Figure 1.
Chromatin loops containing VDR binding sites.
The IGV browser was used to display the chromatin loops around the genes ZFP36 (A), SFT2D1 (B), SEPT3 (C) and SP100 (D). VDR ChIP-seq data from THP-1 cells [7] (unstimulated (−) and treated for 40 min with 1,25(OH)2D3 (+), red) are shown in comparison with CTCF ChIA-PET data from K562 and MCF-7 cells [33] in the looping view (grey horizontal lines). Horizontal red lines indicate the core chromatin loops (as indicated in Table S3). The area of the genomic regions was adapted to the size of the chromatin loops. Gene structures are shown in blue, and VDR peak regions are shaded in grey.
Figure 2.
VDR association and 1,25(OH)2D3-dependent chromatin opening in four chromatin domains.
The IGV browser was used to visualize the loci of the genomic VDR binding regions (+/−15 kb of the peak summit) of the genes ZFP36 (A), SFT2D1 (B), SEPT3 (C) and SP100 (D). The peak tracks display data from a VDR ChIP-seq experiment in THP-1 cells (red, from unstimulated cells and after 40 min 1,25(OH)2D3 (1,25D) treatment [7]) and a time course of FAIRE-seq data from THP-1 cells (grey for EtOH-treated controls and turquoise for 1,25(OH)2D3 treatments for the indicated time periods [35]). Gene structures are shown in blue, and VDR peak regions are shaded in grey. In the bottom lines, the sequences of DR3-type binding sites are indicated.
Figure 3.
ChIP-qPCR was performed to determine VDR association (blue) and unspecific IgG binding (grey) at P3ZFP36 (A), P3SFT2D1 (B), PSEPT3 (C) and PSP100 (D). THP-1 cells were stimulated for 1, 2, 3, 4, 5 and 24 h with 100 nM 1,25(OH)2D3 and chromatin was extracted. The data points represent the means of at least three independent experiments and the bars indicate standard deviations. Unspecific background binding observed to a negative control region of the MB gene (see Fig. S5E) was subtracted. Two-tailed Student’s t-tests were performed to determine the significance of VDR association in reference to IgG background (*p<0.05; **p<0.01; ***p<0.001).
Figure 4.
Detailed time course expression profiling of primary VDR target genes.
qPCR was performed to determine the relative changes of mRNA expression of the genes ZFP36 (A), SFT2D1 (B), SEPT3 (C) and SP100 (D) normalized by the three reference genes B2M, GAPDH and HPRT1. THP-1 cells were incubated with 100 nM 1,25(OH)2D3 for either 2, 4, 6 and 24 h (left) or at 15 min intervals over a time period of 180 min (right). The columns (left) or data points (right) represent the means of three independent experiments (each performed in triplicate) and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the mRNA induction by 1,25(OH)2D3 in reference to solvent-treated cells (*p<0.05; **p<0.01; ***p<0.001).