Additional file 1: Figure S1. of DNA methylation signature of interleukin 1 receptor type II in asthma

Schematic representation of IL1R1 and location of epigenotyped CpG sites. This figure illustrates a simplified schematic representation of IL1R1 and location of selected CpG dinucleotide sites and pairwise correlations between each CpG

Differential hybridization screening.

<p>Representative differential screening results of macroarrays of the SH-Ctls library. Four identical membranes were dot-blotted with PCR products obtained by SSH. The membranes were then hybridized with four different probes: SH-Ctls subtracted cDNAs (A), SH unsubtracted cDNAs (B), Ctls-SH subtracted cDNAs (C) and Ctls unsubtracted cDNAs. The arrow in the top left corner indicates the positive control (<i>LCN2</i>). The arrow head indicates an example of differentially expressed genes in SH compare with Ctls.</p

Evaluation of subtraction efficiency.

<p>A: Reduction of <i>GAPDH</i> cDNA following subtraction in the SH-Ctls sample. PCR was performed on SH-Ctls subtracted and SH unsubtracted samples. <i>GAPDH</i> PCR products (760 pb) were detectable 10 cycles earlier in the unsubtracted sample (18 cycles) than in the subtracted sample (28 cycles). B: Enrichment of <i>LCN2</i> cDNA following subtraction in the SH-Ctls sample. PCR was performed on SH-Ctls and Ctls-SH subtracted samples as well as SH and Ctls unsubtracted samples. <i>LCN2</i> PCR products (210 pb) were detected after 20 cycles for both SH unsubtracted and SH-Ctls subtracted samples, the difference in the intensity of the 2 bands indicate the enrichment compare to Ctls unsubtracted and Ctls-SH subtracted samples.</p

Methodology for SSH.

<p>Schematic representation of the different steps described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029440#s2" target="_blank">Material and Methods</a> (A), the sample pooling (n corresponds to the number of samples) (B), and the different hybridization steps performed with the SSH technique (C).</p

Polymorphic CTCF binding site rs4065275 (C9b) shows genotype dependent CTCF-binding, FAIRE enrichment and variable methylation levels.

<p>(A) Effect of genotype on CTCF-enrichment at the rs4065275 SNP region. Cell lines that carry the rs4065275-G allele show CTCF enrichment, whereas cell lines that carry the rs4065275-A allele do not. (B) Allelic bias in FAIRE enrichment in LCLs. In 3 out of 4 cell lines the rs4065275-G (C in the diagram) is enriched in the nucleosome-free fraction. (C) Methylation levels of the two putative CBS within the C9 CTCF-enriched region, rs4065275 CG (C9b) and the adjacent non-polymorphic CG (C9a), in human epithelial cells were determined using a pyrosequencing methylation assay. Name of cell line and rs4065275 genotype are shown below the x-axis.</p

DNA methylation patterns of the <i>GSDMA</i> promoter in NuLi-1 cells.

<p>(A) Positions of the interrogated CGs with respect to the <i>GSDMA</i> promoter region are shown in the context of the UCSC browser. (B) <i>GSDMA</i> promoter methylation changes after 5-aza-dC treatment. Filled circles represent methylated CGs, open circles represent unmethylated CGs. Each row represents a clone, the number on the right indicates the number of clones with a particular methylation pattern. Data are divided by allele; allelic percent methylation is shown below the diagram. Type of treatment and average DNA methylation are shown on top; hA -haplotype A; hB–haplotype B.</p

5-aza-dC treatment enhances gene expression.

<p>(A) 5-aza-dC treated NuLi-1 cells show reduced proliferation and apoptosis four days after treatment. Arrowheads point to dying/dead cells. (B) Changes in expression levels of 17q12-q21 genes after 5-aza-dC treatment. The y-axis shows fold change in 5-aza-dC treated cells compared to controls. Error bars show standard deviation. Asterisks indicate statistically significant change in expression in 5-aza-dC treated cells compared to controls (* p < 0.05). (C) Allelic expression in 17q12-q21 genes after 5-aza-dC treatment. Arrows show positions of transcribed SNPs in those genes where allelic expression changed post 5-aza-dC treatment. In <i>ZPBP2</i>, 5-aza-dC treatment causes reactivation of the HapA allele. In <i>ORMDL3</i> it causes a switch in allelic preference. (D) Positions of 51 CGs in the <i>ZPBP2</i> promoter region that were assayed using the sodium bisulfite sequencing assay are shown in the context of the UCSC browser. The red box indicates the position of the 11 CGs assayed using the pyrosequencing methylation assay. (E) DNA methylation profiles of the <i>ZPBP2</i> promoter region in control (DMSO) and 5-aza-dC treated cells. Filled circles represent methylated CGs, open circles represent unmethylated CGs. Each row represents a clone. Data are divided by haplotype; allelic percent methylation is shown below the diagram. Type of treatment and average methylation levels are shown on top. Arrow points to CG31 (CG6 in pyrosequencing assays) that has one of the most pronounced allelic differences in methylation.</p

Additional file 2: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Table S1. Primers used in the study. (DOCX 12 kb

Additional file 8: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Table S5. Univariate analysis results, full model multivariate analysis results, as well as post hoc power calculations. (DOCX 14 kb

Additional file 1: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Figure S1. Experimental design. (DOCX 18 kb