Project description:RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins involved in gene expression regulation, although their in vivo targets and activities in biological processes like cell differentiation, that requires reprogramming of gene expression programs at multiple levels, are not well characterized. In this report, we uncovered a new mechanism by which DDX5 and DDX17 cooperate with hnRNP H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We next observed that downregulation of DDX5 and DDX17 protein expression during epithelial to mesenchymal transdifferentiation and during myogenesis contributes to switching splicing programs during these processes. Remarkably, this downregulation is mediated by the production of microRNAs 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 M-bM-^@M-^\master orchestratorsM-bM-^@M-^] of differentiation, that dynamically orchestrate several layers of gene expression. 6 samples of MCF7 cells exposed to different treatments were analyzed: 3 x siCTRLM-BM- ; 3 x si(DDX5-17) AND 6 samples of MCF10 cells exposed to different treatments were analyzed: 3 x siCTRLM-BM- ; 3 x si(DDX5-17)
Project description:RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins involved in gene expression regulation, although their in vivo targets and activities in biological processes like cell differentiation, that requires reprogramming of gene expression programs at multiple levels, are not well characterized. In this report, we uncovered a new mechanism by which DDX5 and DDX17 cooperate with hnRNP H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We next observed that downregulation of DDX5 and DDX17 protein expression during epithelial to mesenchymal transdifferentiation and during myogenesis contributes to switching splicing programs during these processes. Remarkably, this downregulation is mediated by the production of microRNAs 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.
Project description:We aimed at analysing the effect of RNA helicases DDX5 and DDX17 and of the transcription factor REST on gene expression in the human SH-SY5Y neuroblastoma cell line (European Collection of Cell Cultures, ECACC), which was cultured as recommended by the supplier.
Project description:This project includes ChIP of total RNA pol II in human SH-SY5Y neuroblastoma cells following the joint depletion of RNA helicases DDX5 and DDX17 to determine the genome-wide effect of DDX5/DDX17 on RNA Pol II distribution.
Project description:DEAD-box RNA helicases DDX5 and DDX17 regulate gene expression at different levels such as transcription and splicing, but the underlying mechanisms are not fully understood. A calibrated ChIP-seq analysis of total RNA Polymerase II was carried out in DDX5/DDX17-depleted SH-SY5Y cells to analyse the contribution of DDX5/DDX17 to RNAPII distribution. Univ Lyon, ENS de Lyon, CNRS UMR 5239, INSERM U1293, Laboratory of Biology and Modelling of the Cell, 46 Allée d'Italie, F-69007, Lyon, France. Corresponding author: cyril.bourgeois@inserm.fr
Project description:Both epigenetic and splicing regulation contribute to tumor progression, but the potential links between these two levels of gene-expression regulation in pathogenesis are not well understood. Here, we report that the mouse and human RNA helicases ddx17 and ddx5 contribute to tumor-cell invasiveness by regulating alternative splicing of several DNA- and chromatin-binding factors, including the macroH2A1 histone. We show that macroH2A1 splicing isoforms differentially regulate the transcription of a set of genes involved in redox metabolism. In particular, the SOD3 gene that encodes the extracellular superoxide dismutase and plays a part in cell migration is regulated in an inverse manner by macroH2A1 splicing isoforms. These findings reveal a new regulatory pathway in which splicing factors control the expression of histone-variant isoforms that in turn drive a transcription program to switch tumor cells to an invasive phenotype. We analyzed 4T1 cells depleted or not for ddx5 and ddx17 RNA helicases using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Affymetrix Exon Array Computational Tool. Four techinical replicates were performed. We analyzed 4T1 cells depleted for ddx5 and ddx17 RNA helicases and macroH2A1.1 or macroH2A1.2 using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Affymetrix Exon Array Computational Tool. Three techinical replicates were performed.
Project description:DEAD-box RNA helicases DDX5 and DDX17 regulate gene expression at different levels such as transcription and splicing, but the underlying mechanisms are not fully understood. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed was carried out to further characterize the widespread deregulation of alternative splicing. This study also revealed a deregulation of 3’ end processing leading to transcriptional read-through. Univ Lyon, ENS de Lyon, CNRS UMR 5239, INSERM U1293, Laboratory of Biology and Modelling of the Cell, 46 Allée d'Italie, F-69007, Lyon, France. Corresponding author: cyril.bourgeois@inserm.fr
Project description:RNAseq : MCF-7 cells were transfected with siRNA targeting both DDX5 and DDX17 RNA helicases, and total RNA were extracted as described previously (Dardenne Cell Rep 2014).