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2015

A DPP-mediated feed-forward loop canalizes morphogenesis during Drosophila dorsal closure.

Author(s) : Ducuing A, Keeley C, Mollereau B, Vincent S,
Journal : J Cell Biol
2015
Development is robust because nature has selected various mechanisms to buffer the deleterious effects of environmental and genetic variations to deliver phenotypic stability. Robustness relies on smart network motifs such as feed-forward loops (FFLs) that ensure the reliable interpretation of developmental signals. In this paper, we show that Decapentaplegic (DPP) and JNKform a coherent FFL that controls the specification and differentiation of leading edge cells during Drosophila melanogaster dorsal closure (DC). We provide molecular evidence that through repression by Brinker (Brk), the DPP branch of the FFL filters unwanted JNK activity. High-throughput live imaging revealed that this DPP/Brk branch is dispensable for DC under normal conditions but is required when embryos are subjected to thermal stress. Our results indicate that the wiring of DPP signaling buffers against environmental challenges and canalizes cell identity. We propose that the main function of DPP pathway during Drosophila DC is to ensure robust morphogenesis, a distinct function from its well-established ability to spread spatial information.

An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.

Author(s) : Heyer E, Ozadam H, Ricci E, Cenik C, Moore M,
Journal : Nucleic Acids Res
2015
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.

Chaperoning 5S RNA assembly.

Author(s) : Madru C, Lebaron S, Blaud M, Delbos L, Pipoli J, Pasmant E, Rety S, Leulliot N,
Journal : Genes Dev
2015
In eukaryotes, three of the four ribosomal RNAs (rRNAs)-the 5.8S, 18S, and 25S/28S rRNAs-are processed from a single pre-rRNA transcript and assembled intoribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior toits rotation to form the central protuberance found in the mature large ribosomal subunit.

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner.

Author(s) : Nahaboo W, Zouak M, Askjaer P, Delattre M,
Journal : Mol Biol Cell
2015
During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward forcegenerated by the spindle midzone, independently of centrosomes, is sufficient tosegregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase.Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

Cooling-Induced ER Stress is Good for Your Brain.

Author(s) : Mollereau B,
Journal : EBioMedicine
2015

Discovery and characterization of auxiliary proteins encoded by type 3 simian T-cell lymphotropic viruses.

Author(s) : Turpin J, Journo C, Ko N, Sinet F, Carpentier A, Galioot A, Edwards D, Vandamme A, Gazzolo L, Duc Dodon M, Gessain A, Kashanchi F, Balansard I, Lacoste R, Mahieux R,
Journal : J Virol
2015
Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lackof HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and ofan STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar tothat of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked toa bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE: Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes wasunknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Does transcription play a role in creating a condensin binding site?

Author(s) : Bernard P, Vanoosthuyse V,
Journal : Transcription
2015
The highly conserved condensin complex is essential for the condensation and integrity of chromosomes through cell division. Published data argue that high levels of transcription contribute to specify some condensin-binding sites on chromosomes but the exact role of transcription in this process remains elusive.Here we discuss our recent data addressing the role of transcription in establishing a condensin-binding site.

Effect of replication on epigenetic memory and consequences on gene transcription.

Author(s) : Zerihun M, Vaillant C, Jost D,
Journal : Phys Biol
2015
Gene activity in eukaryotes is in part regulated at the level of chromatin through the assembly of local chromatin states that are more or less permissive to transcription. How do these chromatin states achieve their functions and whether or not they contribute to the epigenetic inheritance of the transcriptional program remain to be elucidated. In cycling cells, stability is indeed strongly challenged by the periodic occurrence of replication and cell division. To address this question, we perform simulations of the stochastic dynamics of chromatin states when driven out-of-equilibrium by periodic perturbations. We show how epigenetic memory is significantly affected by the cell cycle length. In addition, we develop a simple model to connect the epigenetic state to the transcriptional state and gene activity. In particular, it suggests that replication may induce transcriptional bursting at repressive loci. Finally, we discuss how our findings-effect of replication and link to gene transcription-have original and deep implications to various biological contextsof epigenetic memory.

