Nouvelle méthode statistique pour l'analyse de données de ChIP-chip

International audienceLa méthode de Chromatin ImmunoPrecipitation on chip (ChIP on chip ou ChIP-chip) a pour but de détecter les sites de fixation des protéines (généralement des facteurs de transcription) sur la molécule d'ADN. L'analyse statistique des données consiste a rechercher des régions de pics significatifs synonymes de sites de fixation. La méthode que nous avons élaborée est issue de la théorie des valeurs extrêmes et particulièrement de la méthode POT (Peaks Over Threshold). Cette méthode consiste à modéliser les données de queues de distribution, en ne retenant que les valeurs dépassant un certain seuil, elle a la particularité de modéliser d'une part les intensités de dépassement de seuil, mais aussi les positions d'occurrences de ces dépassements de seuil. Cette méthode va nous permettre de déterminer un seuil au delà duquel les pics pourront être considérés comme significatifs

Cloning, expression and pharmacology of the mouse 5-HT4L receptor

AbstractSince most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT4L receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT4L) expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT4L receptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT4S) and the long form (5-HT4L) of 5-HT4 receptors are expressed in all mouse and rat brain areas

Zac1 functions through TGFβII to negatively regulate cell number in the developing retina

<p>Abstract</p> <p>Background</p> <p>Organs are programmed to acquire a particular size during development, but the regulatory mechanisms that dictate when dividing progenitor cells should permanently exit the cell cycle and stop producing additional daughter cells are poorly understood. In differentiated tissues, tumor suppressor genes maintain a constant cell number and intact tissue architecture by controlling proliferation, apoptosis and cell dispersal. Here we report a similar role for two tumor suppressor genes, the <it>Zac1 </it>zinc finger transcription factor and that encoding the cytokine TGFβII, in the developing retina.</p> <p>Results</p> <p>Using loss and gain-of-function approaches, we show that <it>Zac1 </it>is an essential negative regulator of retinal size. <it>Zac1 </it>mutants develop hypercellular retinae due to increased progenitor cell proliferation and reduced apoptosis at late developmental stages. Consequently, supernumerary rod photoreceptors and amacrine cells are generated, the latter of which form an ectopic cellular layer, while other retinal cells are present in their normal number and location. Strikingly, <it>Zac1 </it>functions as a direct negative regulator of a rod fate, while acting cell non-autonomously to modulate amacrine cell number. We implicate TGFβII, another tumor suppressor and cytokine, as a <it>Zac1</it>-dependent amacrine cell negative feedback signal. TGFβII and phospho-Smad2/3, its downstream effector, are expressed at reduced levels in <it>Zac1 </it>mutant retinae, and exogenous TGFβII relieves the mutant amacrine cell phenotype. Moreover, treatment of wild-type retinae with a soluble TGFβ inhibitor and TGFβ receptor II (TGFβRII) conditional mutants generate excess amacrine cells, phenocopying the <it>Zac1 </it>mutant phenotype.</p> <p>Conclusion</p> <p>We show here that <it>Zac1 </it>has an essential role in cell number control during retinal development, akin to its role in tumor surveillance in mature tissues. Furthermore, we demonstrate that <it>Zac1 </it>employs a novel cell non-autonomous strategy to regulate amacrine cell number, acting in cooperation with a second tumor suppressor gene, <it>TGFβII</it>, through a negative feedback pathway. This raises the intriguing possibility that tumorigenicity may also be associated with the loss of feedback inhibition in mature tissues.</p

In Vitro Fertilization and Embryo Culture Strongly Impact the Placental Transcriptome in the Mouse Model

BACKGROUND: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART

Genome-wide profiling of G protein-coupled receptors in cerebellar granule neurons using high-throughput, real-time PCR

<p>Abstract</p> <p>Background</p> <p>G protein-coupled receptors (GPCRs) are major players in cell communication, regulate a whole range of physiological functions during development and throughout adult life, are affected in numerous pathological situations, and constitute so far the largest class of drugable targets for human diseases. The corresponding genes are usually expressed at low levels, making accurate, genome-wide quantification of their expression levels a challenging task using microarrays.</p> <p>Results</p> <p>We first draw an inventory of all endo-GPCRs encoded in the murine genome. To profile GPCRs genome-wide accurately, sensitively, comprehensively, and cost-effectively, we designed and validated a collection of primers that we used in quantitative RT-PCR experiments. We experimentally validated a statistical approach to analyze genome-wide, real-time PCR data. To illustrate the usefulness of this approach, we determined the repertoire of GPCRs expressed in cerebellar granule neurons and neuroblasts during postnatal development.</p> <p>Conclusions</p> <p>We identified tens of GPCRs that were not detected previously in this cell type; these GPCRs represent novel candidate players in the development and survival of cerebellar granule neurons. The sequences of primers used in this study are freely available to those interested in quantifying GPCR expression comprehensively.</p

The rise and fall of the ancient northern pike master sex determining gene

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex-linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y-chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions

The 'PUCE CAFE' Project: the First 15K Coffee Microarray, a New Tool for Discovering Candidate Genes correlated to Agronomic and Quality Traits

Background: Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results: The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion: We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Genoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research

Etude des domaines fonctionnels de la sous-unite alpha de Gs, la G proteine responsable de l'activation de l'adenylate cyclase

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78988 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc