Fig. 3: wt ATF1 is present at a majority of EWSR1-ATF1 binding sites.

a Heatmaps depicting EWSR1 N, ATF1 C and ATF1 N ChIP-seq signal intensities at 2011 distal and 374 proximal EWSR1-ATF1 binding sites in SU-CCS-1 and primary CCS1 tumor cells, illustrating genomic co-occupancy by the wt ATF1 TF at the fusion protein binding sites. For each heatmap 20 kb regions centered on the EWSR1-ATF1 peaks are shown. Signals are ranked by ATF1 C intensity. b Composite plots showing changes in ChIP-seq signal for ATF1 N peaks associated (left) or not (right) with EWSR1-ATF1 binding in SU-CCS-1 cells transfected with a siRNA targeting the fusion gene (siEA) or control (siCTL) siRNAs. The loss of the fusion protein is associated with a decrease in ATF1 N signal only at EWSR1-ATF1 bound sites, suggesting wt ATF1 recruitment by the fusion protein. c Co-immunoprecipitation (Co-IP) assay showing the direct interaction between the EWSR1-ATF1 and wt ATF1 proteins in 293 T (left) and DTC1 cells (right). 293 T cells were transfected with either a V5-tagged EWSR1-ATF1 (EA-CV5) or empty vector (CTL) constructs. The IP was performed using an anti-V5 tag antibody, and the western blot revealed using anti-V5 (top) or anti-ATF1 C (bottom) antibodies. DTC1 IPs were performed using anti-ATF1 C or -ATF1 N antibodies, compared to control (CTL) IgG, and the western blot revealed using an anti-ATF1 C antibody. The experiment was independently repeated twice with similar results. d Correlation plot of EWSR1 N, ATF1 N, and H3K27ac ChIP-seq scores at the 2011 distal regions, showing stronger correlation between H3K27ac deposition and wt ATF1 presence, than with EWSR1-ATF1 presence (represented by ATF1 N and EWSR1 N signals, respectively). e Scatter plots of ATF1 N and p300 (left), or EWSR1 N and p300 (right) ChIP-seq scores, illustrating that wt ATF1 presence correlates better with p300 occupancy than EWSR1-ATF1. f Western blot analysis of EWSR1-ATF1 and wt ATF1 protein levels in DTC1 and SU-CCS-1 cell lines infected with a shRNA targeting the wt ATF1 transcript (shATF1) or an unrelated sequence (shCTL) at 72 h post-lentiviral infection. Tubulin protein levels were used as loading control. The experiment was independently repeated twice with similar results. g qPCR analysis showing reduction of wt ATF1 transcripts, as well as a panel of EWSR1-ATF1 / wt ATF1 co-regulated target transcripts, in shATF1- vs shCTL-infected SU-CCS-1 (S) and DTC1 (D) cells at 72 h post-lentiviral infection. Three replicates of each condition were used to calculate mean Ct values, mean relative expression values were then calculated according to the 2^-ΔΔCt method relative to the shCTL sample values, and normalized to the endogenous control gene GAPDH. Error bars show standard deviation of mean values (n = 3 sample replicates). The experiment was independently repeated twice with similar results. Source data are provided in the Source Data file.