Identification et caractérisation de promoteurs bidirectionnels au sein du muscle squelettique de l'espèce Bos taurus

"Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access "Bidirectionnels promoters are definited as regulatory regions that can co-regulate the expression of two neighboring genes arranged in divergent manner. Until now, this type of promoter was identified in all species that have been studied, where they account for 5 to 55% of all promoters. In this maniscript, the study was focused on the identification and characterization of bidirectional promoters involved in the co-expression of two neighboring genes in bovine muscle tissue. More precisely, we performed a large scale survey on protein coding genes, and a RNA-Seq transcriptome analysis to identify co-regulated gene pairs in skeletal muscle. However, since this technology leads to several quantification biases, we developped an integrated normalization method. After correction of the expression date, a study of the biological functions associated with co-expressed gene pairs was conducted, which showed that these genes were generally linked to fundamental cellular functions, with an overrepresentation in mitochondrion compartment. Eleven putative bidirectional promoters were then selected and subject to an in vitro screening, in order to verify that the sequences located between the two genes are necessary and sufficient to drive an expression of two reporter genes. Then, these sequences were analyzed in detail to identify factors potentially involved in transcription. It appeared that these bidirectional promoters probably have a tissue-independent bidirectional activity, due to many ubiquitous transcription factors. Furthermore, a conservation study of these gene pairs within Vertebrates revealed a good conservation particularly among Mammals, suggesting that a selection pressure is acting to maintain these gene pairs, and therefore important functions. Finally, a more detailed characterization of one of these eleven regulatory regions was then performed, which reinforced the hypothesis of a true bidirectional promoter.Les promoteurs bidirectionnels sont définis comme des régions régulatrices pouvant potentiellement réguler l'expression de deux gènes contigus, situés en orientations opposées. A l'heure actuelle, ce type de promoteur a été identifié chez toutes les espèces étudiées, où il représente entre 5 % à 55 % des promoteurs. Dans ce manuscrit, l' étude se focalise sur l'identification et la caractérisation de promoteurs bidirectionnels responsables d'une co-expression de deux gènes au sein du tissu musculaire bovin. Plus précisément, l'intérêt est porté sur les gènes codants pour des protéines, où suite à une analyse in silico à l'échelle du génome, une analyse du transcriptome a été réalisée par RNA-Seq en vue d'identifier les gènes co-régulés dans le muscle squelettique. Toutefois, cette méthode de quantification étant faussée par certains biais, une méthode de normalisation spécifique a été développée. Une fois les données d'expression corrigées, une étude des fonctions biologiques associées aux paires de gènes co-exprimés a été entreprise, montrant que ces gènes étaient généralement impliqués dans des fonctions cellulaires fondamentales, avec une surreprésentation dans les fonctions associées à la mitochondrie. Onze promoteurs bidirectionnels putatifs ont ensuite été sélectionnés afin d'être analysés dans dans un crible in vitro, dans le but de vérifier que les séquences situées entre les deux gènes étaient nécessaires et suffisantes afin d'engendrer l'expression de deux gènes rapporteurs. Puis, ces séquences ont été analysées en détail afin d'identifier les facteurs potentiellement impliqués dans la transcription. Il en ressort que ces promoteurs bidirectionnels présentent probablement une activité bidirectionnelle tissu-indépendante, en raison de l'implication de nombreux facteurs de transcription ubiquitaires. D'autres part, une étude de la conservation de ces paires de gènes au sein des Vertébrés a révélé une bonne conservation notamment au sein des Mammifères, suggérant la présence d'une pression de sélection maintenant ces différents couples de gènes, et donc une importance d'un point de vue fonctionnel. Pour finir, une caractérisation plus fined'une de ces onze régions régulatrices a ensuite été réalisée, ce qui renforça l'hypothèse d'un vrai promoteur bidirectionnel

Identification of large intergenic non-coding RNAs in bovine muscle using next-generation transcriptomic sequencing.

