The effect of STAG2 loss in Ewing sarcoma [HiChIP]

Ewing sarcoma is an aggressive malignancy characterized by oncogenic rearrangements of the EWS gene with an ETS-family transcription factor, most commonly FLI. Recent comprehensive next-generation sequencing efforts have revealed few other highly recurrent mutations in this disease apart from loss-of-function mutations in STAG2 which occur in 15-20% of tumors. STAG2 is a member of the cohesin complex, which regulates sister chromatid alignment during mitosis and epigenetic regulation of gene expression. While some studies suggest that loss of STAG2 is associated with the development of aneuploidy, this is not the case in Ewing sarcoma. To investigate whether STAG2 loss affects epigenetic regulation of gene expression in Ewing sarcoma, we developed isogenic Ewing sarcoma cell lines with STAG2 knockout. We found that Ewing sarcoma cells engineered for loss of STAG2 maintain an intact cohesion complex that alternately incorporates STAG1. HiChIP SMC1A was performed for the Ewing sarcoma cell line A673 under two conditions: (1) A673 cells treated with non-targeting CRISPR Cas9 guides (STAG2 WT) and (2) A673 cells treated with STAG2-targeting CRISPR Cas9 guides (STAG2 KO). Cells treated for gene editing were clonally selected and confirmed to either express STAG2 (control condition) or have a loss of STAG2 expression (STAG2 loss condition). For the control condition, we used the cell clones A673.sgNT-1c4 and the STAG2 knockout clone A673.sgSTAG2-1c6. HiChIP was performed for each clone in duplicate. Data analysis was designed as a 2x2 comparison of differential looping for SMC1A HiChIP in A673 STAG2 KO cells vs. A673 STAG2 WT cells.