Expression of dengue virus NS3 protein in Drosophila alters its susceptibility to infection.

Author(s) : Querenet M, Danjoy M, Mollereau B, Davoust N,
Journal : Fly (Austin)
2015
We developed a Drosophila model in which the dengue virus NS3 protein is expressed in a tissue specific and inducible manner. Dengue virus NS3 is a multifunctional protein playing a major role during viral replication. Both protease and helicase domains of NS3 are interacting with human and insect host proteins including innate immune components of the host machinery. We characterized the NS3 transgenic flies showing that NS3 expression did not affect fly development. To further study the links between NS3 and the innate immune response, we challenge the flies with gram-positive and gram-negative bacteria. Interestingly, the Drosophila transgenic flies expressing NS3 were more susceptible to bacterial infections than control flies. However ubiquitous or immune-specific NS3 expression affected neither the life span nor the response to a non-infectious stress of the flies. In conclusion, we generated a new in vivo system to study the functional impact of DENV NS3 protein on the innate immune response.

Fatty acid transport proteins in disease: New insights from invertebrate models.

Author(s) : Dourlen P, Sujkowski A, Wessells R, Mollereau B,
Journal : Prog Lipid Res
2015
The dysregulation of lipid metabolism has been implicated in various diseases, including diabetes, cardiopathies, dermopathies, retinal and neurodegenerative diseases. Mouse models have provided insights into lipid metabolism. However, progress in the understanding of these pathologies is hampered by the multiplicity of essential cellular processes and genes that modulate lipid metabolism. Drosophila and Caenorhabditis elegans have emerged as simple geneticmodels to improve our understanding of these metabolic diseases. Recent studies have characterized fatty acid transport protein (fatp) mutants in Drosophila andC. elegans, establishing new models of cardiomyopathy, retinal degeneration, fatstorage disease and dermopathies. These models have generated novel insights into the physiological role of the Fatp protein family in vivo in multicellular organisms, and are likely to contribute substantially to progress in understanding the etiology of various metabolic disorders. Here, we describe anddiscuss the mechanisms underlying invertebrate fatp mutant models in the light of the current knowledge relating to FATPs and lipid disorders in vertebrates.

From Immunodeficiency to Humanization: The Contribution of Mouse Models to Explore HTLV-1 Leukemogenesis.

Author(s) : Peres E, Bagdassarian E, This S, Villaudy J, Rigal D, Gazzolo L, Duc Dodon M,
Journal : Viruses
2015
The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in thegeneration of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed.

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

Author(s) : Marza E, Taouji S, Barroso K, Raymond A, Guignard L, Bonneu M, Pallares-Lupon N, Dupuy J, Fernandez-Zapico M, Rosenbaum J, Palladino F, Dupuy D, Chevet E,
Journal : EMBO Rep
2015
The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. TheAAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Histone storage and deposition in the early Drosophila embryo.

Author(s) : Horard B, Loppin B,
Journal : Chromosoma
2015
Drosophila development initiates with the formation of a diploid zygote followedby the rapid division of embryonic nuclei. This syncytial phase of development occurs almost entirely under maternal control and ends when the blastoderm embryo cellularizes and activates its zygotic genome. The biosynthesis and storage of histones in quantity sufficient for chromatin assembly of several thousands of genome copies represent a unique challenge for the developing embryo. In this article, we have reviewed our current understanding of the mechanisms involved in the production, storage, and deposition of histones in the fertilized egg and during the exponential amplification of cleavage nuclei.

How Retroviruses Escape the Nonsense-Mediated mRNA Decay.

