A hierarchical regulatory network analysis of the vitamin D induced transcriptome reveals novel regulators and complete VDR dependency in monocytes

The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D-3 (1,25(OH)(2)D-3). In order to identify pure genomic transcriptional effects of 1,25(OH)(2)D-3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)(2)D-3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)(2)D-3-induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)(2)D-3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)(2)D-3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.