biol
2008
Circadian regulation of gene expression plays a major role in health and disease. The precise role of the circadian system remains to be clarified, but it is known that circadian proteins generate physiological rhythms in organisms by regulating clock-controlled target genes. The estrogen receptor beta (ERbeta) is, together with ERalpha, a member of the nuclear receptor superfamily and a key mediator ofestrogen action. Interestingly, recent studies show that disturbed circadian rhythmicity in humans can increase the risk of reproductive malfunctions, suggesting a link between the circadian system and ER-mediated transcription pathways. Here, we identify a novel level of regulation of estrogen signaling where ERbeta, but not ERalpha, is controlled by circadian clock proteins. We show that ERbeta mRNA levels fluctuate in different peripheral tissues following a robust circadian pattern, with a peak at the light-dark transition, which is maintained under free-running conditions. Interestingly, this oscillation is abolished in clock-deficient BMAL1 knockout mice. Circadian control of ERbeta expression is exerted through a conserved E-box element in the ERbeta promoter region that recruits circadian regulatory factors. Furthermore, using small interfering RNA-mediated knockdown assays, we show that the expression levels ofthe circadian regulatory factors directly influence estrogen signaling by regulating the intracellular levels of endogenous ERbeta.

Hedgehog and Wingless stabilize but do not induce cell fate during Drosophila dorsal embryonic epidermal patterning.

Author(s) : Vincent S, Perrimon N, Axelrod J,
Journal : Development
2008
A fundamental concept in development is that secreted molecules such as Wingless(Wg) and Hedgehog (Hh) generate pattern by inducing cell fate. By following markers of cellular identity posterior to the Wg- and Hh-expressing cells in theDrosophila dorsal embryonic epidermis, we provide evidence that neither Wg nor Hh specifies the identity of the cell types they pattern. Rather, they maintain pre-existing cellular identities that are otherwise unstable and progress stepwise towards a default fate. Wg and Hh therefore generate pattern by inhibiting specific switches in cell identity, showing that the specification and the patterning of a given cell are uncoupled. Sequential binary decisions without induction of cell identity give rise to both the groove cells and their posterior neighbors. The combination of independent progression of cell identity and arrest of progression by signals facilitates accurate patterning of an extremely plastic developing epidermis.

Heterobimetallic Bisphosphonate Titanium Complexes: Carbene-Like Carbanions?

Author(s) : Spichty M, Kulicke K, Neuburger M, Schaffner S, Mueller J,
Journal : European journal of inorganic chemistry
2008
A new class of carbene-like titanium complexes is easily accessible by means of a dilithiation–transmetallation sequence of a bisphosphonate. The X-ray structure of the dimeric, organometallic compound 3 reveals a nano-sized, multi-layer aggregate with titanium-metallated, trigonal planar carbon centers being devoid of Li contacts. DFT calculations show that the metallated carbon is a carbanion with only a slight carbene portion (10 %); the carbene character can be easily strengthened or weakened by varying the counterion. We anticipate that the sensitivity and flexibility to influence the electronic character of these carbene-like carbanions offers a way to design new versatile reagents with variable chemical properties.

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein.

Author(s) : Ricci E, Herbreteau C, Decimo D, Schaupp A, Datta S, Rein A, Darlix J, Ohlmann T,
Journal : RNA
2008
The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease toshow that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production andalso on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.

Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains.

Author(s) : Vitte A, Jalinot P,
Journal : BMC Biotechnol
2008
BACKGROUND: We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form. RESULTS: Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainlythe Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs weredelivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway. CONCLUSION: Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing deliveryof the peptide alone.

Involvement of the TGF-beta and mTOR/p70S6Kinase pathways in the transformation process induced by v-ErbA.

Author(s) : Gonin-Giraud S, Bresson-Mazet C, Gandrillon O,
Journal : Leuk Res
2008
v-ErbA is the oncogenic form of TRalpha/c-ErbA which transforms chicken erythrocytic progenitors by blocking differentiation. The oncogenic property of v-ErbA has been correlated with its ability to antagonize ligand-dependent gene activation by TRalpha/c-ErbA and retinoic acid receptors. Nevertheless, its cytoplasmic retention suggests that v-ErbA could interfere with intracellular signaling pathways. We demonstrate that only the transforming form of v-ErbA confers to chicken erythroid progenitors a TGF-beta independency and induces an activation of the mTOR/p70S6K pathway. In these cells, TGF-beta and mTOR/p70S6K pathways regulate the expression of a known target gene of v-ErbA, band3. This is the first demonstration that v-ErbA is able to modulate specifically some signaling pathways leading to changes in the expression level of a gene involvedin transformation.

Large multiprotein structures modeling and simulation: the need for mesoscopic models.

Author(s) : Coulon A, Beslon G, Gandrillon O,
Journal : Methods Mol Biol
2008
Recent observational techniques based upon confocal microscopy make it possible to observe cells at a scale that has never been probed before: the mesoscopic scale. In the eukaryotic cell nucleus, many objects demonstrating phenomena occurring at this scale, such as nuclear bodies, are current subjects of investigations. But from a modeling perspective, this scale has not been widely explored, and hence there is a lack of suitable models for such studies. By reviewing higher and lower scale modeling techniques, we analyze their relevancein the context of mesoscale phenomena. We emphasize important characteristics that should be included in a mesoscopic model: an explicit continuous three-dimensional space with discrete simplified molecules that still have the characteristics of steric volume exclusion and realistic distant interaction forces. Then we present 3DSPI, a model dedicated to studies of nuclear bodies based on a simple formalism inspired from molecular dynamics and coarse-grained models: particles interacting through a potential energy function and driven by an overdamped Langevin equation. Finally, we present the features expected to beincluded in the model, pointing out the difficulties that might arise.

Lentiviral RNAs can use different mechanisms for translation initiation.

Author(s) : Ricci E, Soto Rifo R, Herbreteau C, Decimo D, Ohlmann T,
Journal : Biochem Soc Trans
2008
The full-length genomic RNA of lentiviruses can be translated to produce proteins and incorporated as genomic RNA in the viral particle. Interestingly, both functions are driven by the genomic 5'-UTR (5'-untranslated region), which harbours structural RNA motifs for the replication cycle of the virus. Recent work has shown that this RNA architecture also functions as an IRES (internal ribosome entry site) in HIV-1 and -2, and in SIV (simian immunodeficiency virus). In addition, the IRES extends to the gag coding region for all these viruses andthis leads to the synthesis of shorter isoforms of the Gag polyprotein from downstream initiation codons. In the present study, we have investigated how different members of the lentivirus family (namely HIV-1 and -2, and SIV) can initiate protein synthesis by distinct mechanisms. For this, we have used the competitive reticulocyte lysate that we have recently described. Our results show that HIV-1 is able to drive the synthesis of the Gag polyprotein both by a classical cap-dependent mechanism and an IRES, whereas HIV-2 and SIV appear to use exclusively an IRES mechanism.

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Author(s) : El-Bchiri J, Guilloux A, Dartigues P, Loire E, Mercier D, Buhard O, Sobhani I, de la Grange P, Auboeuf D, Praz F, Fl?jou J, Duval A,
Journal : PLoS One
2008

Nuclear hormone receptor signaling in amphioxus.

