Figure 4.

Analysis of primary VDR target transcription factors and their secondary vitamin D responses. The DYNAMITE analysis pipeline was used to identify primary VDR target transcription factors, which regulate secondary vitamin D target genes. Normalized absolute classifier coefficients from DYNAMITE are shown for primary VDR target transcription factors inside the top 150. On top of each bar the rank of each transcription factor in the entire DYNAMITE analysis is shown (a). The total numbers of DEGs after 24 h of 1,25(OH)₂D₃ treatment predicted to be targeted by each of the early and late induced primary 1,25(OH)₂D₃ target transcription factors comprised more than 2,000, including ETS1 or Kruppel like factor 4 (KLF4) (b). The ratio of early and late 1,25(OH)₂D₃-regulated genes to which CEBPA or ETS1 bind within + /- 25 kb of the TSS has been determined by ChIP-seq analyses (c). The predicted binding of ETS1 and CEBPA¹³ to the gene loci of the secondary VDR targets COX7A2L, MS4A7 and MS4A14 was confirmed by ChIP-seq assays, while no significant VDR association could be seen. The binding mostly occurred in open chromatin¹⁵ and co-located with the active enhancer mark H3K27ac¹² (d, e). The gene names of all vitamin D target genes are shown in bold.