Figure 4.

Ksp^CreDnmt3a/3b DMRs are enriched for developmental transcription factor binding. (A) Transcription factor motif enrichment (HOMER) of DMRs, during kidney development (P0–P21), in control versus Ksp^CreDnmt3a/3b kidneys. Fragments that gained methylation during development but failed to gain methylation in Dnmt3a/3b knockout mice. (B) The degree of overlap between DMRs that are in enhancer regions and on Six2 binding sites in nephron progenitors (NPC) from ChIP-seq (cyan) compared with background (gray). (C) Methylation differences in control versus Ksp^CreDnmt3a/3b kidneys (left) or adult versus fetal kidneys (right) that overlap with Six2 binding versus background shuffled region. (D) Scatter plot of methylation changes of Six2 binding sites in NPCs, during kidney development (x axis) and in control versus Ksp^CreDnmt3a/3b (y axis). The red dots represent the fragments gaining methylation in adult kidneys, but not in Ksp^CreDnmt3a/3b mice. (E) IGV genome browser view of the Pax2 region (chromosome 19: 44729363–44851852). DNA methylation changes in hypo-DMRs (lower methylation in Ksp^CreDnmt3a/3b), note the methylation pattern in the developing mouse kidney (E15.5, P0), wild-type and Dnmt3a/3b knockout (DKO) mice, H3K27ac enhancer mark, and Six2 binding. Note the failure to increase in methylation of a fetal-enhancer region. (F) Global CpG methylation in NPC Six2 peaks and shuffled regions at different stages of kidney development. (G) Correlation between gene expression of Six2 and CpG methylation of segments overlapped with NPC Six2 peaks and shuffled regions. Wilcoxon signed rank sum test was carried out to calculate the significance of difference between NPC Six2 peaks and shuffled regions, and P values was provided for each comparison. ESC, embryonic stem cell.