Mean DNA methylation levels at the A) H19DMR, B) the distal NNAT-CTCF binding site, and C) the NNAT promoter in normal kidney and tumor tissues.

<p>Red horizontal lines either depict two standard deviations above or below the mean of the normal controls (A) or the median DNA methylation values (B+C).</p

Association of gene expression and mean methylation of A) IGF2 and H19DMR, B) H19 and H19DMR, C) NNAT and NNAT-CTCF, D) NNAT and NNAT promoter, and E) BLCAP_v2a and NNAT-CTCF using Spearman’s rank correlation.

<p>The data from the tumor and normal kidney cases are depicted as black and orange diamonds, respectively. F) Model of the NNAT/BLCAP locus based on our data depicting suppression of BLCAP_v2a and NNAT in normal kidney due to methylated CpGs in the NNAT-CTCF site and NNAT promoter. Consequently, BLCAP_v2a and NNAT upregulation in Wilms tumors is caused by the demethylation of both regulatory regions. The BLCAP gene is expressed in both tumors and normal kidney due to unmethylted CpGs in its promoter.</p

Relative expression of the genes A) IGF2, B) H19, D) NNAT E) BLCAP, G) BLCAP_v1a, and H) BLCAP_v2a in 45 Wilms tumors and 11 normal kidney tissues, as determined by real-time PCR.

<p>The dots represent relative candidate gene expression in relation to the house-keeping gene TBP. Median expression values are given as red horizontal lines. Association of C) IGF2 and H19, F) NNAT and BLCAP, as well as I) NNAT and BLCAP_v2a gene expression in log2 scale using Spearman’s rank correlation. Data from the tumor and normal kidney cases are depicted as black and orange diamonds, respectively.</p

Schematic illustration of the A) IGF2/H19 and B) NNAT/BLCAP locus.

<p>Transcription starts are indicated by arrows and transcript variants are numbered. The CTCF binding sites (yellow bars) within the A) H19DMR and B) CpG-rich intergenic NNAT region as well as the NNAT and BLCAP promoter used for generating the DNA methylation assays are highlighted as magnifications indicating each CpG dinucleotide under investigation. C) Consensus sequence motif of CTCF-binding sites according to Kim et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067605#pone.0067605-Kim1" target="_blank">[28]</a>. The height of each letter represents the relative frequency of occurrence of the nucleotide at each position. CTCF motifs 1–4 within the NNAT regulatory region with scores for similarity to the consensus, as determined by the JASPAR software (jaspar.genereg.net). CpGs under investigation are highlighted in black squares.</p

Mean DNA methylation levels delineated for each CpG at A) and D) the H19DMR, B) and E) the distal NNAT-CTCF binding site, C) and F) the NNAT promoter, and G) the BLCAP promoter in normal kidney (red squares) and tumor tissues (blue diamonds).

<p>A) to C) depict the DNA methylation levels for all tumor and normal kidney cases, whereas D) to F) depict only tumors with >10-fold mean expression of normal kidney for D) IGF2 (n = 22) and E) to F) NNAT (n = 33). Error bars represent the standard deviation of all tumor or kidney sample measurements.</p