Ogy 2014, 15:522 http://genomebiology.com/2014/15/12/Page 11 ofFigure 7 Differential DNA methylation is associated with altered AZD3759MedChemExpress AZD3759 expression of microRNAs and their target genes in female compared with male human islets. (A) Decreased DNA methylation of five CpG sites as well as increased expression (males n = 5, females n = 6) of hsa-miR-660 in female compared with male islets. Also, potential target genes of hsa-miR-660 showed lower gene expression in female compared with male human islets. (B) Decreased DNA methylation as well as increased expression (males n = 4, females n = 7) of hsa-miR-532 in female compared with male islets. Also, potential target genes showed lower gene expression in female compared with male human islets. Data are presented as mean ?standard error of the mean. Asterisks indicate q <0.05 for DNA methylation data and P <0.05 for gene expression data.methylation between males and females. DNA from pancreatic islets from 32 males and 18 females was used for the technical validation. In agreement with the Infinium HumanMethylation450 BeadChip data, all three CpG sites showed differential DNA methylation in pancreatic islets from male compared with female donors and the differences in DNA methylation seen with PyroSequencing were similar to the differences seen with the Infinium HumanMethylation450 BeadChip (Figure 8). Interestingly, we also found significant differences in DNA methylation between sexes for all but one adjacent CpG site only covered by the PyroSequencing assays, that is, sites not included on the Infinium HumanMethylation450 BeadChip (Figure 8A-C).Increased DNA methylation of the proximal promoters of NKAP and SPESP1 decreases reporter gene expressionDNA methylation in the proximal promoter region is generally associated with PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 decreased transcriptional activity [30,31]. We therefore functionally tested if promoter methylation affects expression of two selected genes that exhibited both differential DNA methylation and expression in islets due to sex (Figure 4B,D). While NKAP is located on the X chromosome, SPESP1 is an autosomal gene located on chromosome 15. The promoter region of NKAP contains several differentially methylated CpG sites (q <0.05) and the mRNA expression of NKAP was lower in females compared with males (P <0.05) (Figure 4B). SPESP1 has a higher degree of DNA methylation in the promoter and the first exon, in parallel with lower expression in female compared with male islets (Figure 4D). Tofunctionally test if DNA methylation of the NKAP and SPESP1 promoters could influence their gene expression, we used a luciferase reporter assay. A 1,500 bp DNA sequence upstream of their respective TSSs was inserted into a CpG free vector containing the firefly luciferase gene. The constructs were then either mock-methylated or methylated using two different enzymes, HhaI and SssI, where HhaI methylates the internal CpG site in the GCGC sequence, and SssI methylates all CpG sites in the sequence. The numbers of CpG sites methylated by these enzymes in the 1,500 bp NKAP and SPESP1 promoter sequences are shown in Figure 9. SssI methylation of the NKAP promoter almost completely repressed the transcriptional activity of the reporter gene, while HhaI methylation did not significantly affect the transcriptional activity (P = 0.065), probably due to the low number of GCGC sequences in the promoter sequence of NKAP (Figure 9A). For the SPESP1 promoter, methylation with either enzyme caused a significant red.