Fig 2: Changes in 3D connectivity upon EWSR1-ATF1 depletion point to candidate CCS precursor cells.a Heatmaps depicting H3K27ac signal at Distal (top panels) and Proximal (bottom panels) sites in SU-CCS-1 cells, transfected with either control (siCTL, left) or EWSR1-ATF1 targeting (siEA, right) siRNAs, illustrating the induction of “de novo” sites in EWSR1-ATF1-depleted cells. For each heatmap 20 kb regions centered on the H3K27ac signal are shown. Signals are ranked by H3K27ac intensity in siEA-transfected SU-CCS-1 cells. b Hi-ChIP interaction map (top) and chromatin looping profile (bottom) for the RHOBTB1 genomic locus in siCTL or siEA – treated SU-CCS-1 tumor cells. H3K27ac ChIP-seq signal are also shown (bottom). Chromatin loops in the bottom panel are color-coded depending on their association (EA loops, red) or not (Non-EA loops, blue) with the fusion protein binding sites. c Gene expression z-scores heatmaps in DTC1 and SU-CCS-1 cells for the 157 transcripts identified as targets of “de novo” regulatory sites as in (a), showing upregulation at FC > 1.5 and p-value<0.05 in both cell lines upon EWSR1-ATF1 depletion. d Expression z-scores of genes involved in melanocytic differentiation, showing a higher expression in siEA-transfected DTC1 (D) and SU-CCS-1 (S) cells. Genes marked in bold were among the de novo targets in (c). e Homer motif analysis of the 4063 “de novo” distal sites shown in (a). Binomial p-values are given by the motif enrichment software HOMER. f Functional analysis of the same 157 genes for the Wiki-Pathways (WP), Biological Processes (GO: BP), and Molecular Function (GO: MF), showing the involvement of this gene repertoire in the NCSC differentiation and chromatin remodeling. g UMAP representation for the expression profiles of 168,103 single cells derived from five healthy human skin samples (including both epidermis and dermis layers). Normalized log2 scores were used for UMAP generation. Melanocyte, Schwann and Stromal Schwann cells are marked with dashed circles. h Boxplots depicting the enrichment score for the downregulated EWSR1-ATF1 target genes (bottom, n = 393) or the upregulated “de novo” gene signature (top, n = 154) expressed across 16 different cell groups. Following the removal of the fusion protein the tumor cells acquire a more differentiated phenotype, reminiscent of melanocytes and stromal Schwann cells. Box plots show the 1st to 3rd quartiles (25–75%) of the data with median values indicated.

Fig 3: EWSR1-ATF1 depletion reduces chromatin activation and DNA accessibility.a Western blot analysis of EWSR1-ATF1 protein levels in DTC1 and SU-CCS-1 cell lines transfected with a siRNA targeting the fusion gene (siEA 72 h and 96 h) or an unrelated sequence (siCTL 96 h). Tubulin protein levels were used as loading control. The experiment was independently repeated twice with similar results. Source images are provided in the Source Data file. b Composite plots showing the marked reduction in ATF1 C ChIP-seq signal at 2385 EWSR1-ATF1 bound sites in DTC1 and SU-CCS-1 cells transfected with either siEA or siCTL siRNAs for 96 h. c Left panel: Heatmaps depicting the distribution of ATF1 C ChIP-seq signal across 2011 EWSR1-ATF1 bound distal sites in siEA or siCTL-treated DTC1 and SU-CCS-1 cells at 96 h. Heatmaps show 20 kb genomic regions centered on EWSR1-ATF1 peaks ranked by ATF1 C signal intensity. Middle panel: composite plots of H3K4me1 (upper panels) and H3K27ac (lower panels) signal at 2011 EWSR1-ATF1 bound distal sites in siEA or siCTL-treated DTC1 and SU-CCS-1 cells at 96 h, showing a reduction in both enhancer marks upon EWSR1-ATF1 depletion. Right panel: composite plots of ATAC signal in siEA or siCTL-transfected DTC1 and SU-CCS-1 cells at 96 h, showing the reduction in chromatin accessibility associated with the loss of the fusion protein. d ChIP-seq and ATAC-seq tracks at the MERTK and ID4 genomic loci (as in Fig. 1e) in siEA or siCTL-treated DTC1 cells at 96 h, showing the decrease in chromatin activity and accessibility induced by the loss of EWSR1-ATF1. EWSR1-ATF1 binding regions are highlighted in gray.

