Lionel Spinelli

Tyrosine phosphorylation of RNA Polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001

Clust&See: A Cytoscape plugin for the identification, visualization and manipulation of network clusters ,

BACKGROUND AND SCOPE Large networks, such as protein interaction networks, are extremely difficult to analyze as a whole. We developed Clust&See, a Cytoscape plugin dedicated to the identification, visualization and analysis of clusters extracted from such networks. IMPLEMENTATION AND PERFORMANCE Clust&See provides the ability to apply three different, recently developed graph clustering algorithms to networks and to visualize: (i) the obtained partition as a quotient graph in which nodes correspond to clusters and (ii) the obtained clusters as their corresponding subnetworks. Importantly, tools for investigating the relationships between clusters and vertices as well as their organization within the whole graph are supplied.

BubbleGUM: automatic extraction of phenotype molecular signatures and comprehensive visualization of multiple Gene Set Enrichment Analyses

BackgroundRecent advances in the analysis of high-throughput expression data have led to the development of tools that scaled-up their focus from single-gene to gene set level. For example, the popular Gene Set Enrichment Analysis (GSEA) algorithm can detect moderate but coordinated expression changes of groups of presumably related genes between pairs of experimental conditions. This considerably improves extraction of information from high-throughput gene expression data. However, although many gene sets covering a large panel of biological fields are available in public databases, the ability to generate home-made gene sets relevant to one’s biological question is crucial but remains a substantial challenge to most biologists lacking statistic or bioinformatic expertise. This is all the more the case when attempting to define a gene set specific of one condition compared to many other ones. Thus, there is a crucial need for an easy-to-use software for generation of relevant home-made gene sets from complex datasets, their use in GSEA, and the correction of the results when applied to multiple comparisons of many experimental conditions.ResultWe developed BubbleGUM (GSEA Unlimited Map), a tool that allows to automatically extract molecular signatures from transcriptomic data and perform exhaustive GSEA with multiple testing correction. One original feature of BubbleGUM notably resides in its capacity to integrate and compare numerous GSEA results into an easy-to-grasp graphical representation. We applied our method to generate transcriptomic fingerprints for murine cell types and to assess their enrichments in human cell types. This analysis allowed us to confirm homologies between mouse and human immunocytes.ConclusionsBubbleGUM is an open-source software that allows to automatically generate molecular signatures out of complex expression datasets and to assess directly their enrichment by GSEA on independent datasets. Enrichments are displayed in a graphical output that helps interpreting the results. This innovative methodology has recently been used to answer important questions in functional genomics, such as the degree of similarities between microarray datasets from different laboratories or with different experimental models or clinical cohorts. BubbleGUM is executable through an intuitive interface so that both bioinformaticians and biologists can use it. It is available at http://www.ciml.univ-mrs.fr/applications/BubbleGUM/index.html.

Protein synthesis inhibition and GADD34 control IFN‐β heterogeneous expression in response to dsRNA

In innate immune responses, induction of type‐I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type‐I IFN production is linked to cells ability to enter dsRNA‐activated PKR‐dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti‐viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN‐β mRNA transcription, while GADD34‐dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA‐activated cells.

Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex

Guanabenz attenuates autoimmune inflammation by preventing TLR9 from reaching endosomes and becoming fully activated. From fighting hypertension to inflammation The FDA-approved antihypertensive drug guanabenz also has anti-inflammatory properties, which are thought to be mediated by the inhibition of GADD34, a phosphatase subunit that promotes the production of proinflammatory cytokines and IFN-β. Perego et al. found that guanabenz inhibited the activation of TLR9, a receptor that induces type I IFN secretion from immune cells in response to DNA. TLR9 is implicated in autoimmune diseases in which self-DNA is erroneously recognized by the immune system, and guanabenz reduced symptom severity in a mouse model of lupus. However, guanabenz did not inhibit TLR9 by affecting GADD34 activity, but rather by altering cholesterol metabolism such that TLR9 did not reach endosomes where it is fully activated. The authors note that many lupus patients suffer from hypertension and that guanabenz and related compounds could, thus, have dual benefits for these patients. Endoplasmic reticulum (ER) stress triggers or amplifies inflammatory signals and cytokine production in immune cells. Upon the resolution of ER stress, the inducible phosphatase 1 cofactor GADD34 promotes the dephosphorylation of the initiation factor eIF2α, thereby enabling protein translation to resume. Several aminoguanidine compounds, such as guanabenz, perturb the eIF2α phosphorylation-dephosphorylation cycle and protect different cell or tissue types from protein misfolding and degeneration. We investigated how pharmacological interference with the eIF2α pathway could be beneficial to treat autoinflammatory diseases dependent on proinflammatory cytokines and type I interferons (IFNs), the production of which is regulated by GADD34 in dendritic cells (DCs). In mouse and human DCs and B cells, guanabenz prevented the activation of Toll-like receptor 9 (TLR9) by CpG oligodeoxynucleotides or DNA-immunoglobulin complexes in endosomes. In vivo, guanabenz protected mice from CpG oligonucleotide–dependent cytokine shock and decreased autoimmune symptom severity in a chemically induced model of systemic lupus erythematosus. However, we found that guanabenz exerted its inhibitory effect independently of GADD34 activity on eIF2α and instead decreased the abundance of CH25H, a cholesterol hydroxylase linked to antiviral immunity. Our results therefore suggest that guanabenz and similar compounds could be used to treat type I IFN–dependent pathologies and that CH25H could be a therapeutic target to control these diseases.