International audienceBACKGROUND: The advent of large-scale gene expression technologies has helped to reveal in eukaryotic cells, the existence of thousands of non-coding transcripts, whose function and significance remain mostly poorly understood. Among these non-coding transcripts, long non-coding RNAs (lncRNAs) are the least well-studied but are emerging as key regulators of diverse cellular processes. In the present study, we performed a survey in bovine Longissimus thoraci of lincRNAs (long intergenic non-coding RNAs not overlapping protein-coding transcripts). To our knowledge, this represents the first such study in bovine muscle. RESULTS: To identify lincRNAs, we used paired-end RNA sequencing (RNA-Seq) to explore the transcriptomes of Longissimus thoraci from nine Limousin bull calves. Approximately 14-45 million paired-end reads were obtained per library. A total of 30,548 different transcripts were identified. Using a computational pipeline, we defined a stringent set of 584 different lincRNAs with 418 lincRNAs found in all nine muscle samples. Bovine lincRNAs share characteristics seen in their mammalian counterparts: relatively short transcript and gene lengths, low exon number and significantly lower expression, compared to protein-encoding genes. As for the first time, our study identified lincRNAs from nine different samples from the same tissue, it is possible to analyse the inter-individual variability of the gene expression level of the identified lincRNAs. Interestingly, there was a significant difference when we compared the expression variation of the 418 lincRNAs with the 10,775 known selected protein-encoding genes found in all muscle samples. In addition, we found 2,083 pairs of lincRNA/protein-encoding genes showing a highly significant correlated expression. Fourteen lincRNAs were selected and 13 were validated by RT-PCR. Some of the lincRNAs expressed in muscle are located within quantitative trait loci for meat quality traits. CONCLUSIONS: Our study provides a glimpse into the lincRNA content of bovine muscle and will facilitate future experimental studies to unravel the function of these molecules. It may prove useful to elucidate their effect on mechanisms underlying the genetic variability of meat quality traits. This catalog will complement the list of lincRNAs already discovered in cattle and therefore will help to better annotate the bovine genome

Identification of copy number variation in French dairy and beef breeds using next-generation sequencing

International audienceAbstractBackgroundCopy number variations (CNV) are known to play a major role in genetic variability and disease pathogenesis in several species including cattle. In this study, we report the identification and characterization of CNV in eight French beef and dairy breeds using whole-genome sequence data from 200 animals. Bioinformatics analyses to search for CNV were carried out using four different but complementary tools and we validated a subset of the CNV by both in silico and experimental approaches.ResultsWe report the identification and localization of 4178 putative deletion-only, duplication-only and CNV regions, which cover 6% of the bovine autosomal genome; they were validated by two in silico approaches and/or experimentally validated using array-based comparative genomic hybridization and single nucleotide polymorphism genotyping arrays. The size of these variants ranged from 334 bp to 7.7 Mb, with an average size of ~ 54 kb. Of these 4178 variants, 3940 were deletions, 67 were duplications and 171 corresponded to both deletions and duplications, which were defined as potential CNV regions. Gene content analysis revealed that, among these variants, 1100 deletions and duplications encompassed 1803 known genes, which affect a wide spectrum of molecular functions, and 1095 overlapped with known QTL regions.ConclusionsOur study is a large-scale survey of CNV in eight French dairy and beef breeds. These CNV will be useful to study the link between genetic variability and economically important traits, and to improve our knowledge on the genomic architecture of cattle

Comparative analysis of transcriptome remodeling in plaque-associated and plaque-distant microglia during amyloid-β pathology progression in mice

International audienceBackground: Research in recent years firmly established that microglial cells play an important role in the pathogenesis of Alzheimer's disease (AD). In parallel, a series of studies showed that, under both homeostatic and pathological conditions, microglia are a heterogeneous cell population. In AD, amyloid-β (Aβ) plaque-associated microglia (PAM) display a clearly distinct phenotype compared to plaque-distant microglia (PCM), suggesting that these two microglia subtypes likely differently contribute to disease progression. So far, molecular characterization of PAM was performed indirectly using single cell RNA sequencing (scRNA-seq) approaches or based on markers that are supposedly up-regulated in this microglia subpopulation.Methods: In this study based on a well-characterized AD mouse model, we combined cell-specific laser capture microdissection and RNA-seq analysis to i) identify, without preconceived notions of the molecular and/or functional changes that would affect these cells, the genes and gene networks that are dysregulated in PAM or PCM at three critical stages of the disease, and ii) to investigate the potential contribution of both plaque-associated and plaque-distant microglia.Results: First, we established that our approach allows selective isolation of microglia, while preserving spatial information and preventing transcriptome changes induced by classical purification approaches. Then, we identified, in PAM and PCM subpopulations, networks of co-deregulated genes and analyzed their potential functional roles in AD. Finally, we investigated the dynamics of microglia transcriptomic remodeling at early, intermediate and late stages of the disease and validated select findings in postmortem human AD brain.Conclusions: Our comprehensive study provides useful transcriptomic information regarding the respective contribution of PAM and PCM across the Aβ pathology progression. It highlights specific pathways that would require further study to decipher their roles across disease progression. It demonstrates that the proximity of microglia to Aβ-plaques dramatically alters the microglial transcriptome and reveals that these changes can have both positive and negative impacts on the surrounding cells. These opposing effects may be driven by local microglia heterogeneity also demonstrated by this study. Our approach leads to molecularly define the less well studied plaque-distant microglia. We show that plaque-distant microglia are not bystanders of the disease, although the transcriptomic changes are far less striking compared to what is observed in plaque-associated microglia. In particular, our results suggest they may be involved in Aβ oligomer detection and in Aβ-plaque initiation, with increased contribution as the disease progresses

