EWS-FLI1 and HOXD13 control tumor cell plasticity in Ewing sarcoma [RNA-seq]

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. Activation of this enhancer is EWS-FLI1 dependent and epigenomic silencing of this region leads to loss of HOXD13 expression in Ewing sarcoma cells, but not in unrelated cells. To determine the function of HOXD13 activation in Ewing sarcoma we performed nascent RNA sequencing or RNA sequencing upon HOXD13 knockdown. Overall design: HOXD13 was knocked down by doxy-cycline inducible (0.5 ug/uL) shRNAs in two human Ewing sarcoma cell lines, CHLA10 and A673 for 72 hours. Three replicates each of targeting shRNA (shHOXD13 #1) and non-targeting control (shNS) were subject to nascent RNA sequencing (Bru-seq). HOXD13 was knocked down by doxy-cycline inducible (0.5 ug/uL) shRNAs in the human Ewing sarcoma cell lines, TC32, for 72 hours. Three replicates each of targeting shRNA (shHOXD13 #1) and non-targeting control (shNS) were subject to RNA sequencing