Figure 4.

VDR peak height, location at <400 kb from the TSS, and the presence of de novo DR3-type REs predicts strong upregulation of 1α,25(OH)₂D₃ target genes. (A) Common and treatment-unique VDR binding sites in the neighborhoods of up- (upper panel) and downregulated genes (lower panel). (B) The diagram on top describes the distance categories used for selecting the most likely regulating VDR binding site as follows (distances not to scale). For each differentially expressed gene, the most likely regulating FDR <1% VDR peak was assumed to be the largest peak (by FE) that was within ±400 kb of the gene TSS. The most likely regulators were further divided into proximal and distal at ±30 kb to identify distance-related differences. If no peak was within ±400 kb, then the closest one of any FE was taken to represent a potential regulating site. See Supplementary Figure S3 for the basis of the selected distance limits. The bar graph below shows that the most likely gene regulatory VDR binding sites for up but not downregulated genes are enriched to within 400 kb of the target gene TSS and preferentially use DR3-type REs. Here, up- and downregulated genes were separately split by fold change into tertiles, indicated by increasing intensity of red in the triangle below the x-axis. Peaks were split into two equal sized halves by FE (left and right panels). Gene counts are plotted for up- (upper panel) and downregulated genes (lower panel). The gene counts are further split according to the presence or absence of a de novo DR3-type RE near the summit of the most likely regulating VDR peak, as indicated in the legend. The likely regulatory scenarios are indicated by the circled numbers 1–12, and used as peak labels in Figure 5 and Supplementary Figure S4. Unstim, unstimulated; Prox, proximal; Dist, distal.