Key Vitamin D Target Genes with Functions in the Immune System

Abstract

The biologically active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), modulates innate and adaptive immunity via genes regulated by the transcription factor vitamin D receptor (VDR). In order to identify the key vitamin D target genes involved in these processes, transcriptome-wide datasets were compared, which were obtained from a human monocytic cell line (THP-1) and peripheral blood mononuclear cells (PBMCs) treated in vitro by 1,25(OH)₂D₃, filtered using different approaches, as well as from PBMCs of individuals supplemented with a vitamin D₃ bolus. The led to the genes ACVRL1, CAMP, CD14, CD93, CEBPB, FN1, MAPK13, NINJ1, LILRB4, LRRC25, SEMA6B, SRGN, THBD, THEMIS2 and TREM1. Public epigenome- and transcriptome-wide data from THP-1 cells were used to characterize these genes based on the level of their VDR-driven enhancers as well as the level of the dynamics of their mRNA production. Both types of datasets allowed the categorization of the vitamin D target genes into three groups according to their role in (i) acute response to infection, (ii) infection in general and (iii) autoimmunity. In conclusion, 15 genes were identified as major mediators of the action of vitamin D in innate and adaptive immunity and their individual functions are explained based on different gene regulatory scenarios.

Keywords: PBMCs; VDR; Vitamin D; epigenome; gene regulation; immune system; monocytes; transcriptome; vitamin D target genes.

Figure A1

Vitamin D target genes controlled by super-enhancers. The IGV browser was used to visualize VDR ChIP-seq results (red) obtained in THP-1 cells that had been treated for 24 h with 1,25(OH)₂D₃ (1,25D) or solvent (EtOH) [28]. VDR-bound enhancers are shaded in grey and TSS regions of vitamin D target genes in blue. The type of VDR binding sites at enhancers is indicated as persistent (P), transient (T) or 24 h only (24) [28]. These data are compared with ChIP-seq results obtained under the same conditions for histone markers of active chromatin (H3K27ac, green) [35] and active TSS regions (H3K4me3, purple) [35] as well as with FAIRE-seq data (turquoise) [29]. The peak tracks display merged data from the three biological repeats. Gene structures are shown in blue and the vitamin D target genes NINJ1 (A), THEMIS2 (B) and SRGN (C) are highlighted in red. The genomic regions 1 Mb up- and downstream of the gene’s TSS were inspected but only the areas relevant for 1,25(OH)₂D₃-dependent regulation are displayed.

Figure A2

Vitamin D target genes controlled by ligand-dependent single enhancers. The IGV browser was used to visualize VDR ChIP-seq results (red) obtained in THP-1 cells that had been treated for 24 h with 1,25(OH)₂D₃ (1,25D) or solvent (EtOH) [28]. VDR-bound enhancers are shaded in grey and non-ligand-dependent TSS regions in blue. The type of VDR binding sites at enhancers is indicated as transient (T) or 24 h only (24) [28]. These data are compared with ChIP-seq results obtained under the same conditions for histone markers of active chromatin (H3K27ac, green) [35] and active TSS regions (H3K4me3, purple) [35] as well as with FAIRE-seq data (turquoise) [29]. The peak tracks display merged data from the three biological repeats. Gene structures are shown in blue and the vitamin D target genes FN1 (A) and LILRB4 (B) are highlighted in red. The genomic regions 1 Mb up- and downstream of the gene’s TSS were inspected but only the areas relevant for 1,25(OH)₂D₃-dependent regulation are displayed.

