Figure 4. KLF5 binds to distinct regions in OE19 cells.

(A) Expression Log₂(1+FPKM) of KLF5 in BO and OAC tissue. (B) Heatmap of KLF5 ChIP-seq signal at regions (peak centre ±5 kb) significantly bound in OE19 only (+2x; Q < 0.05), shared regions (no significant change) and regions bound in CP-A only (−2x; Q-value <0.05). (C) Tag density plot of KLF5 ChIP-seq signal at regions (peak centre ±2.5 kb) bound in OE19 only, shared regions and regions bound in CP-A only. (D) Genome browser tracks showing KLF5 ChIP-seq and ATAC-seq in CP-A and OE19 cells at the CCNE1 locus. Differential bound regions are highlighted in red. (E) Bar chart of percentage targets and percentage background of de novo discovered motifs at regions bound in OE19 only, shared regions and regions bound in CP-A only. De novo motifs, called transcription factor with match scores and P-values shown. (F) UPSET plot of DNA motifs found in 371 KLF5 binding regions that are specific to OE19-specific binding regions that are located within loci (+/- 250 kb) containing genes upregulated in OAC and downregulated with KLF5 depletion. The motifs identified in E (KLF5, GATA1, FOXA2, FRA1 and TCF7L2) found within each peak are shown. (G) Venn diagram showing the overlap in genes downregulated in OE19 cells following treatment with siRNAs targeting KLF5 and GATA6. (H) Heatmap of ATAC-seq signal at the KLF5 binding regions from (F) in the indicated cell lines (left) or patient derived tissue (right). Regions were subject to k-means hierarchical clustering (k = 2). See also Figure 4—figure supplements 1 and 2.

Figure 4—figure supplement 1. KLF5 ChIP-seq analysis in CP-A cells.

(A) Immunoblot of CP-A and OE19 protein lysate probed with antibodies against KLF5 and ERK1/2. (B) Immunoblot of KLF5 from CP-A cells following ChIP in different fractions; IgG FT (flow through from non-specific IgG precipitation), KLF5 FT (flow through from KLF5 precipitation), IgG elute (material eluted following IgG precipitation) KLF5 IP (material eluted following KLF5 immunoprecipitation). Arrows indicating immunoprecipitated protein (KLF5) and asterisks are IgG heavy chain. (C) Scatter plots of KLF5 ChIP-seq data from CP-A from two biological replicates. Pearson correlation coefficient shown. (D) Venn diagrams showing overlap of peaks called by each replicate for KLF5 ChIP-seq from CP-A cells. The numbers of peaks in each sector are given. (E) Bar chart of percentage targets and percentage background of de novo discovered motifs at CP-A KLF5 ChIP-seq peaks. De novo motif, called transcription factor with motif match score (in brackets) and p-value shown. (F) Example UCSC genome browser tracks of KLF5 ChIP-seq and ATAC-seq data from CP-A and OE19 cells at the NKX3-1 (left) and JAG1 loci (right). CP-A-specific peaks are highlighted in blue and shared peaks are highlighted in grey.

Figure 4—figure supplement 2. Integrative analysis of KLF5 ChIP-seq data in OE19 and CP-A cells.

(A) Tag density plots of normalised ATAC-seq signal at OE19 specific KLF5 ChIP-seq regions from CP-A (blue) and OE19 (red) cells (top) and merged BO (blue) and merged OAC (red) tissue (bottom). (B) Top GO terms of nearest genes associated with OE19-specific (top) or CP-A-specific (bottom) KLF5 peaks which show either increased (top) or decreased (bottom) expression in OAC. (C) Euler diagram of 299 KLF5-binding regions that are specific to OE19-specific binding regions that are located within loci (+/- 250 kb) containing genes upregulated in OAC and downregulated with KLF5 depletion. The motifs identified in Figure 4E (KLF5, GATA1, FOXA2, FRA1 and TCF7L2) found within each peak are shown (note that an additional 71 regions cannot be depicted due to the small numbers involved). The regions in the white circle contain none of the indicated motifs. (D) Heatmap of KLF5, GATA6 and HNF4A ChIP-seq signal from OE19 cells at OE19 specific KLF5 ChIP-seq peaks nearby KLF5-activated target genes. (E) RT-qPCR analysis of the indicated genes following treatment of CP-A cells with non-targeting (NT) or KLF5 siRNAs for 72 hr (n = 3; P-values **=<0.01; ns = not significant). (F) Tag density plots of normalised ATAC-signal at cluster 1 (left) and cluster 2 (right) regions (see Figure 4G) from CP-A (blue) and OE19 (red) cells (top) and merged BO (blue) and merged OAC (top) samples (bottom). (G) Bar chart of percentage targets and percentage background of de novo discovered motifs in cluster 1 (top) and cluster 2 (bottom) regions. De novo motif, called transcription factor with motif match score and p-value are shown.