Fig. 1
From: A comprehensive enhancer screen identifies TRAM2 as a key and novel mediator of YAP oncogenesis
CRISPR-YAP-enh-lib library design, evaluation, and functional screening. a A schematic representation of the bioinformatics pipeline for the design of short guide RNAs (sgRNA) for CRISPR-YAP-enh-lib library construction. b ChIP-qPCR assays were performed to evaluate CRISPR efficiency in blocking YAP-binding to three selected top YAP-bound regions (Region1 chr19:41729486-41729491; Region2 chr22:41686880-41686885; Region3 chr1:19336594-19336599). Relative YAP binding was calculated and normalized over negative control (NC1 and NC2) regions and input. PC, positive control region - promoter of ANKRD1, as described in [29]. sgRNA-NT is a non-targeting control CRISPR vector. Error bars indicate standard deviation (SD) calculated from three technical replicates. ***P < 0.005, two-tailed Student’s t test. The pie plots are mutational profiles of MCF10A-YAP5SA cells transduced with sgRNAs targeting the TEAD4 motif of the selected regions. c A schematic representation of the setup of the functional screen for YAP enhancers, as detailed in the material and method section “CRISPR library construction and analysis”. d Results of the functional screen of YAP enhancers. Scatter plot showing the average LFC (log fold change) of the sgRNA abundances between T = 20 and T = 0 in both MCF10A-YAP5SA and MCF10A control cells (calculated from 3 biological replicates). Colored in orange are the sgRNAs targeting the YAP5SA (positive controls), blue dots are the negative control sgRNAs, and the green dots are the selected hits showing reduced abundance only in MCF10A-YAP5SA cells. e A representative plot for enhancer A, one of the selected hits, supplemented with genomic information of the targeting sgRNA sites, H3K27ac levels (an active enhancer marker), YAP and TEAD4 binding in MDA-MB-231 cells, and GRO-seq data of MDA-MB-231 and MCF7 cells. The black arrows indicate the production of enhancer RNA (eRNA). The information about the other selected hits is shown in Fig. S2. f Validation of the selected hits by competitive proliferation assays in MCF10A-YAP5SA and MCF10A control cells. Pie plots indicate the mutation status of MCF10A-YAP5SA cells transduced with the indicated sgRNAs. NT Ctrl is a non-targeting sgRNA control vector. YAP5SA-KO is an sgRNA vector targeting YAP5SA. Values from days 6 and 12 were normalized to day 0. Error bars indicate SD from three biological replicates. ***P < 0.005, two-tailed Student’s t test