EWS-FLI1 and HOXD13 control tumor cell plasticity in Ewing sarcoma [CUT&RUN]

Posterior homeobox D genes, in particular HOXD13, are over-expressed by Ewing sarcoma, a tumor driven by the oncogenic fusion protein EWS-FLI1. Here, we have found that EWS-FLI1 maintains HOXD13 expression through a GGAA microsatellite enhancer in the developmental posterior HOXD regulatory domain. RNA-seq of shHOXD13 defined HOXD13-ergulated genes, so we performed CUT&RUN for HOXD13 and histone marks (H3K27ac, H3K4me3, H3K4me1, and H3K27me3) to determine how it regulates target genes. Overall design: A673 and CHLA10 cells were run through the automated CUT&RUN protocol (Janssens et al. 2018 Methodology). In brief, 250,000-500,000 cells were collected and incubated with concavalin A beads for 10min. Cells were then washed and permeabilized with 0.05% digitonin wash buffer solution and incubated with primary antibodies for 24-48 hours at 4C. Samples were then run through the automated platform (https://www.protocols.io/view/autocut-run-genome-wide-profiling-of-chromatin-pro-ufeetje)