Figures and data
DBD-α4 helix of FLI domain is required to restructure chromatin in A673 cell. A. A schematic of DBD and DBD+ constructs used in shRNA knock-down and rescue experiments. B. Multidimensional scaling (MDS) plot of top 1000 interactions (500kb resolution) in each biological replicates. C. Genome-wide interaction frequency (ICE-corrected Micro-C counts) over genomic distance (bp) at 5kb resolution. D. Volcano plot showing differentially interacting regions (DIRs) detected at 500kb resolution for DBD+ replicates versus KD replicates. E. Volcano plot showing DIRs detected at same resolution for DBD replicates versus KD replicates. F. Violin plot of interaction distance (unit length of 500kb) for interactions (p adj < 0.2, log2FC > 0.5) detected in DBD only (mean=54Mbp), in both DBD and DBD+ conditions (mean=39.5Mbp), and in DBD+ only (mean=29.7Mbp). Boxplots depict the minimum, first quartile, median, third quartile, and maximum.* P value < 0.05, *** P value < 0.001
Altered TAD structure in DBD cells is linked to GGAA microsatellite binding. A. Number of TADs detected in DBD and DBD+ compared to KD at resolutions of 10kb, 25kb, 50kb and 100kb. B. Proportion of TADs (compared to KD) bound by FLAG, CTCF, both, or neither. C. Venn diagram of overlap between DBD and DBD+ TADs (compared to KD). D-H. Comparison of DBD and DBD+ unique TADs. D. Binding intensity of unique FLAG peaks (FDR < 0.05, FC > 8, counts > 80, IDR < 0.01) across the width of DBD and DBD+ unique TADs. E. Width of DBD and DBD+ unique TADs in bp. F. Expression level of significantly upregulated genes within unique TADs in DBD and DBD+ bound by FLAG. G. Length of microsatellites bound by unique FLAG peaks in DBD and DBD+ conditions in bp. H. Density of GGAA motif in the microsatellites calculated as (# of motif x 4)/(length of microsatellites) in DBD and DBD+ unique TADs bound by unique FLAG peaks. Boxplots depict the minimum, first quartile, median, third quartile, and maximum. *** P value < 0.001
DBD and DBD+ form loops at GGAA microsatellites, but DBD+ rescues more and shorter loops. A. Number of loops detected in DBD and DBD+ compared to KD at resolutions of 1kb at short-range (<50kb), mid-range (50kb-500kb), and long-range (>500kb). B. Venn diagram of overlap between DBD and DBD+ uniquely gained loops (compared to KD). C. Expression level of significant genes overlapped with uniquely gained loop anchors of DBD and DBD+. Means = 0.45 (DBD) and 0.77 (DBD+) D. Peak intensity of unique FLAG peaks (FDR < 0.05, FC > 8, counts > 80, IDR < 0.01) at anchors of uniquely gained loops in DBD and DBD+ cells. Means = 5.01 and 6.30 per DBD and DBD+ E. Length of microsatellites bound by unique FLAG peaks at the anchors of DBD and DBD+ uniquely gained loops in bp. Means = 22.5bp (DBD) and 38bp (DBD+) F. Density of GGAA motif in the microsatellites calculated as (# of motif x 4)/(length of microsatellites) at the anchors of DBD and DBD+ uniquely gained loops bound by unique FLAG peaks. Means = 0.51 (DBD) and 0.70 (DBD+). Boxplots depict the minimum, first quartile, median, third quartile, and maximum.* P value < 0.05, *** P value < 0.001
DBD+ rescues de-novo enhancer formation at microsatellites. A. PCA plot of H3K27ac peaks in biological replicates of KD, DBD and DBD+. B. Venn diagram of overlap of H3K27ac peaks (FDR < 0.05, FC > 8, counts > 80, IDR < 0.01) of DBD and DBD+. C. Percentage of H3K27ac peaks at microsatellites in common, DBD unique and DBD+ unique peaks. D. Number of H3K27ac peaks constituting typical and super enhancers called in DBD and DBD+ conditions. E. Constituent size (in bp) of typical and super enhancers in DBD and DBD+ conditions. F. Expression level of significantly upregulated genes at DBD and DBD+ super enhancers. Boxplots depict the minimum, first quartile, median, third quartile, and maximum. Circles depict outliers. *** P value < 0.001.
