9 research outputs found
Reporter gene analysis of DRB1 promoter VDRE.
<p>Raji B cells were transiently transfected with pGL3 luciferase constructs as indicated together with pRL_TK to normalise luciferase activity. Open bars indicate resting cells, grey shaded bars results following stimulation of transfected cells with 1,25-dihydroxyvitamin D3. Mean+/−SD of three independent transient transfection experiments are shown, each performed in quadruplicate.</p
VDR is recruited to <i>HLA-DRB1*15</i> VDRE in PGF cells.
<p>Chromatin immunoprecipitation experiment using PGF cells either unstimulated (○) or after stimulation with 1,25-dihydroxyvitamin D3 (•). Input controls are shown (lanes 1 and 2), mock antibody immunoprecipitated controls (lanes 3 and 4) and VDR primary antibody immunoprecipitated DNA (lanes 5 and 6).</p
HLA-DRB1 promoter.
<p>Sequence shown is that for <i>HLA-DRB1*15</i>. Important regulatory elements (S, X and Y Boxes) are highlighted.</p
In vitro binding of VDR protein to the <i>HLA-DRB1*15</i> VDRE.
<p>Electrophoretic mobility shift assay showing binding of recombinant VDR and retinoic acid receptor beta (RXR) to radiolabelled oligoduplex probe corresponding to the VDRE in the proximal <i>HLA-DRB1</i> promoter region for the <i>HLA-DRB*15</i> haplotype. Two specific complexes are indicated, denoted I and II, together with a supershifted complex shown by an * symbol in the presence of antibody to VDR.</p
T<i>rans</i>-associations in CD4<sup>+</sup> and CD8<sup>+</sup> T cells.
<p>(<b>A</b>) The outermost rim of the circos plot shows the histogram of the –log<sub>10</sub>(<i>P</i>-value) of the associations between known genes and SNPs. Per every significant known gene only the highest –log<sub>10</sub>(<i>P</i>-value) is depicted. The innermost network represents the <i>trans</i>-associations between the SNPs and the most significant gene expression probes per gene. The lines are colored by the chromosome of the given SNP, except for the locus on chr12q13.2 with over 100 <i>trans</i>-associations that are colored in grey. An arbitrary selection of genes is depicted. All genes not affected by chr12q13.2 SNPs are colored in black and a set of genes affected by chr12q13.2 <i>trans</i>-acting regulatory SNPs are selected based on their known importance in immune system related processes and are colored in grey. The underlined genes are involved in T1D and mTOR signaling in CD4<sup>+</sup> T cells and CD8<sup>+</sup> T cells, respectively. Two boxed genes among the genes with <i>trans</i>-eQTLs in CD4<sup>+</sup> T cells are associated with the T1D susceptibility variant rs4788084 close to the <i>IL27</i> gene. (<b>B</b>) Heatmap of the correlation coefficients between chr12q13.2 <i>trans</i>-acting regulatory SNP allele dosages and gene expression levels in CD4<sup>+</sup> and CD8<sup>+</sup> T cells are shown. The most significant gene expression probe per gene is chosen. An arbitrary selection of genes based on their importance in immune system related processes are marked with black borders and gene symbol. The depicted SNPs are linked with their role in disease susceptibility based on GWAS studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006643#pgen.1006643.ref030" target="_blank">30</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006643#pgen.1006643.ref033" target="_blank">33</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006643#pgen.1006643.ref040" target="_blank">40</a>] and the numbers indicated in the lower panel refer to the same SNPs listed in the top panel.</p
Validation of neutrophil and lymphoid specific <i>cis</i>-eQTLs in purified cell type eQTL datasets.
<p>A) We validated the neutrophil- and lymphoid-mediated <i>cis</i>-eQTL effects in four purified cell type datasets from the lymphoid lineage (B-cells, CD4+ T-cells, CD8+ T-cells and lymphoblastoid cell lines) and in two datasets from the myeloid lineage (monocytes and neutrophils). Compared to generic <i>cis</i>-eQTLs, large effect sizes were observed for neutrophil-mediated <i>cis</i>-eQTLs in myeloid lineage cell types, and small effect sizes in the lymphoid datasets. Conversely, lymphoid-mediated <i>cis</i>-eQTL effects had large effect sizes specifically in the lymphoid lineage datasets, while having smaller effect sizes in myeloid lineage datasets. These results indicate that our method is able to reliably predict whether a specific <i>cis</i>-eQTL is mediated by cell type. B) Comparison between average gene expression levels between different purified cell type eQTL datasets shows that neutrophil mediated <i>cis</i>-eQTLs have, on average a lower expression in cell types derived from the lymphoid lineage, and a high expression in myeloid cell types, while the opposite is true for lymphocyte mediated <i>cis</i>-eQTLs.</p
Method overview.
<p>I) Starting with a dataset that has cell count measurements, determine a set of probes that have a strong positive correlation to the cell count measurements. Calculate the correlation between these specific probes in the other datasets, and apply principal component analysis to combine them into a single proxy for the cell count measurement. II) Apply the prediction to other datasets lacking cell count measurements. III) Use the proxy as a covariate in a linear model with an interaction term in order to distinguish cell-type-mediated from non-cell-type-mediated eQTL effects.</p
Validation of neutrophil proxy.
<p>There is a strong correlation between the neutrophil proxy and the actual neutrophil percentage measurements in the training dataset (EGCUT, Spearman R = 0.75). Validation of neutrophil prediction in the SHIP-TREND cohort shows a strong correlation (Spearman R = 0.81) between the neutrophil proxy and actual neutrophil percentage measurements in this dataset.</p
Effect of sample size on power to detect cell type specific <i>cis</i>-eQTLs.
<p>We systematically excluded datasets from our meta-analysis in order to determine the effect of sample size on our ability to detect significant interaction effects. The number of significant interaction effects was rapidly reduced when the sample size was decreased (the number of unique significant probes given a Bonferroni corrected P-value < 8.1 x 10<sup>–6</sup> is shown). In general, due to their low abundance in whole blood, lymphoid-mediated <i>cis</i>-eQTL effects are harder to detect than neutrophil-mediated cis-eQTL effects.</p