Author(s) : Mocquet V, Durand S, Jalinot P,
Journal : AIDS Res Hum Retroviruses
2015
Many posttranscriptional processes are known to regulate gene expression and some of them can act as an antiviral barrier. The nonsense-mediated mRNA decay (NMD) was first identified as an mRNA quality control pathway that triggers rapid decay of mRNA containing premature stop codons due to mutations. NMD is now consideredas a general posttranscriptional regulation pathway controlling the expression of a large set of cellular genes. In addition to premature stop codons, many other features including alternative splicing, 5' uORF, long 3' UTR, selenocystein codons, and frameshift are able to promote NMD. Interestingly, many viral mRNAs exhibit some of these features suggesting that virus expression and replication might be sensitive to NMD. Several studies, including recent ones, have shown that this is the case for retroviruses; however, it also appears that retroviruses have developed strategies to overcome NMD in order to protect theirgenome and ensure a true expression of their genes. As a consequence of NMD inhibition, these viruses also affect the expression of host genes that are prone to NMD, and therefore can potentially trigger pathological effects on infected cells. Here, we review recent studies supporting this newly uncovered function of the NMD pathway as a defense barrier that viruses must overcome in order to replicate.

In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

Author(s) : Laustriat D, Gide J, Barrault L, Chautard E, Benoit C, Auboeuf D, Boland A, Battail C, Artiguenave F, Deleuze J, B?nit P, Rustin P, Franc S, Charpentier G, Furling D, Bassez G, Nissan X, Martinat C, Peschanski M, Baghdoyan S,
Journal : Mol Ther Nucleic Acids
2015

In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin.

Author(s) : Laustriat D, Gide J, Barrault L, Chautard E, Benoit C, Auboeuf D, Boland A, Battail C, Artiguenave F, Deleuze J, Benit P, Rustin P, Franc S, Charpentier G, Furling D, Bassez G, Nissan X, Martinat C, Peschanski M, Baghdoyan S,
Journal : Mol Ther Nucleic Acids
2015
Major physiological changes are governed by alternative splicing of RNA, and itsmisregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication anddemonstrate the effects of the biguanide metformin, on alternative splicing. Themechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy.We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. Thedrug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

Author(s) : Cenik C, Cenik E, Byeon G, Grubert F, Candille S, Spacek D, Alsallakh B, Tilgner H, Araya C, Tang H, Ricci E, Snyder M,
Journal : Genome Res
2015
Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves theidentification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of thesedifferences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation.

Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3.

Author(s) : Chen W, Shih H, Liu K, Shih Z, Chen L, Tsai T, Chen M, Liu H, Tan B, Chen C, Lee H, Loppin B, Ait-Ahmed O, Wu J,
Journal : EMBO Rep
2015
Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding ofBRWD3-dependent intellectual disability.

Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death.

Author(s) : Borowiak M, Nahaboo W, Reynders M, Nekolla K, Jalinot P, Hasserodt J, Rehberg M, Delattre M, Zahler S, Vollmar A, Trauner D, Thorn-Seshold O,
Journal : Cell
2015
Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introducethe photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuabletools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

Author(s) : Crauste F, Terry E, Mercier I, Mafille J, Djebali S, Andrieu T, Mercier B, Kaneko G, Arpin C, Marvel J, Gandrillon O,
Journal : J Theor Biol
2015
The primary CD8 T cell immune response constitutes a major mechanism to fight aninfection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developeda mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end ofthe response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential whenlooking at the predictions of the model several months after the infection.

Protection of Drosophila chromosome ends through minimal telomere capping.

Author(s) : Dubruille R, Loppin B,
Journal : J Cell Sci
2015
In Drosophila, telomere-capping proteins have the remarkable capacity to recognize chromosome ends in a sequence-independent manner. This epigenetic protection is essential to prevent catastrophic ligations of chromosome extremities. Interestingly, capping proteins occupy a large telomere chromatin domain of several kilobases; however, the functional relevance of this to end protection is unknown. Here, we investigate the role of the large capping domainby manipulating HOAP (encoded by caravaggio) capping-protein expression in the male germ cells, where telomere protection can be challenged without compromising viability. We show that the exhaustion of HOAP results in a dramatic reduction of other capping proteins at telomeres, including K81 [encoded by ms(3)K81], which is essential for male fertility. Strikingly however, we demonstrate that, although capping complexes are barely detected in HOAP-depleted male germ cells,telomere protection and male fertility are not dramatically affected. Our study thus demonstrates that efficient protection of Drosophila telomeres can be achieved with surprisingly low amounts of capping complexes. We propose that these complexes prevent fusions by acting at the very extremity of chromosomes, reminiscent of the protection conferred by extremely short telomeric arrays in yeast or mammalian systems.