Figure A3

Vitamin D target genes controlled by non-ligand-dependent single enhancers. The IGV browser was used to visualize VDR ChIP-seq results (red) obtained in THP-1 cells that had been treated for 24 h with 1,25(OH)₂D₃ (1,25D) or solvent (EtOH) [28]. VDR-bound enhancers are shaded in grey, ligand-dependent TSS regions in red and non-ligand-dependent TSS regions in blue. The type of VDR binding sites at enhancers is indicated as persistent (P), transient (T) and 24 h only (24) [28]. These data are compared with ChIP-seq results obtained under the same conditions for histone markers of active chromatin (H3K27ac, green) [35] and active TSS regions (H3K4me3, purple) [35] as well as with FAIRE-seq data (turquoise) [29]. The peak tracks display merged data from the three biological repeats. Gene structures are shown in blue and the vitamin D target genes LRRC25 (A), MAPK13 (B) and SEMA6B (C) are highlighted in red. The genomic regions 1 Mb up- and downstream of the gene’s TSS were inspected but only the areas relevant for 1,25(OH)₂D₃-dependent regulation are displayed.

Figure 1

Key vitamin D target genes in immune-related cell types. A Venn diagram represents vitamin D target genes that are common in THP-1 cells stimulated in vitro in three biological repeats for 24 h with 1,25(OH)₂D₃; PBMCs of 12 individuals treated in vitro in a single repeat for 24 h with 1,25(OH)₂D_3; and PBMCs of five individuals supplemented once for 24 h with a vitamin D₃ bolus. Identification of differentially expressed genes was performed in all three datasets by DESeq2, but in vitro treated THP-1 and PBMCs were each filtered by two different approaches. Thirty-four genes were chosen for further inspection, 3 of which were found in all five datasets (center) and further 31 genes overlapped in four of the five lists. Immune-related genes are highlighted in red.

Figure 2

Vitamin D target genes regulated by super-enhancers. The IGV browser was used to visualize VDR ChIP-seq results (red) obtained in THP-1 cells that had been treated for 24 h with 1,25(OH)₂D₃ (1,25D) or solvent (EtOH) [28]. VDR-bound enhancers are shaded in grey and ligand-dependent TSS regions of vitamin D target genes in red. The type of VDR binding sites at enhancers is indicated as persistent (P), transient (T) and 24 h only (24) [28]. These data are compared with ChIP-seq results obtained under the same conditions for histone markers of active chromatin (H3K27ac, green) [35] and active TSS regions (H3K4me3, purple) [35] as well as with FAIRE-seq data (turquoise) [29]. The peak tracks display merged data from the three biological repeats. Gene structures are shown in blue and the vitamin D target genes CD14 (A) and THBD/CD93 (B) are highlighted in red. The genomic regions 1 Mb up- and downstream of the gene’s TSS were inspected but only the areas relevant for 1,25(OH)₂D₃-dependent regulation are displayed.

Figure 3

Vitamin D target genes regulated by single enhancers. The IGV browser was used to visualize VDR ChIP-seq results (red) obtained in THP-1 cells that had been treated for 24 h with 1,25(OH)₂D₃ (1,25D) or solvent (EtOH) [28]. VDR-bound enhancers are shaded in grey, ligand-dependent TSS regions in red and non-ligand-dependent TSS regions in blue. The type of VDR binding sites at enhancers is indicated as persistent (P) or transient (T) [28]. These data are compared with ChIP-seq results obtained under the same conditions for histone markers of active chromatin (H3K27ac, green) [35] and active TSS regions (H3K4me3, purple) [35] as well as with FAIRE-seq data (turquoise) [29]. The peak tracks display merged data from the three biological repeats. Gene structures are shown in blue and the vitamin D target genes CAMP (A), TREM1 (B), ACVRL1 (C) and CEBPB (D) are highlighted in red.

Figure 4

Dynamic response of gene expression to 1,25(OH)₂D₃. Based on published RNA-seq data from THP-1 cells [29], the increase in expression after 1,25(OH)₂D₃ stimulation of the 15 key genes is displayed for basal activity (solvent control) and the time points 2.5, 4 and 24 h. Genes are classified into three groups based on the steepness of the expression curves.

Figure 5

Functional profile of key immune-related vitamin D target genes. Schematic picture of a cell indicating the main location of the proteins encoded by the 15 key genes. The information is based on GeneCards (www.genecards.org) and publications cited in the text. The classification of the proteins (group 1: orange, group 2: green, group 3: blue) is based on their transcriptome profile (Figure 4). The main immune-related function of the protein groups is indicated in red.