Abstract
BackgroundVitamin D insufficiency has been implicated in autoimmunity. ChIP-seq experiments using immune cell lines have shown that vitamin D receptor (VDR) binding sites are enriched near regions of the genome associated with autoimmune diseases. We aimed to investigate VDR binding in primary CD4+ cells from healthy volunteers.MethodsWe extracted CD4+ cells from nine healthy volunteers. Each sample underwent VDR ChIP-seq. Our results were analyzed in relation to published ChIP-seq and RNA-seq data in the Genomic HyperBrowser. We used MEMEChIP for de novo motif discovery. 25-Hydroxyvitamin D levels were measured using liquid chromatography–tandem mass spectrometry and samples were divided into vitamin D sufficient (25(OH)D ≥75 nmol/L) and insufficient/deficient (25(OH)D <75 nmol/L) groups.ResultsWe found that the amount of VDR binding is correlated with the serum level of 25-hydroxyvitamin D (r = 0.92, P= 0.0005). In vivo VDR binding sites are enriched for autoimmune disease associated loci, especially when 25-hydroxyvitamin D levels (25(OH)D) were sufficient (25(OH)D ≥75: 3.13-fold, P<0.0001; 25(OH)D <75: 2.76-fold, P<0.0001; 25(OH)D ≥75 enrichment versus 25(OH)D <75 enrichment: P= 0.0002). VDR binding was also enriched near genes associated specifically with T-regulatory and T-helper cells in the 25(OH)D ≥75 group. MEME ChIP did not identify any VDR-like motifs underlying our VDR ChIP-seq peaks.ConclusionOur results show a direct correlation between in vivo 25-hydroxyvitamin D levels and the number of VDR binding sites, although our sample size is relatively small. Our study further implicates VDR binding as important in gene-environment interactions underlying the development of autoimmunity and provides a biological rationale for 25-hydroxyvitamin D sufficiency being based at 75 nmol/L. Our results also suggest that VDR binding in response to physiological levels of vitamin D occurs predominantly in a VDR motif-independent manner.
Highlights
Vitamin D insufficiency has been implicated in autoimmunity
vitamin D receptor (VDR) binding within 5 kb downstream of genes (r = 0.82, P= 0.007) and within introns (r = 0.79, P= 0.01) was correlated with vitamin D levels, whereas VDR binding in areas with 5 kb upstream (r = -0.14, P= 0.72) or both upstream and downstream (r = 0.44, P= 0.24) of genes, within exons (r = -0.21, P= 0.57), Untranslated region (UTR) (r = -0.05, P= 0.89) or intergenic regions (r = -0.40, P= 0.28) was not
VDR binding motifs We found that there was no significant enrichment of binding sites containing DR3-like motifs either when searching de novo using MEME-ChIP [20], CentriMo [38], Weeder [21] or ChIPmunk [22] and analyzing all binding sites, binding sites grouped by high or low vitamin D, binding sites overlapping with the previous lymphoblastic cell line (LCL) or monocytic cell line (MCL) VDR ChIP-seq studies, binding sites common between multiple samples or binding sites overlapping with previous ChIP-seq studies of retinoid X receptors (RXR) in NB4 cells [23]
Summary
ChIP-seq experiments using immune cell lines have shown that vitamin D receptor (VDR) binding sites are enriched near regions of the genome associated with autoimmune diseases. Two studies have analyzed genome-wide binding of VDR using chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq); one using a B-lymphoblastic cell line (LCL) and another using a monocytic cell line (MCL) [9,10]. Each study determined that the VDR-RXR dimer recognizes a classical motif (DR3) but that this is present only at some of the VDR binding sites detected by ChIP-seq. The LCL ChIP-seq used genetic susceptibility loci drawn from genome-wide association studies to demonstrate significant overlap between autoimmune susceptibility regions and VDR binding sites [9]
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