Survey of allele specific expression in bovine muscle

Abstract Allelic imbalance is a common phenomenon in mammals that plays an important role in gene regulation. An Allele Specific Expression (ASE) approach can be used to detect variants with a cis-regulatory effect on gene expression. In cattle, this type of study has only been done once in Holstein. In our study we performed a genome-wide analysis of ASE in 19 Limousine muscle samples. We identified 5,658 ASE SNPs (Single Nucleotide Polymorphisms showing allele specific expression) in 13% of genes with detectable expression in the Longissimus thoraci muscle. Interestingly we found allelic imbalance in AOX1, PALLD and CAST genes. We also found 2,107 ASE SNPs located within genomic regions associated with meat or carcass traits. In order to identify causative cis-regulatory variants explaining ASE we searched for SNPs altering binding sites of transcription factors or microRNAs. We identified one SNP in the 3’UTR region of PRNP that could be a causal regulatory variant modifying binding sites of several miRNAs. We showed that ASE is frequent within our muscle samples. Our data could be used to elucidate the molecular mechanisms underlying gene expression imbalance

MOESM9 of Identification of copy number variation in French dairy and beef breeds using next-generation sequencing

Additional file 9: Table S8. Summary of validated variants by the different approaches. This is an inventory of validated variants using the two in silico and/or array-based CGH approaches. This dataset was the final subset of variants retained in this study. Information about breeds and validation approaches that support each variant is indicated

Immunocompromised patients with acute respiratory distress syndrome : Secondary analysis of the LUNG SAFE database

The aim of this study was to describe data on epidemiology, ventilatory management, and outcome of acute respiratory distress syndrome (ARDS) in immunocompromised patients. Methods: We performed a post hoc analysis on the cohort of immunocompromised patients enrolled in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) study. The LUNG SAFE study was an international, prospective study including hypoxemic patients in 459 ICUs from 50 countries across 5 continents. Results: Of 2813 patients with ARDS, 584 (20.8%) were immunocompromised, 38.9% of whom had an unspecified cause. Pneumonia, nonpulmonary sepsis, and noncardiogenic shock were their most common risk factors for ARDS. Hospital mortality was higher in immunocompromised than in immunocompetent patients (52.4% vs 36.2%; p < 0.0001), despite similar severity of ARDS. Decisions regarding limiting life-sustaining measures were significantly more frequent in immunocompromised patients (27.1% vs 18.6%; p < 0.0001). Use of noninvasive ventilation (NIV) as first-line treatment was higher in immunocompromised patients (20.9% vs 15.9%; p = 0.0048), and immunodeficiency remained independently associated with the use of NIV after adjustment for confounders. Forty-eight percent of the patients treated with NIV were intubated, and their mortality was not different from that of the patients invasively ventilated ab initio. Conclusions: Immunosuppression is frequent in patients with ARDS, and infections are the main risk factors for ARDS in these immunocompromised patients. Their management differs from that of immunocompetent patients, particularly the greater use of NIV as first-line ventilation strategy. Compared with immunocompetent subjects, they have higher mortality regardless of ARDS severity as well as a higher frequency of limitation of life-sustaining measures. Nonetheless, nearly half of these patients survive to hospital discharge. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013

Immunocompromised patients with acute respiratory distress syndrome: Secondary analysis of the LUNG SAFE database

Background: The aim of this study was to describe data on epidemiology, ventilatory management, and outcome of acute respiratory distress syndrome (ARDS) in immunocompromised patients. Methods: We performed a post hoc analysis on the cohort of immunocompromised patients enrolled in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) study. The LUNG SAFE study was an international, prospective study including hypoxemic patients in 459 ICUs from 50 countries across 5 continents. Results: Of 2813 patients with ARDS, 584 (20.8%) were immunocompromised, 38.9% of whom had an unspecified cause. Pneumonia, nonpulmonary sepsis, and noncardiogenic shock were their most common risk factors for ARDS. Hospital mortality was higher in immunocompromised than in immunocompetent patients (52.4% vs 36.2%; p &lt; 0.0001), despite similar severity of ARDS. Decisions regarding limiting life-sustaining measures were significantly more frequent in immunocompromised patients (27.1% vs 18.6%; p &lt; 0.0001). Use of noninvasive ventilation (NIV) as first-line treatment was higher in immunocompromised patients (20.9% vs 15.9%; p = 0.0048), and immunodeficiency remained independently associated with the use of NIV after adjustment for confounders. Forty-eight percent of the patients treated with NIV were intubated, and their mortality was not different from that of the patients invasively ventilated ab initio. Conclusions: Immunosuppression is frequent in patients with ARDS, and infections are the main risk factors for ARDS in these immunocompromised patients. Their management differs from that of immunocompetent patients, particularly the greater use of NIV as first-line ventilation strategy. Compared with immunocompetent subjects, they have higher mortality regardless of ARDS severity as well as a higher frequency of limitation of life-sustaining measures. Nonetheless, nearly half of these patients survive to hospital discharge. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013