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

Author(s) : Chatagnon A, Veber P, Morin V, Bedo J, Triqueneaux G, Semon M, Laudet V, d'Alche-Buc F, Benoit G,
Journal : Nucleic Acids Res
2015
In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play akey role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast,in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated withcell differentiation status.

Reconstructing and analysing cellular states, space and time from gene expression profiles of many cells and single cells.

Author(s) : Francesconi M, Lehner B,
Journal : Mol Biosyst
2015
Genome-wide gene expression profiling is a fast, cheap and standardised analysisthat provides a high dimensional measurement of the state of a biological sample. In this review we describe computational methods that can be applied to identifyand interpret sources of variance in gene expression in whole organisms, organs,tissues or single cells. This allows the identification of constituent cell types and states in complex mixtures, the reconstruction of temporal trajectories of development, differentiation and progression, and the reconstruction of spatial patterning. When applied to genetically variable samples, these methods allow the efficient investigation of how genetic variation influences gene expression and biological processes in space and time.

Regulation of DEAH/RHA helicases by G-patch proteins.

Author(s) : Robert-Paganin J, Rety S, Leulliot N,
Journal : Biomed Res Int
2015
RNA helicases from the DEAH/RHA family are present in all the processes of RNA metabolism. The function of two helicases from this family, Prp2 and Prp43, is regulated by protein partners containing a G-patch domain. The G-patch is a glycine-rich domain discovered by sequence alignment, involved in protein-protein and protein-nucleic acid interaction. Although it has been shown to stimulate the helicase's enzymatic activities, the precise role of the G-patch domain remains unclear. The role of G-patch proteins in the regulation of Prp43 activity has been studied in the two biological processes in which it is involved: splicing and ribosome biogenesis. Depending on the pathway, the activity of Prp43 is modulated by different G-patch proteins. A particular feature of the structure of DEAH/RHA helicases revealed by the Prp43 structure is the OB-fold domain in C-terminal part. The OB-fold has been shown to be a platform responsible for theinteraction with G-patch proteins and RNA. Though there is still no structural data on the G-patch domain, in the current model, the interaction between the helicase, the G-patch protein, and RNA leads to a cooperative binding of RNA andconformational changes of the helicase.

Repression of somatic cell fate in the germline.

Author(s) : Robert V, Garvis S, Palladino F,
Journal : Cell Mol Life Sci
2015
Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs.

Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

Author(s) : Gely-Pernot A, Raverdeau M, Teletin M, Vernet N, Feret B, Klopfenstein M, Dennefeld C, Davidson I, Benoit G, Mark M, Ghyselinck N,
Journal : PLoS Genet
2015
All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also showthat ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression inspermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

Scaling, selection, and evolutionary dynamics of the mitotic spindle.

Author(s) : Farhadifar R, Baer C, Valfort A, Andersen E, Muller-Reichert T, Delattre M, Needleman D,
Journal : Curr Biol
2015
BACKGROUND: Cellular structures such as the nucleus, Golgi, centrioles, and spindle show remarkable diversity between species, but the mechanisms that produce these variations in cell biology are not known. RESULTS: Here we investigate the mechanisms that contribute to variations in morphology and dynamics of the mitotic spindle, which orchestrates chromosome segregation in all Eukaryotes and positions the division plane in many organisms. We use high-throughput imaging of the first division in nematodes to demonstrate that the measured effects of spontaneous mutations, combined with stabilizing selection on cell size, are sufficient to quantitatively explain both the levelsof within-species variation in the spindle and its diversity over approximately 100 million years of evolution. Furthermore, our finding of extensive within-species variation for the spindle demonstrates that there is not just one"wild-type" form, rather that cellular structures can exhibit a surprisingly broad diversity of naturally occurring behaviors. CONCLUSIONS: Our results arguethat natural selection acts predominantly on cell size and indirectly influencesthe spindle through the scaling of the spindle with cell size. Previous studies have shown that the spindle also scales with cell size during early development.Thus, the scaling of the spindle with cell size controls its variation over bothontogeny and phylogeny.