Protein complex scaffolding predicted as a prevalent function of long non-coding RNAs

Abstract The human transcriptome contains thousands of long non-coding RNAs (lncRNAs). Characterizing their function is a current challenge. An emerging concept is that lncRNAs serve as protein scaffolds, forming ribonucleoproteins and bringing proteins in proximity. However, only few scaffolding lncRNAs have been characterized and the prevalence of this function is unknown. Here, we propose the first computational approach aimed at predicting scaffolding lncRNAs at large scale. We predicted the largest human lncRNA–protein interaction network to date using the catRAPID omics algorithm. In combination with tissue expression and statistical approaches, we identified 847 lncRNAs (∼5% of the long non-coding transcriptome) predicted to scaffold half of the known protein complexes and network modules. Lastly, we show that the association of certain lncRNAs to disease may involve their scaffolding ability. Overall, our results suggest for the first time that RNA-mediated scaffolding of protein complexes and modules may be a common mechanism in human cells.

Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure.

Perturbed human sub-networks by Fusobacterium nucleatum candidate virulence proteins

BackgroundFusobacterium nucleatum is a gram-negative anaerobic species residing in the oral cavity and implicated in several inflammatory processes in the human body. Although F. nucleatum abundance is increased in inflammatory bowel disease subjects and is prevalent in colorectal cancer patients, the causal role of the bacterium in gastrointestinal disorders and the mechanistic details of host cell functions subversion are not fully understood.ResultsWe devised a computational strategy to identify putative secreted F. nucleatum proteins (FusoSecretome) and to infer their interactions with human proteins based on the presence of host molecular mimicry elements. FusoSecretome proteins share similar features with known bacterial virulence factors thereby highlighting their pathogenic potential. We show that they interact with human proteins that participate in infection-related cellular processes and localize in established cellular districts of the host–pathogen interface. Our network-based analysis identified 31 functional modules in the human interactome preferentially targeted by 138 FusoSecretome proteins, among which we selected 26 as main candidate virulence proteins, representing both putative and known virulence proteins. Finally, six of the preferentially targeted functional modules are implicated in the onset and progression of inflammatory bowel diseases and colorectal cancer.ConclusionsOverall, our computational analysis identified candidate virulence proteins potentially involved in the F. nucleatum—human cross-talk in the context of gastrointestinal diseases.

Pasha: a versatile R package for piling chromatin HTS data

UNLABELLED We describe an R package designed for processing aligned reads from chromatin-oriented high-throughput sequencing experiments. Pasha (preprocessing of aligned sequences from HTS analyses) allows easy manipulation of aligned reads from short-read sequencing technologies (ChIP-seq, FAIRE-seq, MNase-Seq, …) and offers innovative approaches such as ChIP-seq reads elongation, nucleosome midpoint piling strategy for positioning analyses, or the ability to subset paired-end reads by groups of insert size that can contain biologically relevant information. AVAILABILITY AND IMPLEMENTATION Pasha is a multi-platform R package, available on CRAN repositories under GPL-3 license (https://cran.r-project.org/web/packages/Pasha/). CONTACTS rfenouil@gmail.com or jean-christophe.andrau@igmm.cnrs.fr SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Human germinal center transcriptional programs are de-synchronized in B cell lymphoma

Most adult B cell lymphomas originate from germinal center (GC) B cells, but it is unclear to what extent B cells in overt lymphoma retain the functional dynamics of GC B cells or are blocked at a particular stage of the GC reaction. Here we used integrative single-cell analysis of phenotype, gene expression and variable-region sequence of the immunoglobulin heavy-chain locus to track the characteristic human GC B cell program in follicular lymphoma B cells. By modeling the cyclic continuum of GC B cell transitional states, we identified characteristic patterns of synchronously expressed gene clusters. GC-specific gene-expression synchrony was lost in single lymphoma B cells. However, distinct follicular lymphoma–specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B cell state but might adopt new dynamic modes of functional diversity, which opens the possibility of novel definitions of lymphoma identity.Human follicular lymphomas arise from germinal center B cells. Milpied and colleagues use single-cell transcriptomic analysis to show that follicular lymphoma cells lose synchronized gene-expression patterns that characterize normal germinal center B cells.