Fig 4: EWSR1-ATF1-associated connectivity dominates the 3D landscape of CCS tumor cells.a Three-dimensional connectivity of EWSR1-ATF1-associated (EA loops, red) or –independent (Non EA loops, blue) chromatin loops in SU-CCS-1 tumor cells, showing a more distal-to-distal interaction pattern for fusion protein-connected loops. b Left: circos plot depicting all EWSR1-ATF1-associated or -independent chromatin loops genome-wide. The height of the blue and red signals inside the circos corresponds to the loop’s length depicted in log10 scale. Right: higher magnification of chromosome 20 illustrating increased length for EWSR1-ATF1-associated loops, as compared to all other loops. c Bar plots showing the association of H3K27ac peaks (left) and chromatin loops (right) with EA loops (5121 and 38095, respectively) or Non-EA loops (22654 and 41402, respectively). d Box plots depicting the median number (left) and intensity (right) of chromatin loops associated (n = 38095) or not (n = 41402) with EWSR1-ATF1 binding sites. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Statistical significance was calculated by two-sided t-test (n = 4181 forATF1 anchors, n = 16756 for No ATF1 anchors). e Venn diagram presenting the number of direct target genes identified by Hi-ChIP as connected to the 2385 EWSR1-ATF1 binding sites in SU-CCS-1 and DTC1 tumor cells. f Hi-ChIP interaction map (top) and chromatin looping profile (bottom) for the RRM2 genomic locus in SU-CCS-1 tumor cells. EWSR1-ATF1 (EA) binding sites and H3K27ac ChIP-seq signal are also shown (middle). Chromatin loops in the bottom panel are color-coded depending on their association (EA loops, red) or not (Non EA loops, blue) with the fusion protein binding sites.

Fig 5: EWSR1-ATF1 displays a distinct binding pattern compared to wt ATF1.a Genomic distribution of 2385 consensus EWSR1-ATF1 binding sites shared between DTC1 and SU-CCS-1 cell lines, illustrating preferential EWSR1-ATF1 binding to distal genomic regions. b DNAse I hypersensitivity profile comparison between 2011 distal EWSR1-ATF1 sites and 516 wt ATF1 bound distal sites across 113 different cell types, showing a more restricted DNA accessibility pattern for the fusion protein binding sites in other cell types. c Top panel: heatmaps depicting EWSR1 N, ATF1 C, H3K4me1, H3K27ac, and ATAC signal intensities at 2011 EWSR1-ATF1 bound distal sites in DTC1 and SU-CCS-1 cell lines, as well as three primary CCS tumors. Bottom panel: EWSR1 N, ATF1 C, H3K4me3, H3K27ac, and ATAC signal intensities at 374 TSS-associated EWSR1-ATF1 binding sites. For each heatmap 20 kb regions centered on the EWSR1-ATF1 peaks are shown. Signals are ranked by ATF1 C intensity. d De novo motif enrichment analysis for 2011 distal EWSR1-ATF1 binding regions. The top three motifs identified are shown. Binomial p-values are given by the motif enrichment software HOMER. e, ChIP-seq track at MERTK and ID4 genomic loci, illustrating similar EWSR1-ATF1 binding and chromatin activity profiles between DTC1 and primary CCS tumor cells, and the presence of TFAP2A, SOX10, and MITF at EWR1-ATF1 sites.