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. 2018 Aug 1;32(15-16):1008–1019. doi: 10.1101/gad.315192.118

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© 2018 Boulay et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 5. — Silencing a Ewing sarcoma-specific GGAA repeat enhancer impairs de novo induction of NKX2-2 in MSCs. (A) ChIP-seq for FLI1 (EWS-FLI1) in A673 cells and FLI1 (EWS-FLI1), H3K27ac, H3K4me3, and H3K4me1 in MSCs lentivirally transduced with a control vector or EWS-FLI1. EWS-FLI1 can activate a GGAA repeat enhancer near NKX2-2. (B) Schematic showing the experimental strategy used in our assays. MSCs were lentivirally transduced with dCas9-KRAB and either sgRNA control (GFP) or a sgRNA targeting the EWS-FLI1-bound GGAA repeat enhancer near NKX2-2 before EWS-FLI1 lentiviral induction. (C) Immunoblotting confirms EWS-FLI1 expression in MSCs upon lentiviral induction. GAPDH was used as loading control. (D) RT-qPCR shows the specific decreased mRNA levels of NKX2-2 in MSCs infected with dCas9-KRAB and a sgRNA targeting the Ewing sarcoma-specific GGAA repeat enhancer site near NKX2-2 before EWS-FLI1 induction. SOX2, EZH2, and NR0B1 are other EWS-FLI1-induced genes not affected in these experiments. XRN2 was used as a control gene. (**) P-value < 0.01.