Author(s) : Schubert M, Brunet F, Paris M, Bertrand S, Benoit G, Laudet V,
Journal : Dev Genes Evol
2008
The nuclear hormone receptors (NRs) form a superfamily of transcription factors unified by conserved protein structure and mode of function. While most members of this superfamily are activated by ligands, such as thyroid hormones, steroids, vitamin D or retinoic acid, other NRs are called orphan receptors because they have no known ligand. NR-dependent signaling is crucial for vertebrate development with the majority of receptors being expressed in the developing embryo. Due to massive gene duplications during vertebrate diversification, there are usually more NRs in vertebrates than in invertebrates. In this study, we examine the evolutionary diversification of the NR superfamily and of NR-dependent signaling in chordates (vertebrates, tunicates, and amphioxus). We take advantage of the unique features of the genome of the invertebrate amphioxus, which is characterized by a vertebrate-like gene content without having undergone massive duplications, to assess the NR signaling complement (NRs and NR coregulators) of the ancestral chordate. We find 33 NRs in amphioxus, which are more NRs than originally anticipated. This increase is mainly due to an amphioxus-specific duplication of genes encoding receptors of the NR1H group. Inaddition, there are three heterologous NRs in amphioxus that could not be placedwithin the framework of the NR superfamily. Apart from these exceptions, there is usually one amphioxus NR or NR signaling coregulator for each paralogous group of two, three, or four human receptors suggesting that the ancestral chordate had aset of 22 different NRs plus one copy of each NR coregulator.

Patterning of palatal rugae through sequential addition reveals an anterior/posterior boundary in palatal development.

Author(s) : Pantalacci S, Prochazka J, Martin A, Rothova M, Lambert A, Bernard L, Charles C, Viriot L, Peterkova R, Laudet V,
Journal : BMC Dev Biol
2008
BACKGROUND: The development of the secondary palate has been a main topic in craniofacial research, as its failure results in cleft palate, one of the most common birth defects in human. Nevertheless, palatal rugae (or rugae palatinae),which are transversal ridges developing on the secondary palate, received littleattention. However, rugae could be useful as landmarks to monitor anterior/posterior (A/P) palatal growth, and they provide a simple model of mesenchymal-epithelial structures arranged in a serial pattern. RESULTS: We first determined in which order the nine mouse rugae appear during development. Our results revealed a reiterative process, which is coupled with A/P growth of palatal shelves, and by which rugae 3 to 7b are sequentially interposed, in the increasing distance between the second most anterior ruga, ruga 2, and the two most posterior rugae, rugae 8 and 9. We characterized the steps of ruga interposition in detail, showing that a new ruga forms from an active zone of high proliferation rate, next to the last formed ruga. Then, by analyzing the polymorphism of wild type and Eda(Ta) mutant mice, we suggest that activation-inhibition mechanisms may be involved in positioning new rugae, like for other skin appendages. Finally, we show that the ruga in front of which new rugae form, i.e. ruga 8 in mouse, coincides with an A/P gene expression boundaryin the palatal shelves (Shox2/Meox2-Tbx22). This coincidence is significant, since we also found it in hamster, despite differences in the adult ruga patternof these two species. CONCLUSION: We showed that palatal rugae are sequentially added to the growing palate, in an interposition process that appears to be dependent on activation-inhibition mechanisms and reveals a new developmental boundary in the growing palate. Further studies on rugae may help to shed light on both the development and evolution of structures arranged in regular patterns. Moreover, rugae will undoubtedly be powerful tools to further study the anteroposterior regionalization of the growing palate.

Preferential subfunctionalization of slow-evolving genes after allopolyploidization in Xenopus laevis.

Author(s) : Semon M, Wolfe K,
Journal : Proc Natl Acad Sci U S A
2008
As paleopolyploid genomes evolve, the expression profiles of retained gene pairsare expected to diverge. To examine this divergence process on a large scale in a vertebrate system, we compare Xenopus laevis, which has retained approximately 40% of loci in duplicate after a recent whole-genome duplication (WGD), with itsunduplicated relative Silurana (Xenopus) tropicalis. This comparison of ingroup pairs to an outgroup allows the direction of change in expression profiles to beinferred for a set of 1,300 X. laevis pairs, relative to their single orthologs in S. tropicalis, across 11 tissues. We identify 68 pairs in which X. laevis is inferred to have undergone a significant reduction of expression in at least twotissues since WGD. Of these pairs, one-third show evidence of subfunctionalization, with decreases in the expression levels of different gene copies in two different tissues. Surprisingly, we find that genes with slow rates of evolution are particularly prone to subfunctionalization, even when the tendency for highly expressed genes to evolve slowly is controlled for. We interpret this result to be an effect of allopolyploidization. We then compare the outcomes of this WGD with an independent one that happened in the teleost fish lineage. We find that if a gene pair was retained in duplicate in X. laevis, the orthologous pair is more likely to have been retained in duplicate in zebrafish, suggesting that similar factors, among them subfunctionalization, determined which gene pairs survived in duplicate after the two WGDs.

Reconstructing networks of pathways via significance analysis of their intersections

Author(s) : Francesconi M, Remondini D, Neretti N, Sedivy J, Cooper L, Verondini E, Milanesi L, Castellani G,
Journal : BMC Bioinformatics
2008
Significance analysis at single gene level may suffer from the limited number of samples and experimental noise that can severely limit the power of the chosen statistical test. This problem is typically approached by applying post hoc corrections to control the false discovery rate, without taking into account prior biological knowledge. Pathway or gene ontology analysis can provide an alternative way to relax the significance threshold applied to single genes and may lead to a better biological interpretation.

SQUAT: A web tool to mine human, murine and avian SAGE data.

Author(s) : Leyritz J, Schicklin S, Blachon S, Keime C, Robardet C, Boulicaut J, Besson J, Pensa R, Gandrillon O,
Journal : BMC Bioinformatics
2008
BACKGROUND: There is an increasing need in transcriptome research for gene expression data and pattern warehouses. It is of importance to integrate in these warehouses both raw transcriptomic data, as well as some properties encoded in these data, like local patterns. DESCRIPTION: We have developed an application called SQUAT (SAGE Querying and Analysis Tools) which is available at: http://bsmc.insa-lyon.fr/squat/. This database gives access to both raw SAGE data and patterns mined from these data, for three species (human, mouse and chicken). This database allows to make simple queries like "In which biological situationsis my favorite gene expressed?" as well as much more complex queries like: <<what are the genes that are frequently co-over-expressed with my gene of interest in given biological situations?>>. Connections with external web databases enrich biological interpretations, and enable sophisticated queries. To illustrate the power of SQUAT, we show and analyze the results of three different queries, one of which led to a biological hypothesis that was experimentally validated. CONCLUSION: SQUAT is a user-friendly information retrieval platform, which aims at bringing some of the state-of-the-art mining tools to biologists.

Stem cell antigen 2: a new gene involved in the self-renewal of erythroid progenitors.

Author(s) : Bresson-Mazet C, Gandrillon O, Gonin-Giraud S,
Journal : Cell Prolif
2008
OBJECTIVES: Stem cell antigen 2 (SCA2), also known as TSA1 and LY6E, is a glycosylphosphatidylinositol-anchored molecule that belongs to the Ly-6 family and whose function remains largely unknown. We have previously shown that SCA2 is overexpressed in self-renewing avian erythroid progenitors (T2ECs) as opposed todifferentiating T2ECs. The aim of this study was to define the role of SCA2 in the switch between self-renewal and differentiation of erythroid progenitors. MATERIALS AND METHODS: We have investigated the cellular processes controlled bySCA2 in T2ECs by RNA interference and overexpression approaches. Moreover, we have used a SAGE Querying and analysis tools developed in our laboratory, to investigate the expression level of SCA2 gene in different human cell types. RESULTS: We demonstrate the regulation of SCA2 expression by TGF-beta, a growth factor essential for self-renewal of T2ECs. We establish that SCA2 knockdown by RNA interference reduced the proliferation and promoted the differentiation of T2ECs. In contrast, SCA2 overexpression inhibited differentiation of T2ECs only.Furthermore, by using a bioinformatic approach, we found that SCA2 is highly expressed in a variety of human cancer cells. We confirmed this result by quantitative PCR on human colon and kidney tissues. CONCLUSIONS: Altogether, these findings imply that SCA2 may function in a dose-dependent manner to support the self-renewal state and that its deregulation might contribute to the development of some human cancers.
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2007

Coregulators: transducing signal from transcription to alternative splicing

Author(s) : Auboeuf D, Batsch? E, Dutertre M, Muchardt C, O'Malley B,
Journal : Trends Endocrinol Metab
2007

Disruption of the palatal rugae pattern in Tabby (eda) mutant mice.

