Fig. 4

Transcriptional response to methylation gain is linked to differential transcription factor motifs. Distribution of A CpG density, B DMR width, C weighted average mCG % gain, and D number of distal regulatory sites per gene for promoter-DMR associated genes. Red depicts downregulated genes, orange depicts not differentially expressed genes (DEseq2 FDR > 0.05), and turquoise depicts upregulated genes in the noDox versus Dox comparison (Fig. 3E). In all four panels, an asterisk indicates a significant one-sided Wilcoxon rank-sum test (p < 0.01), and lack of asterisk indicates a higher p-value. E Proportion of reads at promoter-DMRs classified by per-read mCG %. Only reads spanning at least 5 CpGs were used. F ATAC-seq signal on DMRs across different samples classified by transcriptional response. ATAC-seq signal normalized using DEseq2. One asterisk indicates a p-value < 0.01 in a two-sided Wilcoxon rank-sum test between two distributions, and two asterisks indicates a p-value < 0.001. Boxplot center lines are medians, box limits are quartiles 1 (Q1) and 3 (Q3), whiskers are 1.5 × interquartile range (IQR), and points are outliers. G Transcription factor binding motif enrichments in promoter-DMRs classified by transcriptional change. Bonferroni-corrected p-values obtained from HOMER2 represented by circle size and color depicts log fold level of enrichment versus background. Colored rectangles indicate the transcriptional status of the associated transcription factor, where gray indicates that there are many TFs associated with the motif or low expression level (<50 baseMean DEseq2 normalized level). Red and blue dots indicate TFs shown to be repelled or attracted by 5mC respectively. Highlighted in bold are SOX2 (original target of the ZF) and the ZF-D3A motif. H Highest scoring motifs associated with the TF footprints depleted upon Dox activation at promoter-DMRs, derived from ATAC-seq data. CpGs on the core-binding motifs are highlighted with black lines, and red circles indicate those previously reported to be repelled by methylation