DBD-α4 helix of FLI promotes binding at longer and denser GGAA microsatellites. A. Venn diagram of overlap between FLAG peaks (FDR < 0.05, FC > 8, counts > 80, IDR < 0.01) of DBD and DBD+ cells. B. Percentage of FLAG peaks bound at microsatellites in common, DBD unique and DBD+ unique peaks. C. Intensity of peaks in DBD unique (mean=5.75), DBD common (mean=7.76), DBD+ common (mean=7.75) and DBD+ unique (mean=6.83) FLAG peaks. D. Length (in bp) of GGAA microsatellites bound by DBD unique (mean=36.61), common in both (mean=50.94), and DBD+ unique (mean=43.25) FLAG peaks. E.Total number of GGAA motifs in microsatellites bound by DBD unique (mean=4.66), common in both (mean=8.70), and DBD+ unique (mean=7.78) FLAG peaks. F. Maximum consecutive number of GGAA motifs in microsatellites bound by DBD unique (mean=1.48), common in both (mean=5.21), and DBD+ unique (mean=5.32) FLAG peaks G. Percent of GGAA motif in the microsatellites calculated as (# of motif x 4)/(length of microsatellites) bound by DBD unique (mean=0.54), common in both (mean=0.65), and DBD+ unique (mean=0.69) FLAG peaks. H. Maximum number of insertion (gaps in bp) in microsatellites bounds by DBD unique (mean=10.1), common (mean=9.1), and DBD+ unique (mean=8.3) FLAG peaks. Boxplots depict the minimum, first quartile, median, third quartile, and maximum. * P value < 0.05, ** P value < 0.01, and *** P value < 0.001
DBD-α4 helix promotes formation of transcription hubs by effective binding at microsatellites. A. 250kb region on chr 19 containing FCGRT and other genes. TADs are depicted on 1kb matrices (DBD/KD and DBD+/KD). Uniquely gained loops are shown as red inverted arcs. FLAG CUT&Tag bigwig tracks depicted in magenta. GGAA microsatellites in hg19. CTCF CUT&Tag track is in blue middle row. H3K27ac tracks are in green. Enhancers and super-enhancers are shown as green bars. Gene expression is in black tracks. B. FCGRT promoter region containing GGAA microsatellites. C. NOSIP promoter region containing GGAA repeats. D. Model of EWS::FLI-driven transcription hub
A. DBD-α4 helix of FLI1 depicted on dsDNA (PDB) B. Knock-down of endogenous EWS::FLI detected with FLI1 ab and rescue of wtEF, DBD, and DBD+ detected with FLAG ab. C. A PCA plot of RNA-seq experiments in A673 cells. D. Representative image of soft agar colony plates and quantification of three biological replicates.
A. Expression level of significant genes ovarlapped with unique TADs in DBD (mean=-0.64) and DBD+ (mean=-1.16) bound by FLAG at GGAA microsatellites. B. Proportion of TAD boundaries bound by FLAG, CTCF or neither. C-G. Comparison of DBD and DBD+ unique TAD boundaries. C. Binding intensity of unique FLAG peaks (FDR < 0.05, FC > 8, counts > 80, IDR < 0.01) at boundaries of DBD and DBD+ unique TADs. D. Expression level of significantly upregulated genes ovarlapped with boundaries of unique TADs in DBD and DBD+ bound by FLAG at GGAA microsatellites. E. Expression level of significantly downregulated genes ovarlapped with boundaries of unique TADs in DBD and DBD+ bound by FLAG at GGAA microsatellites. F. Length of microsatellites bound by unique FLAG peaks at the boundaries of DBD and DBD+ conditions in bp. G. Percent of GGAA motif in the microsatellites calculated as (of motif x 4)/(length of microsatellites) at the boundaries of DBD and DBD+ unique TADs bound by unique FLAG peaks. Boxplots depict the minimum, first quartile, median, third quartile, and maximum. *** P value < 0.001
A. Venn diagram of overlap between DBD and DBD+ uniquely lost loops (compared to KD). B. Expression level of downregulated genes overlapped with uniquely gained loop anchors of DBD and DBD+. Means = -0.68, -1.25, 0.35, -1.08 * P value < 0.05, *** P value < 0.001. Boxplots depict the minimum, first quartile, median, third quartile, and maximum.
A. Expression level of downregulated genes at DBD and DBD+ super enhancers. *** P value < 0.001 Boxplots depict the minimum, first quartile, median, third quartile, and maximum.
A. CCND1 hub in 700kb region on chr 11 in DBD cells. TADs are depicted on 1kb matrices (DBD/KD). Uniquely gained loops are shown as red inverted arcs. FLAG CUT&Tag bigwig tracks depicted in magenta. GGAA microsatellites in hg19. CTCF CUT&Tag track is in blue middle row. H3K27ac tracks are in green. Enhancers and super-enhancers are shown as green bars. Gene expression is in black tracks.
B. CCND1 hub in 700kb region on chr 11 in DBD+ cells. TADs are depicted on 1kb matrices (DBD+/KD). Uniquely gained loops are shown as red inverted arcs. FLAG CUT&Tag bigwig tracks depicted in magenta. GGAA microsatellites in hg19. CTCF CUT&Tag track is in blue middle row. H3K27ac tracks are in green. Enhancers and super-enhancers are shown as green bars. Gene expression is in black tracks.