Author(s) : Charles C, Pantalacci S, Peterkova R, Peterka M, Laudet V, Viriot L,
Journal : Eur J Oral Sci
2007
The eda mouse gene is linked with anomalies of ectodermal derivatives, such as hair, glands, and teeth. The palatal rugae (oral mucosa foldings on the hard palate) are also ectodermal derivatives. Therefore, we searched for and comparedpalatal rugae anomalies of Tabby mice bearing a mutation in the eda gene with their wild-type counterparts. We compared the number and shape of palatal rugae in 179 mutant and 102 wild-type mice from four different stocks of Tabby mice. Palatal rugae anomalies were documented at a low frequency in wild-type mice of different backgrounds, which may reflect a lack of robustness of palatal rugae development. However, the proportion of anomalies observed in the C57BL/6J background makes us recommend avoiding its use in further palate studies. We showed statistically that the phenotypic variability seen in wild-type animals is further increased in Tabby mutants. The anomalies mainly included various forms of reduction, with rugae IV-VI being more frequently affected. Those rugae were shortened, dotted or absent (mainly ruga V). By analogy to the role played by eda in other ectodermal derivatives, we propose that it might play a role in defining the pattern of the palatal rugae.

Efficient use of DNA molecular markers to construct industrial yeast strains.

Author(s) : Marullo P, Yvert G, Bely M, Aigle M, Dubourdieu D,
Journal : FEMS Yeast Res
2007
Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phaseduration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.

Genetic complexity and quantitative trait loci mapping of yeast morphological traits.

Author(s) : Nogami S, Ohya Y, Yvert G,
Journal : PLoS Genet
2007
Functional genomics relies on two essential parameters: the sensitivity of phenotypic measures and the power to detect genomic perturbations that cause phenotypic variations. In model organisms, two types of perturbations are widelyused. Artificial mutations can be introduced in virtually any gene and allow thesystematic analysis of gene function via mutants fitness. Alternatively, naturalgenetic variations can be associated to particular phenotypes via genetic mapping. However, the access to genome manipulation and breeding provided by model organisms is sometimes counterbalanced by phenotyping limitations. Here weinvestigated the natural genetic diversity of Saccharomyces cerevisiae cellular morphology using a very sensitive high-throughput imaging platform. We quantified 501 morphological parameters in over 50,000 yeast cells from a cross between twowild-type divergent backgrounds. Extensive morphological differences were found between these backgrounds. The genetic architecture of the traits was complex, with evidence of both epistasis and transgressive segregation. We mapped quantitative trait loci (QTL) for 67 traits and discovered 364 correlations between traits segregation and inheritance of gene expression levels. We validated one QTL by the replacement of a single base in the genome. This study illustrates the natural diversity and complexity of cellular traits among natural yeast strains and provides an ideal framework for a genetical genomics dissection of multiple traits. Our results did not overlap with results previously obtainedfrom systematic deletion strains, showing that both approaches are necessary forthe functional exploration of genomes.

HTLV-1 HBZ cooperates with JunD to enhance transcription of the human telomerase reverse transcriptase gene (hTERT).

Author(s) : Kuhlmann A, Villaudy J, Gazzolo L, Castellazzi M, Mesnard J, Duc Dodon M,
Journal : Retrovirology
2007
BACKGROUND: Activation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells displaya high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription. RESULTS: Here, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain anyAP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter. CONCLUSION: These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.

Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle.

Author(s) : Buchsbaum S, Morris C, Bochard V, Jalinot P,
Journal : Oncogene
2007
The mouse int6 gene is a frequent integration site of the mouse mammary tumor virus and INT6 silencing by RNA interference in HeLa cells causes an increased number of cells in the G2/M phases of the cell cycle, along with mitotic defects. In this report, we investigated the functional significance of the interaction between INT6 and MCM7, which was observed in a two-hybrid screen performed with INT6 as bait. It was found that proteasome inhibition strengthens interaction between both proteins and that INT6 stabilizes MCM7. Removal of MCM7 from chromatin as replication proceeds was accelerated in INT6-silenced cells and reduced amounts of protein were transiently observed, followed by a correction resulting from stimulation of mcm7 gene expression. Synchronized cells depleted for either INT6 or MCM7 display a reduction in thymidine incorporation and a reinforced association of RPA and claspin with chromatin. These data show that INT6 stabilizes chromatin-bound MCM7 and that alteration of this effect is associated with replication deficiency.

Human INT6/eIF3e is required for nonsense-mediated mRNA decay.

Author(s) : Morris C, Wittmann J, Jack H, Jalinot P,
Journal : EMBO Rep
2007
The mammalian integration site 6 (INT6) protein has been implicated in breast carcinogenesis and characterized as the eIF3e non-core subunit of the translation initiation factor eIF3, but its role in this complex is not known. Here, we showthat INT6 knockdown by RNA interference strongly inhibits nonsense-mediated messenger RNA decay (NMD), which triggers degradation of mRNAs with premature stop codons. In contrast to the eIF3b core subunit, which is required for both NMD and general translation, INT6 is only necessary for the former process. Consistent with such a role, immunoprecipitation experiments showed that INT6 co-purifies with CBP80 and the NMD factor UPF2. In addition, several transcriptsknown to be upregulated by UPF1 or UPF2 depletion were also found to be sensitive to INT6 suppression. From these observations, we propose that INT6, in association with eIF3, is involved in routing specific mRNAs for degradation.

Immunological changes and cytokine gene expression during primary infection with human T-cell leukaemia virus type 1 in squirrel monkeys (Saimiri sciureus)

Author(s) : Heraud J, Merien F, Mortreux F, Mahieux R, Kazanji M,
Journal : Virology
2007

Influence of the transposable element neighborhood on human gene expression in normal and tumor tissues.

Author(s) : Lerat E, Semon M,
Journal : Gene
2007
Transposable elements (TEs) are genomic sequences able to replicate themselves, and to move from one chromosomal position to another within the genome. Many TEscontain their own regulatory regions, which means that they may influence the expression of neighboring genes. TEs may also be activated and transcribed in various cancers. We therefore tested whether gene expression in normal and tumortissues is influenced by the neighboring TEs. To do this, we associated all human genes to the nearest TEs. We analyzed the expression of these genes in normal and tumor tissues using SAGE and EST data, and related this to the presence and typeof TEs in their vicinity. We confirmed that TEs tend to be located in antisense orientation relative to their hosting genes. We found that the average number oftissues where a gene is expressed varies depending on the type of TEs located near the gene, and that the difference in expression level between normal and tumor tissues is greatest for genes that host SINE elements. This deregulation increases with the number of SINE copies in the gene vicinity. This suggests that SINE elements might contribute to the cascade of gene deregulation in cancer cells.

Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action.

Author(s) : Bresson C, Keime C, Faure C, Letrillard Y, Barbado M, Sanfilippo S, Benhra N, Gandrillon O, Gonin-Giraud S,
Journal : BMC Genomics
2007
BACKGROUND: The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R). v-ErbA transforms erythroid progenitors in vitro by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor). However, v-ErbA target genes involved in its transforming activity still remain to be identified. RESULTS: By using Serial Analysis of Gene Expression (SAGE), we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysisof promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription) and unexpected (protein metabolism) functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCRthat in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA. CONCLUSION: This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.

Parasitic inhibition of cell death facilitates symbiosis.

