Journal: eLife

Article Title: NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency

doi: 10.7554/eLife.54993

Figure Lengend Snippet: ( A ) Representation of NuRD complex according to and ( B ) Expression levels, as normalized counts, of the displayed NuRD subunits (CHD4 in orange, HDAC2 in pink, MTA2 in dark pink, and RBBP4 in dark orange) obtained from RNA-seq data of RH4 cells expressing a tetracycline-inducible shRNA scramble construct at 24 and 48hrs of doxycycline treatment (n=3, see Materials and methods for details). ( C ) Violin plot depicting the expression levels of NuRD subunits in FP-RMS tumor tissue. Displayed are microarray data from three independent studies ( ; ; ) available on the R2 gene expression database (r2.aml.nl). ( D ) Immunoblot depicting Cas9 expression in RH4-Cas9 cells. GAPDH was used as a loading control and wildtype RH4 cells (WT) served as negative control. ( E ) Box plot depicting the tumor dependency scores, calculated as CERES, of the indicated NuRD members in 6 FP-RMS cell lines (CRISPR Avana Public 19Q2, depmap.org ). ( F ) Results of CRISPR/Cas9 double knockoutsdisplayed as ratio between the indicated NuRD members double knockout (DKO) population and the control population (sgAAVS1) at day 12 normalized to day 2. Each point represents the average of 3 biological replicates.

Article Snippet: RH4 cells stably expressing Cas9 were obtained by transducing wildtype cells with the expression vector lentiCRISPRv2 puro (#98290, Addgene) followed by puromycin selection (1 μg/mL).

Techniques: Expressing, RNA Sequencing, shRNA, Construct, Microarray, Gene Expression, Western Blot, Control, Negative Control, CRISPR, Double Knockout

Journal: eLife

Article Title: NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency

doi: 10.7554/eLife.54993

Figure Lengend Snippet: ( A ) Illustrative scheme of the NuRD centered CRISPR/Cas9-based screen. Briefly, RH4 cells stably expressing Cas9 were transduced with lentiviral expression vectors containing either a BFP-labelled control sgRNA (sgAAVS1) or a RFP-la-belled sgRNA targeting a certain NuRD subunit. Two days after transduction, the blue and red populations were mixed 1:1 and their evolution was analyzed by flow cytometry at day 2 and 12 after transduction. ( B ) CRISPR/Cas9 screen results displayed as ratio between the indicated NuRD member knockout (KO) population and the control population (RFP/BFB ratio) at day 12 normalized to day 2. Each point represents the average of 3 biological replicates. Five sgRNAs were used per NuRD member. ( C ) Representative phase-contrast images of RH4 cells 5 days after doxycycline-mediated (Dox) RBBP4, CHD4, and PAX3-FOXO1 (P3F) depletion by shRNA. A scramble shRNA was used as negative control. Scale bar - 100μm. ( D ) Percentage of dead cells, measured by 7-AAD staining, observed in the same samples described in ( C ). Data are represented as mean ± SD (n=3; *p< 0.1, **p < 0.01, ***p < 0.001, ratio paired t test). ( E, F and G ) Expression levels (relative to GAPDH) of the indicated P3F target genes quantified by qPCR in RH4 cells at 48hrs upon RBBP4, P3F and CHD4 induced knockdown by doxycycline treatment. Data were normalized to untreated cells and are represented as mean ± SD (n=3; *p<0.1, **p < 0.01, ***p < 0.001, ratio paired t test). Figure 1—source data 1. Raw data and statistics related to and its supplements.

Article Snippet: RH4 cells stably expressing Cas9 were obtained by transducing wildtype cells with the expression vector lentiCRISPRv2 puro (#98290, Addgene) followed by puromycin selection (1 μg/mL).

Techniques: CRISPR, Stable Transfection, Expressing, Transduction, Control, Flow Cytometry, Knock-Out, shRNA, Negative Control, Staining, Knockdown

Journal: eLife

Article Title: NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency

doi: 10.7554/eLife.54993

Figure Lengend Snippet: ( A ) Illustrative scheme of the affinity purification-mass spectrometry (AP-MS) studies performed to identify CHD4 interactors. CRISPR/Cas9-mediated repair was used to endogenously Flag tag CHD4 on RH4 cells at the N- and C-terminus, creating two new clonal cell lines (N-CHD4 and C-CHD4) (left). Endogenous CHD4 was immunoprecipitated from the N- and C-CHD4 cell lines using an anti-Flag antibody and interactors were identified by liquid chromatography-mass spectrometry (LC-MS)(right). ( B ) Overlap of CHD4 putative interactors identified in the Flag pull-downs of CHD4. ( C ) Top 10 gene ontology terms found enriched on CHD4 interactome by Metascape online tool. ( D ) Distribution of the putative CHD4 interactors according to their protein class. ( E and F ) Western blots of Flag immunoprecipitation assays (IP). Figure 2—source data 1. List of CHD4 candidate interactors.

Article Snippet: RH4 cells stably expressing Cas9 were obtained by transducing wildtype cells with the expression vector lentiCRISPRv2 puro (#98290, Addgene) followed by puromycin selection (1 μg/mL).

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, CRISPR, FLAG-tag, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: eLife

Article Title: NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency

doi: 10.7554/eLife.54993

Figure Lengend Snippet:

Article Snippet: RH4 cells stably expressing Cas9 were obtained by transducing wildtype cells with the expression vector lentiCRISPRv2 puro (#98290, Addgene) followed by puromycin selection (1 μg/mL).

Techniques: Recombinant, Plasmid Preparation, Expressing, Construct, shRNA, Sequencing, CRISPR, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Sample Prep, Software