A global understanding of the actions of the nuclear hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and its vitamin D receptor (VDR) requires a genome-wide analysis of VDR binding sites. In THP-1 human monocytic leukemia cells we identified by ChIP-seq 2340 VDR binding locations, of which 1171 and 520 occurred uniquely with and without 1α,25(OH)2D3 treatment, respectively, while 649 were common. De novo identified direct repeat spaced by 3 nucleotides (DR3)-type response elements (REs) were strongly associated with the ligand-responsiveness of VDR occupation. Only 20% of the VDR peaks diminishing most after ligand treatment have a DR3-type RE, in contrast to 90% for the most growing peaks. Ligand treatment revealed 638 1α,25(OH)2D3 target genes enriched in gene ontology categories associated with immunity and signaling. From the 408 upregulated genes, 72% showed VDR binding within 400 kb of their transcription start sites (TSSs), while this applied only for 43% of the 230 downregulated genes. The VDR loci showed considerable variation in gene regulatory scenarios ranging from a single VDR location near the target gene TSS to very complex clusters of multiple VDR locations and target genes. In conclusion, ligand binding shifts the locations of VDR occupation to DR3-type REs that surround its target genes and occur in a large variety of regulatory constellations.

The physiological functions of vitamin D are mediated by its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) activating the transcription factor vitamin D receptor (VDR). In THP-1 human monocytes we demonstrated epigenome-wide effects of 1,25(OH)2D3 at 8979 loci with significantly modulated chromatin accessibility. Maximal chromatin opening was observed after 24 h, while after 48 h most sites closed again. The chromatin-organizing protein CTCF bound to 14% of the 1,25(OH)2D3-sensitive chromatin regions. Interestingly, 1,25(OH)2D3 affected the chromatin association of CTCF providing an additional mechanism for the epigenome-wide effects of the VDR ligand. The 1,25(OH)2D3-modulated transcriptome of THP-1 cells comprised 1284 genes, 77.5% of which responded only 24 h after stimulation. During the 1,25(OH)2D3 stimulation time course the proportion of down-regulated genes increased from 0% to 44.9% and the top-ranking physiological function of the respective genes shifted from anti-microbial response to connective tissue disorders. The integration of epigenomic and transcriptomic data identified 165 physiologically important 1,25(OH)2D3 target genes, including HTT and NOD2, whose expression can be predicted primarily from epigenomic data of their genomic loci. Taken together, a large number of 1,25(OH)2D3-triggered epigenome-wide events precede and accompany the transcriptional activation of target genes of the nuclear hormone.

Vitamin D deficiency has been associated with an increased risk of developing a number of diseases. Here we investigated samples from 71 pre-diabetic individuals of the VitDmet study, a 5-month high dose vitamin D3 intervention trial during Finnish winter, for their changes in serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations and the expression of primary vitamin D target genes in peripheral blood mononuclear cells and adipose tissue. A negative correlation between serum concentrations of parathyroid hormone and 25(OH)D3 suggested an overall normal physiological vitamin D response among the participants. The genes CD14 and thrombomodulin (THBD) are up-regulated primary vitamin D targets and showed to be suitable gene expression markers for vitamin D signaling in both primary tissues. However, in a ranking of the samples concerning their expected response to vitamin D only the top half showed a positive correlation between the changes of CD14 or THBD mRNA and serum 25(OH)D3 concentrations. Interestingly, this categorization allows unmasking a negative correlation between changes in serum concentrations of 25(OH)D3 and the inflammation marker interleukin 6. We propose the genes CD14 and THBD as transcriptomic biomarkers, from which the effects of a vitamin D3 supplementation can be evaluated. These biomarkers allow the classification of subjects into those, who might benefit from a vitamin D3 supplementation, and others who do not.

The transcription factor vitamin D receptor (VDR) is the exclusive nuclear target of the biologically active form of vitamin D (1,25(OH)D). In THP-1 human monocytes we obtained a highly accurate VDR cistrome after 2 and 24h ligand stimulation comprising >11,600 genomic loci, 78% of which were detected exclusively after 24h. In contrast, a group of 510 persistent VDR sites occurred at all conditions and some 2100 VDR loci were only transiently occupied. Machine learning and statistical analysis as well as a comparison with the re-analyzed B cell VDR cistrome indicated a subgroup of 339 highly conserved persistent VDR sites that were suited best for describing vitamin D-triggered gene regulatory scenarios. The 1,25(OH)D-dependent transcriptome of THP-1 cells comprised 587 genes, 311 of which were primary targets with main functions in the immune system. More than 97% of the latter genes were located within 1,25(OH)D-modulated topologically associated domains (TADs). The number of persistent and transient VDR sites was found to be the main discriminator for sorting these TADs into five classes carrying vitamin D target genes involved in distinct biological processes. In conclusion, specific regulation of biological processes by vitamin D depends on differences in time-dependent VDR binding.