Fig 2: Knockdown of FRA1 and FLI1 downregulates the expression of genes involved in epithelial cell migration.a Barplots show the overlap rates of MIBC and NMIBC enhancers with FRA1 and FLI1 peaks. b Regulatory network denoting the transcription factors, FRA1 and FLI1 and their target genes involved in epithelium migration, cell junction organization and regulation of epithelial cell proliferation. For simplicity similar terms were left out of the visualized image (please see Supplementary Data 3, ‘epithelium migration’ and ‘cell junction organization’, and ‘regulation of epithelial cell proliferation’ related terms are highlighted in yellow). c, d Plots showing the downregulation of FRA1 (c) and FLI1 (d) at mRNA and protein level. Top panels display the relative mRNA levels, bottom panels show the western blot images. e, f Barplots display the relative mRNA levels of FLOT1 (e) and MAP4K4 (f) (top panels). Snapshots showing H3K27ac, FRA1 and FLI1 signals at the MIBC enhancers regulating FLOT1 and MAP4K4 genes (bottom panels). Scale of the snapshots are adjusted to 25. Error bars show the standard deviation of two biological replicates. Each biological replicate is analyzed in three technical replicates.* p-value < 0.05, **p-value < 0.01.

Fig 3: DNA methylation analysis of FPDMM-mimicking HPCs. (a) Overlaps of hypermethylated (left) and hypomethylated (right) CpGs between RUNX1WT/R201Q (blue) and RUNX1WT/Y287X (red) HPCs. (b) (Left) The known HOCOMOCO v11 binding motif for GABPA (top), FEV (middle), and ELF1 (bottom). (Middle and right) Distribution of ETS family TF-binding motif-enrichment. The X- and Y-axes show the distance from DMC (bp) and probability of TF-binding motifs, respectively. Solid lines are probabilities at ± 5 kb for hypermethylated DMCs in RUNX1WT/R201Q (middle) and RUNX1WT/Y287X (right) HPCs, and dashed lines are probabilities at ± 5 kb from randomly selected CpGs. Data that are significantly enriched (E-value of the Fisher’s exact test < 0.05) and ranked in the top-20 of Fisher E-value rankings are presented. Blue: GABPA-binding motif, red: FEV-binding motif, and pink: ELF1-binding motif. (c) Expression of ELF1 (left) and FLI1 (right) in RUNX1WT/R201Q (blue) and RUNX1WT/Y287X (red) HPCs compared to wild-type cells, confirmed using qRT-PCR. The X-axis shows the target genes, and the Y-axis represents the fold-change. Data are presented as the mean ± SD of four biological replicates. Asterisks denote significant difference: *P < 0.05 and **P < 0.01. (d) Distribution of enrichment for FLI1-binding sites, as determined by CUT&RUN sequencing for FLI1 in wild-type HPCs, showing the regions within ± 5 kb of the hypermethylated DMCs in RUNX1WT/Y287X HPCs (red line). Gray line represents FLI1-binding site-enrichment at regions around randomly selected CpGs.

Fig 4: Rescue of deficient megakaryocytic differentiation in FPDMM-mimicking HPCs by FLI1 overexpression. (a) Schematic representation of the megakaryocytic differentiation method. FPDMM-mimicking iPSCs were transfected with a lentiviral vector containing the DOX-inducible FLI1 expression system. (b) Confirmation of the expression of FLI1 in Y287X-mock (red) and Y287X-FLI1 (orange) HPCs using qRT-PCR. Data are presented as the mean ± SD of six biological replicates. The asterisk denotes significant difference: *P < 0.05. (c) Percentages of CD41+CD42b+ Mks per 20,000 wild-type (WT, gray), Y287X-mock (red), and Y287X-FLI1 (orange) HPCs. Data are presented as the mean ± SD of six biological replicates. The asterisk denotes significant difference: **P < 0.01 and ns, not significant.

Fig 5: Transcription factors enriched at MIBC and NMIBC enhancers.a, c Top transcription factor motifs enriched at MIBC (a) and NMIBC (c) enhancers. b, d Boxplots showing the expression of FRA1 and FLI1 across the NMIBC (n = 2) and MIBC cell lines (n = 4) (b) and the expression of TP63, GRHL2, and PPARG (d). *p-value < 0.05.