Author(s) : Pannebakker B, Loppin B, Elemans C, Humblot L, Vavre F,
Journal : Proc Natl Acad Sci U S A
2007
Symbiotic microorganisms have had a large impact on eukaryotic evolution, with effects ranging from parasitic to mutualistic. Mitochondria and chloroplasts areprime examples of symbiotic microorganisms that have become obligate for their hosts, allowing for a dramatic extension of suitable habitats for life. Out of the extraordinary diversity of bacterial endosymbionts in insects, most are facultative for their hosts, such as the ubiquitous Wolbachia, which manipulateshost reproduction. Some endosymbionts, however, have become obligatory for host reproduction and/or survival. In the parasitoid wasp Asobara tabida the presenceof Wolbachia is necessary for host oogenesis, but the mechanism involved is yet unknown. We show that Wolbachia influences programmed cell death processes (a host regulatory feature typically targeted by pathogens) in A. tabida, making its presence essential for the wasps' oocytes to mature. This suggests that parasitestrategies, such as bacterial regulation of host apoptosis, can drive the evolution of host dependence, allowing for a swift transition from parasitism tomutualism.

Regulation of H-ras splice variant expression by cross talk between the p53 and nonsense-mediated mRNA decay pathways

Author(s) : Barbier J, Dutertre M, Bittencourt D, Sanchez G, Gratadou L, de la Grange P, Auboeuf D,
Journal : Mol Cell Biol
2007

SAGE analysis of mosquito salivary gland transcriptomes during Plasmodium invasion.

Author(s) : Rosinski-Chupin I, Briolay J, Brouilly P, Perrot S, Gomez S, Chertemps T, Roth C, Keime C, Gandrillon O, Couble P, Brey P,
Journal : Cell Microbiol
2007
Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune-related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulatedduring salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strains.

Author(s) : Marullo P, Aigle M, Bely M, Masneuf-Pomarede I, Durrens P, Dubourdieu D, Yvert G,
Journal : FEMS Yeast Res
2007
Natural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possible to identify single-feature polymorphisms among divergent yeast strains. This tool offers thepossibility of applying quantitative genetics to wild yeast strains. In this instance, we studied the genetic basis for variations in acetic acid production using progeny derived from two strains from grape must isolates. The trait was quantified during alcoholic fermentation of the two strains and 108 segregants derived from their crossing. A genetic map of 2212 markers was generated using oligonucleotide microarrays, and a major quantitative trait locus (QTL) was mapped with high significance. Further investigations showed that this QTL was due to a nonsynonymous single-nucleotide polymorphism that targeted the catalytic core of asparaginase type I (ASP1) and abolished its activity. This QTL was onlyeffective when asparagine was used as a major nitrogen source. Our results link nitrogen assimilation and CO(2) production rate to acetic acid production, as well as, on a broader scale, illustrating the specific problem of quantitative genetics when working with nonlaboratory microorganisms.

The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization.

Author(s) : Bonnefoy E, Orsi G, Couble P, Loppin B,
Journal : PLoS Genet
2007
In many animal species, the sperm DNA is packaged with male germ line--specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected inHira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.

The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction

Author(s) : Skrisovska L, Bourgeois C, Stefl R, Grellscheid S, Kister L, Wenter P, Elliott D, Stevenin J, Allain F,
Journal : EMBO Rep
2007

Unexpected observations after mapping LongSAGE tags to the human genome.

Author(s) : Keime C, Semon M, Mouchiroud D, Duret L, Gandrillon O,
Journal : BMC Bioinformatics
2007
BACKGROUND: SAGE has been used widely to study the expression of known transcripts, but much less to annotate new transcribed regions. LongSAGE produces tags that are sufficiently long to be reliably mapped to a whole-genome sequence. Here we used this property to study the position of human LongSAGE tags obtainedfrom all public libraries. We focused mainly on tags that do not map to known transcripts. RESULTS: Using a published error rate in SAGE libraries, we first removed the tags likely to result from sequencing errors. We then observed that an unexpectedly large number of the remaining tags still did not match the genome sequence. Some of these correspond to parts of human mRNAs, such as polyA tails,junctions between two exons and polymorphic regions of transcripts. Another non-negligible proportion can be attributed to contamination by murine transcripts and to residual sequencing errors. After filtering out our data withthese screens to ensure that our dataset is highly reliable, we studied the tagsthat map once to the genome. 31% of these tags correspond to unannotated transcripts. The others map to known transcribed regions, but many of them (nearly half) are located either in antisense or in new variants of these known transcripts. CONCLUSION: We performed a comprehensive study of all publicly available human LongSAGE tags, and carefully verified the reliability of these data. We found the potential origin of many tags that did not match the human genome sequence. The properties of the remaining tags imply that the level of sequencing error may have been under-estimated. The frequency of tags matching once the genome sequence but not in an annotated exon suggests that the human transcriptome is much more complex than shown by the current human genome annotations, with many new splicing variants and antisense transcripts. SAGE data is appropriate to map new transcripts to the genome, as demonstrated by the highrate of cross-validation of the corresponding tags using other methods.

2006

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching.

Author(s) : Vitte A, Buchsbaum S, Jalinot P,
Journal : FEBS Lett
2006
The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to argininereduces markedly the steady state amount of the protein, but does not impair itsability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.

Optimal estimates of free energies from multistate nonequilibrium work data.

Author(s) : Maragakis P, Spichty M, Karplus M,
Journal : Phys Rev Lett
2006
We derive the optimal estimates of the free energies of an arbitrary number of thermodynamic states from nonequilibrium work measurements; the work data are collected from forward and reverse switching processes and obey a fluctuation theorem. The maximum likelihood formulation properly reweights all pathways contributing to a free energy difference and is directly applicable to simulations and experiments. We demonstrate dramatic gains in efficiency by combining the analysis with parallel tempering simulations for alchemical mutations of model amino acids.

Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency

Author(s) : Kim Y, Bourgeois C, Pearson R, Tyagi M, West M, Wong J, Wu S, Chiang C, Karn J,
Journal : EMBO J
2006

The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development.

Author(s) : Coustham V, Bedet C, Monier K, Schott S, Karali M, Palladino F,
Journal : Dev Biol
2006
HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters by corepressor proteins including TIF1 and Rb. The Caenorhabditis elegans HP1 homologue HPL-2 acts in the "synMuv" (synthetic multivulval) pathway, which defines redundant negative regulators of a Ras signaling cascade required for vulval induction. Several synMuv genes encode for chromatin-associated proteins involved in transcriptional regulation, including Rb and components of the Mi-2/NuRD and TIP60/NuA4 chromatin remodeling complexes. Here, we show that HPL-2 physically interacts in vitro and in vivo with the multiple zinc finger protein LIN-13, another member of the synMuv pathway. A variant of the conservedPXVXL motif found in many HP1-interacting proteins mediates LIN-13 binding to the CSD of HPL-2. We further show by in vivo localization studies that LIN-13 is required for HPL-2 recruitment in nuclear foci. Our data suggest that the LIN-13/HPL-2 complex may physically link a subset of the Rb related synMuv proteins to chromatin.

The efficacy of combined therapy of arsenic trioxide and alpha interferon in human T-cell leukemia virus type-1-infected squirrel monkeys (Saimiri sciureus)

Author(s) : Heraud J, Mortreux F, Merien F, Contamin H, Mahieux R, Pouliquen J, Wattel E, Gessain A, de Th? H, Bazarbachi A, Hermine O, Kazanji M,
Journal : Antiviral Res
2006

Unique and redundant functions of C. elegans HP1 proteins in post-embryonic development.

