Pierre-Olivier Angrand

A physical and functional map of the human TNF-α/NF-κB signal transduction pathway

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α triggers a signalling cascade, converging on the activation of the transcription factor NF-κB, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-α/NF-κB pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-α/NF-κB pathway and is generally applicable to other pathways relevant to human disease.

Transgenic Mouse Proteomics Identifies New 14-3-3-associated Proteins Involved in Cytoskeletal Rearrangements and Cell Signaling

Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction. Here we describe, for the first time, the application of the tandem affinity purification (TAP) and LC-MS method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3ζ protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3ζ-binding proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3ζ in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor β (βPIX).

The role of long non-coding RNAs in genome formatting and expression

Long non-coding RNAs (lncRNAs) are transcripts without protein-coding potential but having a pivotal role in numerous biological functions. Long non-coding RNAs act as regulators at different levels of gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. Misregulation of lncRNAs expression has been found to be associated to cancer and other human disorders. Here, we review the different types of lncRNAs, their mechanisms of action on genome formatting and expression and emphasized on the multifaceted action of the H19 lncRNA.

Cell adhesion properties on chemically micropatterned boron-doped diamond surfaces.

The adhesion properties of living cells were investigated on a range of chemically modified boron-doped diamond (BDD) surfaces. We studied the influence of oxidized, H-, amine- (NH(2)-), methyl- (CH(3)-), trifluoromethyl- (CF(3)-) and vinyl- (CH(2)═CH-) terminated BDD surfaces on human osteosarcoma U2OS and mouse fibroblast L929 cells behavior. Cell-surface interactions were analyzed by fluorescence microscopy in terms of cell attachment, spreading and proliferation. U2OS cells poorly adhered on hydrophobic surfaces and their growth was blocked. In contrast, L929 cells were mainly influenced by the presence of perfluoroalkyl chains in regard to their morphology. The results were subsequently applied to selectively micropattern U2OS cells on dual hydrophobic/hydrophilic surfaces prepared by a UV/ozone lithographic approach. U2OS cells colonized preferentially hydrophilic (oxide-terminated) motifs, forming confluent arrays with distinguishable edges separating the alkyl regions.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2–1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2–2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2–2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

The Polycomb Group Protein Pcgf1 Is Dispensable in Zebrafish but Involved in Early Growth and Aging.

Polycomb Repressive Complex (PRC) 1 regulates the control of gene expression programs via chromatin structure reorganization. Through mutual exclusion, different PCGF members generate a variety of PRC1 complexes with potentially distinct cellular functions. In this context, the molecular function of each of the PCGF family members remains elusive. The study of PCGF family member expression in zebrafish development and during caudal fin regeneration reveals that the zebrafish pcgf genes are subjected to different regulations and that all PRC1 complexes in terms of Pcgf subunit composition are not always present in the same tissues. To unveil the function of Pcgf1 in zebrafish, a mutant line was generated using the TALEN technology. Mutant pcgf1-/- fish are viable and fertile, but the growth rate at early developmental stages is reduced in absence of pcgf1 gene function and a significant number of pcgf1-/- fish show signs of premature aging. This first vertebrate model lacking Pcgf1 function shows that this Polycomb Group protein is involved in cell proliferation during early embryogenesis and establishes a link between epigenetics and aging.

PRC1 components exhibit different binding kinetics in Polycomb bodies

Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined.

The histone lysine methyltransferase Ezh2 is required for maintenance of the intestine integrity and for caudal fin regeneration in zebrafish

The histone lysine methyltransferase EZH2, as part of the Polycomb Repressive Complex 2 (PRC2), mediates H3K27me3 methylation which is involved in gene expression program repression. Through its action, EZH2 controls cell-fate decisions during the development and the differentiation processes. Here, we report the generation and the characterization of an ezh2-deficient zebrafish line. In contrast to its essential role in mouse early development, loss of ezh2 function does not affect zebrafish gastrulation. Ezh2 zebrafish mutants present a normal body plan but die at around 12 dpf with defects in the intestine wall, due to enhanced cell death. Thus, ezh2-deficient zebrafish can initiate differentiation toward the different developmental lineages but fail to maintain the intestinal homeostasis. Expression studies revealed that ezh2 mRNAs are maternally deposited. Then, ezh2 is ubiquitously expressed in the anterior part of the embryos at 24 hpf, but its expression becomes restricted to specific regions at later developmental stages. Pharmacological inhibition of Ezh2 showed that maternal Ezh2 products contribute to early development but are dispensable to body plan formation. In addition, ezh2-deficient mutants fail to properly regenerate their spinal cord after caudal fin transection suggesting that Ezh2 and H3K27me3 methylation might also be involved in the process of regeneration in zebrafish.