Splicing Programs and Cancer

Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets

Control of Flowering and Cell Fate by LIF2, an RNA Binding Partner of the Polycomb Complex Component LHP1

Polycomb Repressive Complexes (PRC) modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2). LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA processing and Polycomb regulation

Interaction de la protéine LIKE HETEROCHROMATIN PROTEIN 1 avec la chromatine chez Arabidopsis thaliana

La chromatine permet la compaction de l information génétique chez les eucaryotes et joue un rôle central dans la régulation de l accès à cette information. Structure et fonction sont étroitement liées et reposent sur des modifications chimiques de l ADN, sur des composants ARN et une grande diversité protéique. Les protéines à chromodomaine (CD) de la famille HP1 interviennent dans la lecture du code des histones et permettent d établir et propager des états chromatiniens spécifiques. Chez Arabidopsis, la protéine LHP1 est impliquée dans la régulation génique et dans le contrôle du développement. Afin de déterminer les cibles chromatiniennes de LHP1 in planta et à l échelle génomique, nous avons adapté la technique DamID. 2354 sites de fixation euchromatiques ont été identifiés, colocalisant avec les résidus triméthylés de la lysine 27 de l histone H3. Il a été montré que le CD de LHP1 reconnaît et se fixe à cette marque épigénétique. Ces résultats suggèrent que LHP1 possède des fonctions similaires à celles de la protéine Polycomb chez les animaux. Nous nous sommes intéressés aux complexes LHP1 en étudiant deux de ses partenaires, les protéines LIF1 et LIF2. Ces protéines se caractérisent par la présence de motifs RRM de fixation à l ARN simple brin, suggérant la participation d une composante ARN dans les complexes LHP1. Nous avons confirmé l interaction LIF2/LHP1 in vitro et in vivo, étudié la localisation de ces protéines, leurs expressions et analysé les phénotypes des mutants lif1 et lif2. LIF1 et LIF2 jouent un rôle dans la transition florale et dans le développement. La recherche de leurs partenaires ARN permettra de mieux comprendre leurs modes d action.In eukaryotes, chromatin packages genetic information and plays a crucial role in regulation of access to the underlying genome, influencing many biological processes. The level of compaction of the DNA and the ability of biological processes to occur are closely related to one another. This relationship relies on biochemical modifications of the DNA and histones (histone code), on RNA components and on a broad diversity of proteins. Chromodomain (CD) proteins from the HP1 family are involved in reading the histone code, in establishing and spreading specific chromatin states. In Arabidopsis, LHP1 is involved in gene regulation and in development control. To characterize LHP1 chromatin targets in planta at genomic scale, we adapted DamID technique to use in plants. 2354 euchromatic fixation sites have been identified, which colocalize with trimethylated lysine 27 in histone H3 (H3K27me3). Moreover, it has been shown that CD of LHP1 recognizes and binds to this epigenetic mark. These results suggest that LHP1 has similar functions to those of Polycomb (Pc) protein in animals, and might participate to a PcG repressive complex-1 type in plants. To analyze LHP1 complexes, we focused our study on two LHP1 partners, LIF1 and LIF2. These proteins are made of RRM motifs known to be involved in single-stranded RNA fixation. We confirmed LIF2/LHP1 in vitro (pull down) and in vivo (BiFC), studied protein localization, expression and analysed phenotypes of lif1 and lif2 mutants. LIF1 and LIF2 play a role in floral transition and in development. Further research to study their RNA partners will allow a better understanding of their detailed functions.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation

International audiencePolycomb proteins are required for maintenance of silent chromatin states via histone H3 Lys27 trimethylation ( H3K27me3) in animals, but homologs are not found in plant genomes. Using a DamID- chip method, we found that the Arabidopsis thaliana chromodomain- containing protein LHP1 colocalizes with H3K27me3 genome- wide. The LHP1 chromodomain also binds H3K27me3 with high affinity, suggesting that LHP1 has functions similar to those of Polycomb

RNA helicases DDX5 and DDX17 dynamically orchestrate transcription, miRNA, and splicing programs in cell differentiation.

SummaryThe RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins “master orchestrators” of differentiation that dynamically orchestrate several layers of gene expression

Tracking the sterol biosynthesis pathway of the diatom Phaeodactylum tricornutum

© 2014 New Phytologist Trust. Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the pathway leading to sterol biosynthesis in diatoms has remained hitherto unidentified. Through the DiatomCyc database we reconstructed the mevalonate and sterol biosynthetic pathways of the model diatom Phaeodactylum tricornutum in silico. We experimentally verified the predicted pathways using enzyme inhibitor, gene silencing and heterologous gene expression approaches. Our analysis revealed a peculiar, chimeric organization of the diatom sterol biosynthesis pathway, which possesses features of both plant and fungal pathways. Strikingly, it lacks a conventional squalene epoxidase and utilizes an extended oxidosqualene cyclase and a multifunctional isopentenyl diphosphate isomerase/squalene synthase enzyme. The reconstruction of the P. tricornutum sterol pathway underscores the metabolic plasticity of diatoms and offers important insights for the engineering of diatoms for sustainable production of biofuels and high-value chemicals