Methylation of 5' gene upstream regions. A, Methylated regions in LNCaP (left) and PrEC (middle) cells, and hypermethylated (right) regions in LNCaP vs. PrEC cells are significantly enriched within 2 kbp upstream of transcriptional start sites. The expected probability distribution for (hyper)methylated regions to overlap with 5' gene upstream regions is shown (gray bars and blue line). The red line indicates the observed fraction of base pairs overlapping 5' gene upstream regions in our actual dataset. B, DNA methylation signals (smoothed adjusted log2(M/T)) surrounding a representative 5' gene upstream region hypermethylated in LNCaP compared to PrEC. Annotations include chromosome coordinates (top), CpG density (number of CpGs in sliding 250 bp windows), Refseq genes, and CpG islands. The box indicates a region that was verified by bisulfite sequencing. C, Bisulfite verification of a hypermethylated region (boxed region from panel (B)) upstream of ADAMTS1. Circles represent position of CpGs. In the top line for each cell line the color of each circle represents the fraction of sequenced alleles that were methylated at that CpG according to the color scale (bottom). Each subsequent line represents the methylation pattern for each sequenced clone; black and white circles indicate methylated and unmethylated CpGs respectively. D, AZAdC induces re-expression of ADAMTS1 in LNCaP cells. Expression of ADAMTS1 with respect to that of GAPDH was measured by real time RT-PCR in LNCaP cells treated with vehicle (DMSO) or 1 μM AZAdC for 3 or 7 days.