Open Access Research article

Vitamin D receptor ChIP-seq in primary CD4+ cells: relationship to serum 25-hydroxyvitamin D levels and autoimmune disease

Adam E Handel12, Geir K Sandve3, Giulio Disanto1, Antonio J Berlanga-Taylor1, Giuseppe Gallone1, Heather Hanwell1, Finn Drabløs4, Gavin Giovannoni2, George C Ebers1 and Sreeram V Ramagopalan12*

Author Affiliations

1 Medical Research Council Functional Genomics Unit and Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK

2 Blizard Institute, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK

3 Department of Informatics, University of Oslo, Gaustadalléen 23 B, N-0373, Oslo, Norway

4 Bioinformatics & Gene Regulation, Norwegian University of Science and Technology, Erling Skjalgsons gt. 1, N-7006, Trondheim, Norway

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BMC Medicine 2013, 11:163  doi:10.1186/1741-7015-11-163

Published: 12 July 2013

Additional files

Additional file 1:

Supplementary dataset. VDR binding sites in samples with high and low 25-hydroxyvitamin D. Genomic co-ordinates are shown in hg19. 25(OH)D≥75 = samples with 25-hydroxyvitamin D ≥75 nM, 25(OH)D<75 = samples with 25-hydroxyvitamin D <75 nM.

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Additional file 2: Figure S1:

Heirarchical clustering of VDR ChIP-seq peaks for individual samples. (A) Distance matrix computing distances as the inverse of overlap-enrichment pairwise similarity between samples (color scheme ranges from red for most similar to white for least similar). (B) Dendrogram incorporating the distances from the distance matrix. V1-5, VDR_1 to VDR_5; 25-hydroxyvitamin D ≥75 nM. HB, PD, SP and SR, 25-hydroxyvitamin D <75 nM.

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Additional file 3: Table S1:

Top gene ontology terms by binomial FDR Q-value. Values are derived from GREAT. 25(OH)D≥75 = subjects with vitamin D levels ≥75 nM, 25(OH)D<75 = subjects with vitamin D levels <75 nM.

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Additional file 4: Figure S2:

Top MEME-ChIP motifs for VDR ChIP-seq peaks. This figure shows the top two motifs for each set of VDR ChIP-seq peaks by E-score as established by MEME-ChIP [15]: VD ≥ 75, samples with 25-hydroxyvitamin D ≥75 nM; VD <75, samples with 25-hydroxyvitamin D <75 nM. CD4+ VDR ChIP-seq peaks overlapping LCL VDR ChIP-seq peaks and CD4+ VDR ChIP-seq peaks overlapping NB4 RXR ChIP-seq peaks.

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Additional file 5: Figure S3:

Sequence logos for VDR-like binding site motifs. The motifs were identified for each data set by searching the full ChIP-Seq regions with the Jaspar/TRANSFAC RXRA::VDR motif using MAST, followed by de novo motif discovery with MEME on the positive regions from MAST [15]. The resulting VDR-like matrix was used for another round of MAST searching on the full ChIP-Seq regions and MEME motif discovery on the positive set. The final matrices are shown for (A) LCL (434 sites used by MEME), (B) MCL (288 sites), (C) CD4+ (56 sites), and (D) Jaspar/TRANSFAC RXRA::VDR. The observation that the LCL and MCL logos are more similar to each other than to the RXRA::VDR logo, whereas the logo for CD4+ is more similar to the RXRA::VDR logo, may reflect the fact that the two first logos are based on a much larger number of sites and are, therefore, more likely to represent the true binding site motif for strong binding.

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Additional file 6: Figure S4:

Number of sequences retrieved from each data set by motif-based searches. Motif occurrences were identified using FIMO with (A) the RXRA::VDR motif, or (B) individually optimal matrices for each data set (LCL, MCL and CD4+) [20]. The number of sequences with at least one motif is plotted as a function of motif P-value. Each P-value is corrected for data set size by multiplying it with the number of tests.

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Additional file 7: Table S2:

Enrichment of genomic features within VDR binding sites. This shows the enrichment within VDR binding intervals for VDR binding intervals in lymphoblastoid cell lines (LCL VDR) and monocytic cell lines (MCL VDR), and other genomic features drawn from Cistrome and ENCODE [18,25,44]. 25(OH)D≥75 = subjects with vitamin D levels ≥75 nM, 25(OH)D<75 = subjects with vitamin D levels <75 nM, O/E = observed/expected overlap of genomic intervals, p = p-value calculated from 10,000 Monte-Carlo randomisations.

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Additional file 8: Table S3:

Enrichment of autoimmune disease susceptibility regions within VDR binding sites. Autoimmune susceptibility regions were defined as those within 100kb of SNPs associated with autoimmune disease [27]. 25(OH)D≥75 = subjects with vitamin D levels ≥75 nM, 25(OH)D<75 = subjects with vitamin D levels <75 nM, O/E = observed/expected overlap of genomic intervals, p = p-value calculated from 10,000 Monte-Carlo randomisations, p(genes) = p-value controlling for position of genes calculated from 1,000 Monte-Carlo randomisations, p(immune genes) = p-value controlling for position of immune-related genes calculated from 1,000 Monte-Carlo randomisations. P(genes) and p(immune genes) are calculated only for enrichment with uncontrolled p<0.05.

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Additional file 9: Table S5:

Enrichment of autoimmune disease associated regions with and without other transcription factors present at each VDR binding site. O/E, = observed/expected overlap of genomic intervals; P, = P-value calculated from 10,000 Monte-Carlo randomizations.

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Additional file 10: Figure S5:

Overlap with autoimmune disease association regions with variable distances around autoimmune single nucleotide polymorphisms. Enrichment is shown for different distances in base-pairs around SNPs from genome-wide association studies (GWAS) implicated in autoimmune diseases with P<10-7[27].

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Additional file 11: Table S4:

Autoimmune disease-associated single nucleotide polymorphisms located within VDR ChIP-seq peaks. RegulomeDB score: 1a eQTL + TF binding + matched TF motif + matched DNase Footprint + DNase peak; 1b eQTL + TF binding + any motif + DNase Footprint + DNase peak; 1c eQTL + TF binding + matched TF motif + DNase peak; 1d eQTL + TF binding + any motif + DNase peak; 1e eQTL + TF binding + matched TF motif; 1f eQTL + TF binding/DNase peak; 2a TF binding + matched TF motif + matched DNase Footprint + DNase peak; 2b TF binding + any motif + DNase Footprint + DNase peak; 2c TF binding + matched TF motif + DNase peak; 3a TF binding + any motif + DNase peak; 3b TF binding + matched TF motif; 4 TF binding + DNase peak; 5 TF binding or DNase peak; 6 other; 7 no functional annotation.25(OH)D≥75 = samples with 25-hydroxyvitamin D ≥75 nM, 25(OH)D<75 = samples with 25-hydroxyvitamin D <75 nM, TF = transcription factor.

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