Author(s) : Schott S, Coustham V, Simonet T, Bedet C, Palladino F,
Journal : Dev Biol
2006
HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent the activity of these different family members isredundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While HPL-2 functions in vulval and germline development, no function has so far been attributed to HPL-1. Here we report thecharacterization of an hpl-1 null allele. We show that while the absence of hpl-1 alone results in no obvious phenotype, hpl-1;hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that hpl-1 has an unexpected role in vulval development by acting redundantly with hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 localization does not completely overlap. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development.

2005

Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1

Author(s) : Debacq C, H?raud J, Asquith B, Bangham C, Merien F, Moules V, Mortreux F, Wattel E, Burny A, Kettmann R, Kazanji M, Willems L,
Journal : Oncogene
2005

Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins.

Author(s) : Seeliger M, Spichty M, Kelly S, Bycroft M, Freund S, Karplus M, Itzhaki L,
Journal : J Biol Chem
2005
Cks proteins are adapter molecules that coordinate the assembly of multiprotein complexes. They share the ability to domain swap by exchanging a beta-strand, beta4. Here we use NMR spectroscopy and molecular dynamics simulations to investigate the dynamic properties of human Cks1 and its response on assembly with components of the SCF(Skp2) ubiquitin ligation machinery. In the NMR experiment with the free form of Cks1, a subset of residues displayed elevated R2 values and the cross-peaks of neighboring residues were missing from the spectrum, indicating a substantial conformational exchange contribution on the microsecond to millisecond time scale. Strikingly the region of greatest conformational variability was the beta4-strand that domain swaps to form the dimer. Binding of the ligand common to all Cks proteins, Cdk2, suppressed the conformational heterogeneity. This response was specific to Cdk2 binding; in contrast, binding of Skp2, a ligand unique to human Cks1, did not alter the dynamic behavior. Short time (<5 ns) molecular dynamics simulations indicate that residues of Cks1 that form the binding site for phosphorylated ligands are considerably more flexible in the free form of Cks1 than they are in the Cdk2-Cks1 complex. A cooperative interaction between Cdk2 and Cks1 is suggested,which reduces the configurational entropy of Cks1 and therefore facilitates phosphoprotein binding. Indications of an unusual dynamic behavior of strand beta4 in the free form of Cks1 were obtained from longer time scale (50 ns) dynamics simulations. A spontaneous reversible unzipping of hydrogen bonds between beta4 and beta2 was observed, suggesting an early intermediate structurefor unfolding and/or domain swapping. We propose that the dynamic properties of the beta-sheet and its modification upon ligand binding underlie the domain swapping ability and the adapter function of Cks proteins.

Serial analysis of gene expression in the silkworm, Bombyx mori.

Author(s) : Huang J, Miao X, Jin W, Couble P, Mita K, Zhang Y, Liu W, Zhuang L, Shen Y, Keime C, Gandrillon O, Brouilly P, Briolay J, Zhao G, Huang Y,
Journal : Genomics
2005
The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmentallife cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, ofwhich 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, andadult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.

Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B-Cdk1 activation.

Author(s) : Morris C, Jalinot P,
Journal : Oncogene
2005
The Int-6 protein has been originally identified as the product of a mouse gene being a frequent integration site of the mouse mammary tumour virus. Here, we show that reducing Int-6 expression by RNA interference in HeLa cells markedly alters mitosis progression. Defects in spindle formation, chromosome segregationand cytokinesis were observed. These abnormalities of mitosis completion are correlated with an inhibition of cyclin B-Cdk1 kinase activity, due to a prolonged inhibitory phosphorylated state of Cdk1. In line with this observation, the Wee1 tyrosine kinase that negatively controls Cdk1 was less efficiently inactivated during G2 in Int-6-depleted cells. These findings support the notionthat the oncogenic properties associated with alteration of Int-6 originate fromchromosomal instability.

Steroid hormone receptor coactivation and alternative RNA splicing by U2AF65-related proteins CAPERalpha and CAPERbeta

Author(s) : Dowhan D, Hong E, Auboeuf D, Dennis A, Wilson M, Berget S, O'Malley B,
Journal : Mol Cell
2005

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus.

Author(s) : Loppin B, Bonnefoy E, Anselme C, Laurencon A, Karr T, Couble P,
Journal : Nature
2005
In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competentmale pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sesame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosomeassembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show thatnucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.

Up-regulation of the ubiquitous alternative splicing factor Tra2beta causes inclusion of a germ cell-specific exon

Author(s) : Venables J, Bourgeois C, Dalgliesh C, Kister L, Stevenin J, Elliott D,
Journal : Hum Mol Genet
2005

2004

Reduced proviral loads during primo-infection of sheep by Bovine Leukemia virus attenuated mutants

Author(s) : Debacq C, Sanchez Alcaraz M, Mortreux F, Kerkhofs P, Kettmann R, Willems L,
Journal : Retrovirology
2004

Regulation of R7 and R8 differentiation by the spalt genes.

Author(s) : Domingos P, Brown S, Barrio R, Ratnakumar K, Frankfort B, Mardon G, Steller H, Mollereau B,
Journal : Dev Biol
2004
Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of thedeveloping ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specificationor differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt andsenseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8differentiation during pupal stages, including the regulation of rhodopsin expression.

Spalt transcription factors are required for R3/R4 specification and establishment of planar cell polarity in the Drosophila eye.

Author(s) : Domingos P, Mlodzik M, Mendes C, Brown S, Steller H, Mollereau B,
Journal : Development
2004
The establishment of planar cell polarity in the Drosophila eye requires correctspecification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it as R4. Here, we show thatthe spalt zinc-finger transcription factors (spalt major and spalt-related) are part of the molecular mechanisms regulating R3/R4 specification and planar cell polarity establishment. In mosaic analysis, we find that the spalt genes are specifically required in R3 for the establishment of correct ommatidial polarity. In addition, we show that spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4, and for the upregulation of Delta in R3. These requirements are very similar to those of frizzled during R3/R4 specification. We show that spalt genes are required cell-autonomously for the expression of seven-up in R3 and R4, and that seven-up is downstream of spalt genes in the genetic hierarchy of R3/R4 specification. Thus, spalt and seven-up are necessary for the correct interpretation of the Frizzled-mediated polarity signal in R3. Finally, we show that, posterior to row seven, seven-up represses spalt in R3/R4 in order to maintain the R3/R4 identity and to inhibit the transformation of these cells to the R7 cell fate.

2003

Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors.

Author(s) : Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker N, Perlmann T,
Journal : Nature
2003
Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminalligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 A resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a 'classical' binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stableconformation. Our findings highlight a unique structural class of NRs and definea model for ligand-independent NR function.

The MEK-1/ERKs signalling pathway is differentially involved in the self-renewal of early and late avian erythroid progenitor cells.

Author(s) : Dazy S, Damiola F, Parisey N, Beug H, Gandrillon O,
Journal : Oncogene
2003
Making decisions between self-renewal and differentiation is a central ability of stem cells. Elucidation of molecular networks governing this decision is therefore of prime importance. A model of choice to explore this question is represented by chicken erythroid progenitors, in which self-renewal versus differentiation as well as progenitor maturation are regulated by external factor combinations. We used this system to study whether similar or different signalling pathways were involved in the self-renewal of early, immature or moremature erythroid progenitors. We show that a transforming growth factor (TGF)-alpha-activated Ras/MEK-1/ERK1/2 pathway is strictly required for immatureself-renewing cells but becomes fully dispensable when those cells are induced to differentiate. Consequently, pharmacological inhibition of this pathway led to spontaneous differentiation, only dependent on the presence of survival signals.Conversely, ectopic expression of a constitutive form of MEK-1 stimulates renewal and arrests differentiation process. Finally, we demonstrate that the ERK/MAPK signalling pathway is required in early but not in late primary erythroid progenitors, which can be turned into each other by different growth factor combinations specifically driving their renewal. To the best of our knowledge, this is the first description of a central role of ERK/MAPK signalling in regulating progenitor plasticity in the same cell type under different environmental conditions.

