FIGURE 3 from Targeting the Clear Cell Sarcoma Oncogenic Driver Fusion Gene EWSR1::ATF1 by HDAC Inhibition

Vorinostat alters EWSR1 promoter activity and histone modification at the promoter region. A, CCS cells were treated with 0, 0.3, 3.0, or 30.0 μmol/L vorinostat for 6 hours. Relative EWSR1 promoter activity was measured via a reporter assay (n = 3). B, MP-CCS-SY and SU-CCS1 cells were treated with 0–10 μmol/L vorinostat for 24 hours, and the protein expression levels of the active acetyl-histone markers H3K27ac and H3K9ac were detected using Western blotting. C, Genome browser screenshot of the epigenome maps at EWSR1. Tracks visualize CUT&RUN-seq data for two acetyl-histone markers (H3K27ac and K3K9ac) (n = 2), and scATAC-seq data are shown for treatments with 3 μmol/L vorinostat or vehicle for 24 hours. Blue highlights indicate the inserted DNA regions used in the reporter assay. Four CCS cells were treated with 3 μmol/L vorinostat or vehicle for 24 hours, and the levels of H3K27ac (D), H3K9ac (E), and H3K4 me3 (F) at three sites within the EWSR1 promoter region were analyzed using CUT&RUN-qPCR (n = 3). The values shown are relative to the input. Data in A and D–F are means ± SDs. *, P < 0.05; **, P < 0.01 (Student t test).