title: Vitamin D receptor ChIP-seq in primary CD4+ cells: relationship to serum 25-hydroxyvitamin D levels and autoimmune disease.
abstract: BACKGROUND
  Vitamin 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Medical Research Council Functional Genomics Unit and Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
authors.forename: Adam E
authors.initials: AE
authors.lastname: Handel
authors.forename: Geir K
authors.initials: GK
authors.lastname: Sandve
authors.forename: Giulio
authors.initials: G
authors.lastname: Disanto
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Berlanga-Taylor
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Gallone
authors.forename: Heather
authors.initials: H
authors.lastname: Hanwell
authors.forename: Finn
authors.initials: F
authors.lastname: Drabløs
authors.forename: Gavin
authors.initials: G
authors.lastname: Giovannoni
authors.forename: George C
authors.initials: GC
authors.lastname: Ebers
authors.forename: Sreeram V
authors.initials: SV
authors.lastname: Ramagopalan
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001327:Autoimmune Diseases / Q000097:blood* / Q000235:genetics / Q000473:pathology; D001665:Binding Sites / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D023281:Genomics / Q000379:methods; D006801:Humans; D061251:Primary Cell Culture; D040081:Protein Array Analysis / Q000379:methods*; D018167:Receptors, Calcitriol / Q000097:blood* / Q000235:genetics; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D018167:Receptors, Calcitriol; D014807:Vitamin D; C104450:25-hydroxyvitamin D