The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila.

Author(s) : Pantalacci S, Tapon N, Leopold P,
Journal : Nat Cell Biol
2003
Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) gene, which encodes a scaffold protein, has been shown to restrict cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Here we identify Hippo (Hpo), the Drosophila orthologue of the mammalian MST1 and MST2 serine/threonine kinases, as a partner of Sav. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1, overexpression of Hpo destabilizes DIAP1 in cell culture. We show that DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, we show that Sav is an unstable protein that is stabilized by Hpo. We propose that Hpo and Sav function together to restrict tissue growth in vivo.

2002

Spt5 cooperates with human immunodeficiency virus type 1 Tat by preventing premature RNA release at terminator sequences

Author(s) : Bourgeois C, Kim Y, Churcher M, West M, Karn J,
Journal : Mol Cell Biol
2002

Strong-association-rule mining for large-scale gene-expression data analysis: a case study on human SAGE data.

Author(s) : Becquet C, Blachon S, Jeudy B, Boulicaut J, Gandrillon O,
Journal : Genome Biol
2002
BACKGROUND: The association-rules discovery (ARD) technique has yet to be applied to gene-expression data analysis. Even in the absence of previous biological knowledge, it should identify sets of genes whose expression is correlated. The first association-rule miners appeared six years ago and proved efficient at dealing with sparse and weakly correlated data. A huge international research effort has led to new algorithms for tackling difficult contexts and these are particularly suited to analysis of large gene-expression matrices. To validate the ARD technique we have applied it to freely available human serial analysis of gene expression (SAGE) data. RESULTS: The approach described here enables us to designate sets of strong association rules. We normalized the SAGE data before applying our association rule miner. Depending on the discretization algorithm used, different properties of the data were highlighted. Both common and specific interpretations could be made from the extracted rules. In each and every case the extracted collections of rules indicated that a very strong co-regulation ofmRNA encoding ribosomal proteins occurs in the dataset. Several rules associating proteins involved in signal transduction were obtained and analyzed, some pointing to yet-unexplored directions. Furthermore, by examining a subset of these rules, we were able both to reassign a wrongly labeled tag, and to proposea function for an expressed sequence tag encoding a protein of unknown function.CONCLUSIONS: We show that ARD is a promising technique that turns out to be complementary to existing gene-expression clustering techniques.

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

Author(s) : Mey A, Gandrillon O, McNagny K, Clegg D, Samarut J,
Journal : Oncogene
2002
T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which mayaccount for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our datasuggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

The v-erbA oncogene. Assessing its differentiation-blocking ability using normal chicken erythrocytic progenitor cells.

Author(s) : Gandrillon O,
Journal : Methods Mol Biol
2002

Two Na,K-ATPase beta 2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis.

Author(s) : Rajarao J, Canfield V, Loppin B, Thisse B, Thisse C, Yan Y, Postlethwait J, Levenson R,
Journal : Dev Dyn
2002
We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase beta 2 subunit. The beta 2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish beta 2a subunit. By using a zebrafish meiotic mapping panel, we determined that the beta2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the beta 2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. beta 2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, beta 2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These resultssuggest that the beta 2a and beta 2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian beta 2 gene may be partitioned between the two zebrafish beta 2 orthologs.

2001

Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites.

Author(s) : Idres N, Benoit G, Flexor M, Lanotte M, Chabot G,
Journal : Cancer Res
2001
The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid(18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRAand all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required todifferentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Lineage restriction of the RARalpha gene expression in myeloid differentiation.

Author(s) : Zhu J, Heyworth C, Glasow A, Huang Q, Petrie K, Lanotte M, Benoit G, Gallagher R, Waxman S, Enver T, Zelent A,
Journal : Blood
2001
To better understand the role of retinoids in myelopoiesis, expression of the retinoid receptor genes (retinoic acid receptors [RARs] and retinoid X receptors[RXRs]) were examined during differentiation of factor-dependent cell-Paterson (FDCP)-mixA4 murine progenitor cells. The major receptor expressed in undifferentiated A4 cells was RARalpha (primarily the RARalpha1 isoform). Following induction of myelomonocytic differentiation with granulocyte and granulocyte-macrophage colony-stimulating factors, a dramatic increase in RARalpha expression (particularly the RARalpha2 isoform) was seen. In contrast, expression of both RARalpha isoforms was rapidly extinguished upon induction of erythroid differentiation with erythropoeitin (EPO). A modest induction of RXRalpha expression was seen, particularly during differentiation in the myelomonocytic lineage. Low expression levels of RARgamma2 and RXRbeta remained unchanged, irrespective of differentiation pathway. Consistent with the gene expression patterns, RARalpha agonists and antagonists stimulated myelomonocyticand erythroid differentiation of FDCP-mixA4 cells, respectively. Taken together,these results suggest that erythropoiesis and granulopoiesis require diminished and enhanced RARalpha activities, respectively, which at physiological all-trans-retinoic acid (RA) concentrations may be accomplished by reciprocal effects of EPO and myelomonocytic growth factors on its expression. This hypothesis is corroborated by data showing that RA, which positively regulates RARalpha2 expression, can exert inhibitory effects on erythroid differentiation.

Long-distance charge transport through DNA. An extended hopping model

Author(s) : Giese B, Spichty M, Wessely S,
Journal : Pure and Applied Chemistry
2001
Long-distance transfer of a positive charge through DNA can be described by a hopping model. In double strands where the (A:T)n bridges between the guanines are short (n 3), the charge hops only between guanines, and each hopping step depends strongly upon the guanine to guanine distances. In strands where the (A:T)n sequences between the guanines are rather long (n 4), also the adenines act as charge carriers. To predict the yields of the H2O-trapping products one has to take into account not only the charge-transfer rates but also the rates of H2O-trapping reactions.

Orchestration of multiple arrays of signal cross-talk and combinatorial interactions for maturation and cell death: another vision of t(15;17) preleukemic blast and APL-cell maturation.

Author(s) : Benoit G, Roussel M, Pendino F, Segal-Bendirdjian E, Lanotte M,
Journal : Oncogene
2001
Despite intensive molecular biology investigations over the past 10 years, and an important breakthrough on how PML-RARalpha, the fusion protein resulting from t(15;17), can alter RARalpha and PML functions, no definitive views on how leukemia is generated and by what mechanism(s) the normal phenotype is restored,are yet available. 'Resistances' to pharmacological levels of all-trans-retinoicacid (ATRA) have been observed in experimental in vivo and in vitro models. In this review, we emphasize the key role played by signal cross-talk for both normal and neoplastic hemopoiesis. After an overview of reported experimental data on APL-cell maturation and apoptosis, we apply our current knowledge on signaling pathways to underline those which might generate signal cross-talks. The design of biological models suitable to decipher the integration of signal cross-talks at the transcriptional level should be our first priority today, to generate some realistic therapeutic approaches After 'Ten Years of Molecular APL', we still know very little about how the disease develops and how effectivemedicines work.

Paternal chromosome incorporation into the zygote nucleus is controlled by maternal haploid in Drosophila.

Author(s) : Loppin B, Berger F, Couble P,
Journal : Dev Biol
2001
maternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternalchromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia.

Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp.

Author(s) : Dedeine F, Vavre F, Fleury F, Loppin B, Hochberg M, Bouletreau M,
Journal : Proc Natl Acad Sci U S A
2001
Wolbachia are bacteria that live in the cells of various invertebrate species towhich they cause a wide range of effects on physiology and reproduction. We investigated the effect of Wolbachia infection in the parasitic wasp, Asobara tabida Nees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected by Wolbachia. The removal of Wolbachia by antibiotic treatment had a totally unexpected effect-aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treated Asobara citri, a closely related species that does not harbor Wolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents)are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production and Wolbachia density infemales. We conclude that Wolbachia is necessary for oogenesis in these A. tabida strains, and this association would seem to be the first example of a transitionfrom facultative to obligatory symbiosis in arthropod-Wolbachia associations.

