The DBD-α4 helix of FLI1 domain is required to restructure chromatin in A-673 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. Boxplots depict the minimum, first quartile, median, third quartile, and maximum.* P value < 0.05, *** P value < 0.001

Altered TAD structure in A-673 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 in A-673 cells.

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-673 cells.

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 FLI1 promotes binding at longer and denser GGAA microsatellites in A-673 cells.

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

The 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 EWSR1::FLI1-driven transcription hub

Supplementary Table 1. Differential expression of FCGRT hub genes in DBD and DBD+ compared to KD.

A. The DBD-α4 helix of FLI1 depicted on dsDNA (PDB) B. Knock-down of endogenous EWSR1::FLI1 detected with FLI1 ab and rescue of wtEF, DBD, and DBD+ detected with FLAG ab. C. A PCA plot of RNA-seq experiments in A-673 cells. D. Representative image of soft agar colony plates and quantification of three biological replicates.

A. Genome-wide interaction frequency (ICE-corrected Micro-C counts) over genomic distance (bp) at 5kb resolution for Replicate 1 of A-673. B. Genome-wide interaction frequency (ICE-corrected Micro-C counts) over genomic distance (bp) at 5kb resolution for Replicate 2 of A-673.

A. Expression level of significant genes overlapped 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 A-673 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 A-673 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.

A. NKX2-2 hub on chr 20 in A-673 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. NKX2-2 hub on chr 20 in A-673 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.

A. GSTM4 hub chr 1 in A-673 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. GSTM4 hub chr 1 in A-673 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.

A. Knock-down of endogenous EWSR1::ERG detected with ERG ab and rescue of wtEF, DBD and DBD+ detected with FLAG ab. B. A PCA plot of RNA-seq experiments in TTC-466 cells. C. Representative images of soft agar colony plates.

DBD-a4 helix of FLI1 domain is required to restructure chromatin in TTC-466 cell. A. Multidimensional scaling (MDS) plot of top 1000 interactions (500kb resolution) in each biological replicates. B. Genome-wide interaction frequency of combined replciates (ICE-corrected Micro-C counts) over genomic distance (bp) at 5kb resolution. C. Volcano plot showing differentially interacting regions (DIRs) detected at 500kb resolution for DBD+ replicates versus KD replicates. D. Volcano plot showing DIRs detected at same resolution for DBD replicates versus KD replicates.

TAD analysis in TTC-466 cells. A. Number of TADs detected in DBD and DBD+ compared to KD at resolutions of 10kb, 25kb, 50kb and 100kb. B. Venn diagram of overlap between DBD and DBD+ TADs (compared to KD).

Loop analysis in TTC-466 cells. 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. Venn diagram of overlap between DBD and DBD+ uniquely lost loops (compared to KD).

H3K27ac and FLAG CUT&Tag analysis in TTC-466 cells. A. PCA plot of H3K27ac peaks in biological replicates of KD, DBD and DBD+. B. Venn diagram of overlap between FLAG peaks (FDR < 0.05, FC > 8, counts > 80, IDR < 0.01) of DBD and DBD+ cells. C. Percentage of FLAG peaks bound at microsatellites in common, DBD unique and DBD+ unique peaks. D. Length (in bp) of GGAA microsatellites bound by DBD unique (mean=30.28), common in both (mean=37.79), and DBD+ unique (mean=22.74) FLAG peaks. E.Total number of GGAA motifs in microsatellites bound by DBD unique (mean=3.70), common in both (mean=6.05), and DBD+ unique (mean=3.00) FLAG peaks. F. Maximum consecutive number of GGAA motifs in microsatellites bound by DBD unique (mean=1.35), common in both (mean=3.44), and DBD+ unique (mean=1.57v) FLAG peaks G. Percent of GGAA motif in the microsatellites calculated as (# of motif x 4)/(length of microsatellites) bound by DBD unique (mean=0.53), common in both (mean=0.60), and DBD+ unique (mean=0.54) FLAG peaks. H. Maximum number of insertion (gaps in bp) in microsatellites bounds by DBD unique (mean=10.0), common (mean=9.36), and DBD+ unique (mean=9.77) FLAG peaks. * P value < 0.05, ** P value < 0.01, and *** P value < 0.001.

A. FCGRT hub in 250kb region on chr 19 in TTC-466 DBD cells. TADs are depicted on 1kb matrices (DBD/KD). 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. Gene expression is in black tracks.

B. FCGRT hub in 250kb region on chr 19 in TTC-466 DBD+ cells. TADs are depicted on 1kb matrices (DBD+/KD). 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. Gene expression is in black tracks.

A. CCND1 hub in 700kb region on chr 11 in TTC-466 DBD cells. TADs are depicted on 1kb matrices (DBD/KD). 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. Gene expression is in black tracks.

B. CCND1 hub in 700kb region on chr 11 in TTC-466 DBD+ cells. TADs are depicted on 1kb matrices (DBD+/KD). 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. Gene expression is in black tracks.

A. NKX2-2 hub chr 20 in TTC-466 DBD cells. TADs are depicted on 1kb matrices (DBD/KD). 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. Gene expression is in black tracks.

B. NKX2-2 hub on chr 20 in TTC-466 DBD+ cells. TADs are depicted on 1kb matrices (DBD+/KD). 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. Gene expression is in black tracks.

A. GSTM4 hub chr 1in TTC-466 DBD cells. TADs are depicted on 1kb matrices (DBD/KD). 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. Gene expression is in black tracks.

B. GSTM4 hub on chr 1 in TTC-466 DBD+ cells. TADs are depicted on 1kb matrices (DBD+/KD). 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. Gene expression is in black tracks.

A-673 and TTC-466 cell line comparison. A. PCA plot of RNA-Seq replicates of A-673 and TTC-466 cells. B. Sequencing depth of Micro-C replicates of A-673 cells. C. Sequencing depth of Micro-C replicates of TTC-466 cells.