...:: TALKS ::...
Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia using a Genome-Scale CRISPR Library.
Matthieu Lewis, Valérie Prouzet-Mauléon, Elodie Richard, Bruno Cardinaud, Richard Iggo, Béatrice Turcq, François-Xavier Mahon
INSERM U1218 Université de Bordeaux
Understanding resistance to targeted therapy in cancer remains of utmost importance in regards to improving future therapeutic strategies. In spite of the efficacy of tyrosine kinase inhibitors (TKIs) to target and inhibit the BCR-ABL1 oncoprotein in chronic myeloid leukaemia (CML), resistance to treatment occurs in approximately 10-20% of patients. Additional unknown mutations, harboured in certain genes of CML cells, may decrease the sensitivity to TKIs. To discover such genes, we utilized a Genome-scale CRISPR Knock-Out (GeCKO) library, containing 121,413 different sgRNAs (6 sgRNAs per gene) that target 20,914 protein coding genes and miRNAs, to screen for resistance to a specific TKI, imatinib, in a CML cell line. After transducing the cells, we obtained a heterogeneous population of cells with single random gene knock-outs (one sgRNA per cell) and we challenged these cells to imatinib selection. The sgRNAs in treated and control samples were sequenced by next generation sequencing. Then, MAGeCK software analysis allowed us to rank them according to their enrichment. The most enriched sgRNAs (FDR<0.01) targeted genes involved in transcriptional and translational regulation, apoptosis and cell cycle regulation. A subset of these genes has already been correlated to CML progression and/or TKI resistance in the past. These results highlighted the successful use of CRISPR libraries to study resistance mechanisms. These genes were then validated using the best ranked sgRNA for individual knock-out experiments. Gene set enrichment and gene ontology analyses of the ranked sgRNA list showed the importance of genes involved in intrinsic apoptosis, negative regulation of the cell cycle, mRNA processing, protein ubiquitinylation and the mediator complex. The utilization of CRISPR libraries may help identify novel genes involved in resistance for a myriad of different processes.
Dissecting the functional importance of Polycomb Response Element (PRE) interactions for the formation of epigenetic chromatin domains in Drosophila
Bernd Schuettengruber, Yuki Ogiyama, Giacomo Cavalli
IGH, UMR9002, CNRS, UM, 141, Rue de la cardonille, 34396 Montpellier
Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In Drosophila, cis-regulatory regions termed Polycomb response elements (PREs) act as nucleation sites for PcG proteins to create large repressive Polycomb domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3). Recent advances in HiC technologies have revealed that PREs can form looping interactions within Polycomb domains leading to the clustering of PcG target genes and their regulatory regions. However the functional importance of these 3D chromatin interactions is largely unknown. Therefore we are using the CRISPR/Cas9 technology to mutate PREs in their endogenous chromatin context and study the functional importance of PRE interactions for the formation of Polycomb domains and PcG target gene regulation.
...:: POSTERS ::...
Identification of genes involved in oncogenic cooperation with BCR-ABL1
Emilie Rousseau, Stéphanie Durrieu-Gaillard, Clémence Melon and Martin Teichmann
University of Bordeaux ; Inserm U1212 -CNRS UMR 5320 146, rue Léo Saignat, 33800 Bordeaux
Waiting for permission to display the summary
Role of alternative splicing in the regulation of Yki activity in Drosophila
Diwas Srivastava, Jamal Tazi, François Juge
Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Montpellier, France.
The Hippo pathway is a conserved signaling pathway involved in tissue growth control, stem cell function, regeneration and tumor suppression. This pathway represses the activity of the transcription co-activator Yki in flies and YAP in mammals. Both Yki and YAP proteins exist as two isoforms containing one (Yki1/YAP1) or two (Yki2/YAP2) WW domains, by alternative splicing of their RNAs. The function and regulation of this alternative splicing event is unknown. We identified the splicing factor B52 as necessary for inclusion of yki alternative exon in flies: depletion of B52 favors skipping of yki alternative exon and expression of Yki1 at the expense of Yki2. We analyzed the differences between Yki1 and Yki2 isoforms in flies and S2 cells. We show that (1) Yki1 overexpression induces weaker phenotypes that Yki2, (2) Yki1 is a weaker transcription co-activator compared to Yki2, (3) the absence of a WW domain in Yki1 lowers its interaction with several partners of the Hippo pathway, (4) Yki1 can compete with Yki2 in vivo. These results highlight alternative splicing of yki as a new level of modulation of the Hippo pathway. To directly address the importance of yki alternative splicing in vivo, we try to abrogate it. By using CRISPR/Cas9-mediated genome engineering we modify endogenous yki locus to create two mutants flies than can produce, either only Yki1, or only Yki2 isoform. We are using a non-tagged strategy based on PCR screening of adult transgenic flies. We hope to reveal the developmental role of yki alternative splicing by this strategy.