Requirement of heterochromatin for cohesion at centromeres.

Author(s) : Bernard P, Maure J, Partridge J, Genier S, Javerzat J, Allshire R,
Journal : Science
2001
Centromeres are heterochromatic in many organisms, but the mitotic function of this silent chromatin remains unknown. During cell division, newly replicated sister chromatids must cohere until anaphase when Scc1/Rad21-mediated cohesion is destroyed. In metazoans, chromosome arm cohesins dissociate during prophase, leaving centromeres as the only linkage before anaphase. It is not known what distinguishes centromere cohesion from arm cohesion. Fission yeast Swi6 (a Heterochromatin protein 1 counterpart) is a component of silent heterochromatin.Here we show that this heterochromatin is specifically required for cohesion between sister centromeres. Swi6 is required for association of Rad21-cohesin with centromeres but not along chromosome arms and, thus, acts to distinguish centromere from arm cohesion. Therefore, one function of centromeric heterochromatin is to attract cohesin, thereby ensuring sister centromere cohesion and proper chromosome segregation.

Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo

Author(s) : Mortreux F, Leclercq I, Gabet A, Leroy A, Westhof E, Gessain A, Wain-Hobson S, Wattel E,
Journal : J Natl Cancer Inst
2001

The Drosophila maternal gene sesame is required for sperm chromatin remodeling at fertilization.

Author(s) : Loppin B, Berger F, Couble P,
Journal : Chromosoma
2001
The spermatozoon features an extremely condensed and inactive nucleus. The unique sperm chromatin organization is acquired during the late stages of spermatid differentiation by the replacement of somatic histones with sperm-specific chromosomal proteins. At fertilization, the inactive sperm nucleus must be rapidly transformed into a DNA replication competent male pronucleus before the formation of the zygote. The sequential events of this crucial process are well conserved among animals and are controlled by molecules present in the egg. We have previously identified a Drosophila maternal effect mutation called sesame, which specifically arrests male pronucleus formation at a late stage of chromatin decondensation. In this study, we show that sesame affects maternal histone incorporation in the male pronucleus, a situation that is expected to prevent nucleosomal organization of the paternal chromatin. As an apparent consequence, the male pronucleus is arrested before the first S-phase and does not condense mitotic chromosomes. However, centromeric heterochromatin is present on paternalcentromeres, which occasionally interact with microtubules. The abnormal chromatin organization of the male pronucleus does not prevent the formation of a male pronuclear envelope, which breaks down and reassembles in synchrony with maternally derived nuclei present in the same cytoplasm.

The v-ErbA oncoprotein quenches the activity of an erythroid-specific enhancer.

Author(s) : Braliou G, Ciana P, Klaassen W, Gandrillon O, Stunnenberg H,
Journal : Oncogene
2001
v-ErbA is a mutated variant of thyroid hormone receptor (TRalpha/NR1A1) borne bythe Avian Erythroblastosis virus causing erythroleukemia. TRalpha is known to activate transcription of specific genes in the presence of its cognate ligand, T3 hormone, while in its absence it represses it. v-ErbA is unable to bind ligand, and hence is thought to contribute to leukemogenesis by actively repressing erythroid-specific genes such as the carbonic anhydrase II gene (CA II). In the prevailing model, v-ErbA occludes liganded TR from binding to its cognate elements and constitutively interacts with the corepressors NCoR/SMRT. We previously identified a v-ErbA responsive element (VRE) within a DNase I hypersensitive region (HS2) located in the second intron of the CA II gene. We now show that HS2 fulfils all the requirements for a genuine enhancer that functions independent of its orientation and position with a profound erythroid-specific activity in normal erythroid progenitors (T2ECs) and in leukemic erythroid cell lines. We find that the HS2 enhancer activity is governed by two adjacent GATA-factor binding sites. v-ErbA as well as unliganded TR prevent HS2 activity by nullifying the positive function of factors bound to GATA-sites. However, v-ErbA, in contrast to TR, does not convey active repression to silence the transcriptional activity intrinsic to a heterologous tk promoter.We propose that depending on the sequence and context of the binding site, v-ErbA contributes to leukemogenesis by occluding liganded TR as well as unliganded TR thereby preventing activation or repression, respectively.

Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

Author(s) : Debec A, Grammont M, Berson G, Dastugue B, Sullivan W, Couderc J,
Journal : Genesis
2001
The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al.,1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at thespindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results stronglysuggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

Two-step nature of human T-cell leukemia virus type 1 replication in experimentally infected squirrel monkeys (Saimiri sciureus)

Author(s) : Mortreux F, Kazanji M, Gabet A, de Thoisy B, Wattel E,
Journal : J Virol
2001

Two-step process for photoreceptor formation in Drosophila.

Author(s) : Mollereau B, Dominguez M, Webel R, Colley N, Keung B, de Celis J, Desplan C,
Journal : Nature
2001
The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1-R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulatorsand sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1-R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.

2000

Giese B, Spichty M
Long distance charge transport through DNA: quantification and extension of the hopping model.
Chemphyschem, 23696320
2000

Glatthar R, Spichty M, Gugger A, Batra R, Damm W, Mohr M, Zipse H, Giese B

Tetrahedron,
2000

Liu X, Grammont M, Irvine K
Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge.
Dev Biol, 11112330
2000

Loppin B, Docquier M, Bonneton F, Couble P
The maternal effect mutation sesame affects the formation of the male pronucleus in Drosophila melanogaster.
Dev Biol, 10837127
2000

Del Gatto-Konczak F, Bourgeois C, Le Guiner C, Kister L, Gesnel M, Stevenin J, Breathnach R
The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site.
Mol Cell Biol, 10938105
2000

Del Gatto-Konczak F, Bourgeois C, Le Guiner C, Kister L, Gesnel M, St?venin J, Breathnach R

