|Public on May 14, 2021
|The effect of STAG2 loss in Ewing sarcoma [ChIP-seq]
|Genome binding/occupancy profiling by high throughput sequencing
|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 effects 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.
|ChIP-Seq profiling was performed as follows:
(i) EWS/FLI1, SMC1A, STAG1, STAG2, H3K27me3, H3K27ac and Input on isogenic clonally-selected A673 cells treated with STAG2 targeting CRISPR Cas9, and on A673 cells with STAG2 wild-type.
(ii) EWS/FLI1 and Input on A673 untreated clones.
(iii) H3K4me3 on A673 clones with STAG2 wild-type.
For each mark, the reads mapped on the STAG2 wild-type clones A673.sgNT-1c4 and A673.sgNT-2c3 were merged and labeled as "STAG2 WT". Similarly, for each mark, the reads mapped on the STAG2 knockout clones A673.sgSTAG2-1c6, A673.sgSTAG2-4c5 were merged and labeled as "STAG2 KO" with the exception of H3K27me3 for which only the A673.sgSTAG2-1c6 clone was available and labeled as "STAG2 sg1KO".
Drosophila melanogaster (Active Motif) was used as a spike-in control for all non-input samples profiled on both the STAG2 knockout and STAG2 wild-type clones, with the exception of H3K27ac. The Active Motif Spike-in Normalization protocol was applied to each sample by multiplying the human tag counts with the normalization factors derived from the uniquely mapped Drosophila reads vs. the dm6 counts for that sample. The dm6 normalization factors for individual clones and for the merged clones were very close to 1.
|Stegmaier K, Alexe G
|Jan 29, 2021
|Last update date
|Jun 16, 2021
|Computational Biology and Bioinformatics
|415 Main St.
|Illumina HiSeq 2000 (Homo sapiens)
|Illumina NovaSeq 6000 (Homo sapiens)
|This SubSeries is part of SuperSeries:
|The effect of STAG2 loss in Ewing sarcoma