Mol Cell Biol,
2000

Spichty M, Fragale G, Wirth T

Journal of the American Chemical Society,
2000

2016 et 2017

Article Reference Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.
INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample tobe detected and quantified. Modern lipidomics techniques are ultra-sensitive assaysthat enable the discovery of novel biomarkers in a variety of fields and provide newinsight in mechanistic investigations. Despite much progress in lipidomics, thereremains, as for all high throughput "omics" strategies, the need to developstrategies to standardize and integrate quality control into studies in order toenhance robustness, reproducibility, and usability of studies within specific fieldsand beyond. OBJECTIVES: We aimed to understand how much results from lipid profilingin the model organism Caenorhabditis elegans are influenced by different cultureconditions in different laboratories. METHODS: In this work we have undertaken aninter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans anddaf-2(e1370) mutants lacking a functional insulin receptor. Sample were collectedfrom worms grown in four separate laboratories under standardized growth conditions.We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MSanalysis. RESULTS: We found common qualitative changes in several marker lipids insamples from the individual laboratories. On the other hand, even in this controlledexperimental system, the exact fold-changes for each marker varied betweenlaboratories. CONCLUSION: Our results thus reveal a serious limitation to thereproducibility of current lipid profiling experiments and reveal challenges to theintegration of such data from different laboratories.
Article Reference A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.
Optogenetics enables genome manipulations with high spatiotemporal resolution,opening exciting possibilities for fundamental and applied biological research.Here, we report the development of LiCre, a novel light-inducible Cre recombinase.LiCre is made of a single flavin-containing protein comprising the AsLOV2photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizingmutations in its N-terminal and C-terminal domains. LiCre can be activated withinminutes of illumination with blue light, without the need of additional chemicals.When compared to existing photoactivatable Cre recombinases based on two splitunits, LiCre displayed faster and stronger activation by light as well as a lowerresidual activity in the dark. LiCre was efficient both in yeast, where it allowedus to control the production of β-carotene with light, and in human cells. Given itssimplicity and performances, LiCre is particularly suited for fundamental andbiomedical research, as well as for controlling industrial bioprocesses.
Article Reference Auxin confers protection against ER stress in Caenorhabditis elegans.
Auxins are plant growth regulators that influence most aspects of plant developmentthrough complex mechanisms. The development of an auxin-inducible degradation (AID)system has enabled rapid, conditional protein depletion in yeast and cultured cells.More recently, the system was successfully adapted to C aenorhabditis elegans toachieve auxin-dependent degradation of targets in all tissues and developmentalstages. Whether auxin treatment alone has an impact on nematode physiology is anopen question. Here we show that indole-3-acetic acid (IAA), the auxin most commonlyused to trigger AID in worms, functions through the conserved IRE-1/XBP-1 branch ofthe Unfolded Protein Response (UPR) to promote resistance to endoplasmic reticulum(ER) stress. Because the UPR not only plays a central role in restoring ERhomeostasis, but also promotes lipid biosynthesis and regulates lifespan, we suggestthat extreme caution should be exercised when using the AID system to study theseand related processes.
Article Reference A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization.
Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed bythe SET1/COMPASS complex and is associated with context-dependent effects on geneexpression and local changes in chromatin organization. The role of SET1/COMPASS inshaping chromosome architecture has not been investigated. Here we usedCaenorhabditis elegans to address this question through a live imaging approach andgenetic analysis. Using quantitative FRET (Förster resonance energy transfer)-basedfluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histoneseGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosomeorganization, with marked effects on the close proximity between nucleosomes. Wefurther show that inactivation of set-2, encoding the C. elegans SET1 homologue, orCFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germlinechromosome organization defects and sterility of condensin-II depleted animals.set-2 loss also aggravates germline defects resulting from conditional inactivationof topoisomerase II, another structural component of chromosomes. Expressionprofiling of set-2 mutant germlines revealed only minor transcriptional changes,suggesting that the observed effects are at least partly independent oftranscription. Altogether, our results are consistent with a role for SET1/COMPASSin shaping meiotic chromosomes in C. elegans, together with the non-histone proteinscondensin-II and topoisomerase. Given the high degree of conservation, our findingsexpand the range of functions attributed to COMPASS and suggest a broader role ingenome organization in different species.
Article Reference Caenorhabditis elegans SET1/COMPASS Maintains Germline Identity by Preventing Transcriptional Deregulation Across Generations.
Chromatin regulators contribute to the maintenance of the germline transcriptionalprogram. In the absence of SET-2, the Caenorhabditis elegans homolog of theSET1/COMPASS H3 Lys4 (H3K4) methyltransferase, animals show transgenerational lossof germline identity, leading to sterility. To identify transcriptional signaturesassociated with progressive loss of fertility, we performed expression profiling ofset-2 mutant germlines across generations. We identify a subset of genes whosemisexpression is first observed in early generations, a step we refer to as priming;their misexpression then further progresses in late generations, as animals reachsterility. Analysis of misregulated genes shows that down-regulation of germlinegenes, expression of somatic transcriptional programs, and desilencing of theX-chromosome are concurrent events leading to loss of germline identity in bothearly and late generations. Upregulation of transcription factor LIN-15B, the C/EBPhomolog CEBP-1, and TGF-β pathway components strongly contribute to loss offertility, and RNAi inactivation of cebp-1 and TGF-β/Smad signaling delays the onsetof sterility, showing they individually contribute to maintenance of germ cellidentity. Our approach therefore identifies genes and pathways whose misexpressionactively contributes to the loss of germ cell fate. More generally, our data showshow loss of a chromatin regulator in one generation leads to transcriptional changesthat are amplified over subsequent generations, ultimately leading to loss ofappropriate cell fate.
Article Reference Histone Variants: The Nexus of Developmental Decisions and Epigenetic Memory.
Nucleosome dynamics and properties are central to all forms of genomic activities.Among the core histones, H3 variants play a pivotal role in modulating nucleosomestructure and function. Here, we focus on the impact of H3 variants on variousfacets of development. The deposition of the replicative H3 variant following DNAreplication is essential for the transmission of the epigenomic information encodedin posttranscriptional modifications. Through this process, replicative H3 maintainscell fate while, in contrast, the replacement H3.3 variant opposes celldifferentiation during early embryogenesis. In later steps of development, H3.3 andspecialized H3 variants are emerging as new, important regulators of terminal celldifferentiation, including neurons and gametes. The specific pathways that regulatethe dynamics of the deposition of H3.3 are paramount during reprogramming eventsthat drive zygotic activation and the initiation of a new cycle of development.
Article Reference Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors.
PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs essential for fertility. In adult mouse testes, most piRNAs are derived from long single-stranded RNAs lacking annotated open reading frames (ORFs). The mechanisms underlying how piRNA sequences are defined during the cleavages of piRNA precursors remain elusive. Here, we show that 80S ribosomes translate the 5'-proximal short ORFs (uORFs) of piRNA precursors. The MOV10L1/Armitage RNA helicase then facilitates the translocation of ribosomes into the uORF downstream regions (UDRs). The ribosome-bound UDRs are targeted by piRNA processing machinery, with the processed ribosome-protected regions becoming piRNAs. The dual modes of interaction between ribosomes and piRNA precursors underlie the distinct piRNA biogenesis requirements at uORFs and UDRs. Ribosomes also mediatepiRNA processing in roosters and green lizards, implying that this mechanism is evolutionarily conserved in amniotes. Our results uncover a function for ribosomes on non-coding regions of RNAs and reveal the mechanisms underlying howpiRNAs are defined.
Article Reference Diversification and hybrid incompatibility in auto-pseudogamous species of Mesorhabditis nematodes.
BACKGROUND: Pseudogamy is a reproductive system in which females rely on the spermof males to activate their oocytes, generally parasitizing males of other species,but do not use the sperm DNA. The nematode Mesorhabditis belari uses a specific formof pseudogamy, where females produce their own males as a source of sperm. Malesdevelop from rare eggs with true fertilization, while females arise by gynogenesis.Males thus do not contribute their genome to the female offspring. Here, we exploredthe diversity of reproductive mode within the Mesorhabditis genus and addressedspecies barriers in pseudogamous species. RESULTS: To this end, we established acollection of over 60 Mesorhabditis strains from soil and rotting vegetal matter. Wefound that males from pseudogamous species displayed a reduced size of their body,male tail and sperm cells compared to males of sexual Mesorhabditis species, asexpected for males that face little competition. Using rDNA sequences and crosses,we could define 11 auto-pseudogamous biological species, with closely relatedspecies pairs and a possible single origin of pseudogamy in the Mesorhabditis genus.Most crosses between males and females of different species did not even producefemale progeny. This surprising species barrier in pseudogamous egg activation waspre or postcopulatory depending on the species pair. In the latter case, when hybridembryos were produced, most arrested before the first embryonic cell division.Hybrid incompatibility between auto-pseudogamous species was due to defectiveinteraction between sperm and oocyte as well as defective reconstitution of zygoticcentrosomes. CONCLUSIONS: We established a collection of sexual and pseudo-sexualspecies which offer an ideal framework to explore the origin and consequences oftransition to asexuality. Our results demonstrate that speciation occurs in thepseudogamous state. Whereas genomic conflicts are responsible for hybridincompatibility in sexual species, we here reveal that centrosomes constitute keyorganelles in the establishment of species barrier.
Article Reference A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pM to 17 µM. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.
Article Reference A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pM to 17 µM. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.