These additional filters resulted in 12 genes remaining for initial follow up (COL1A2, CRABP2, CRIP1, FAM46B, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D, UCHL1 and VAMP8). The genes were further filtered by validating the differences in their expression levels pre- and post-treatment. Transcript levels of these 12 genes were assessed by qRT-PCR in the 5 cell lines with the highest expression differences before and after treatment. Selecting only the genes with a .4–fold average qRT-PCR expression change and the presence of CpG islands in the promoter gave a final list of 10 genes: COL1A2, CRABP2, CRIP1, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D and UCHL1. While the absolute value of the fold-changes varied somewhat between the two methodologies, there was generally good agreement between the microarray and qRT-PCR results (Figure S1). Importantly, for each of these 10 genes both techniques showed a .4–fold average change in expression after drug treatment. We then quantitated the degree of methylation of these 10 candidate genes using mass spectrometry of base-specific cleaved amplification products (Sequenom Epityper assay [15,20]). This technique was applied to a panel of 45 melanoma cell lines, 30 fresh melanoma tumor samples, and 2 independent pools of cultured melanocytes for comparison as the non-malignant control cell type. Each gene promoter was divided into one or more amplicons, within a region comprising 2500 bp upstream of the transcription start site and covering the CpG islands described in the UCSC genome browser. These amplicons were then amplified by PCR (Table S1) and subjected to the Epityper assay. Three genes were not considered further as they either failed to give any analysable data, probably due to the high CpG density of the region (CRABP2), or showed no differences in methylation profiles between melanocytes and the melanoma cell lines (LOX and RGC32). For each gene, the amplicons included in the analysis were those with different average methylation values of .20% in the entire panel of melanoma cell lines and with ,10% methylation in melanocytes (Figure S2). These informative CpG sites were then scored in each amplicon defining the CpG island for each gene. Only the CpG sites presenting high methylation ratios (as defined below) in melanoma cell lines were averaged for the final percentage of methylation (% of methylation). This allowed grouping of the melanoma cell lines following their % of methylation: no/low methylation (0–20%), medium (20–50%) and high methylation (.50%). We then defined the average % of methylation for each gene as the average value across the melanoma cell line panel which was then compared to melanocytes. Integrating the degree of methylation and the level of mRNA expression, as assessed by previous Affymetrix microarray analysis [19] gave high correlation coefficients (Spearman coefficients = 20.75/20.82/20.6/20.52 respectively, p,0.005) for four genes: COL1A2, THBS1, TNFRSF10D and UCHL1. Expression of the COL1A2, THBS1, TNFRSF10D and UCHL1 genes is inhibited by CpG island methylation in melanoma cell lines Following 5AzadC+TSA treatment, COL1A2 and THBS1 transcript levels were increased by an average of 58-fold in 9 of 12 cell lines and 40-fold in 8 of 12 cell lines respectively (Table S3). For TNFRSF10D, 5 cell lines showed a 5-fold average increase in expression. In melanoma cell lines, the average % of methylation for these genes was 24%, 31% and 66% respectively. The methylation levels of these genes in the melanocyte pools were close to background (between 2 to 9%). Epigenetic Gene Silencing in Human Melanoma The 59UTR regions around the transcription start sites of COL1A2 and THBS1 were both divided into 5 amplicons, while 4 amplicons were designed to cover the 59UTR and first exon of TNFRSF10D (Figure S2). For each of the three genes, the amplicons closest to the transcription start (within 1000 bp) showed high levels of methylation in the melanoma samples, which inversely correlated with mRNA expression (Table S1). Table S4 summarizes the number of CpG sites scored for the definition of the % of methylation for each gene (19, 15, 37 and 19 for COL1A2, THBS1, TNFRSF10D and UCHL1 respectively). Over fifty percent (21/40) of the melanoma cell lines had no COL1A2 mRNA expression, which correlated with a high degree of COL1A2 promoter methylation in 67% of this subset. The other 19 cell lines with detectable COL1A2 mRNA expression all showed ,20% of methylation, with the exception of 3 lines (MM415, MM229 and D05). (Figure 2a - Figure S3a). For TNFRSF10D, 31 of 43 (72%) melanoma cell lines had no mRNA expression. Of the 31 lines not expressing TNFRSF10D, 28 (90%) showed .60% of methylation (Figure 2b - Figure S3b). For THBS1, 15 of 43 (35%) melanoma cell lines had no mRNA expression. Eight of the 15 (53%) lines with no THBS1 mRNA showed a high .50% methylation (Figure 2c - Figure S3c). Following 5AzadC+TSA treatment, UCHL1 expression was increased by an average of 18-fold in 10 out of 12 cell lines. For the Epityper assay, the region around the 59UTR and transcription start site of UCHL1 was divided in to 4 amplicons, also covering both exons 1 and 2 (Figure S2). While amplicons 2 and 6 did not show any differential methylation between melanoma cell lines and melanocytes (Table S1), amplicons 4 and 8 showed specific profiles of methylation in melanoma cell lines inversely correlated with mRNA expression (Spearman = 20.53). A majority (56%) of the melanoma cell lines had no UCHL1 mRNA expression, which correlated with a high degree of UCHL1 promoter methylation in 79% of them (5% of methylation in melanocytes compared to an average of 42% in melanoma cell lines, range = 29–95%). The melanoma cell lines with high UCHL1 mRNA expression have ,20% methylation (Figure 2d - Figure S3d). Next, we assessed protein levels for THBS1 and UCHL1 (since robust target-specific commercial antibodies are available) by western blot analysis (Figure 3). There was a high correlation between mRNA and protein expression (Pearson = 0.84 and 0.51 for UCHL1 and THBS1 respectively), and an inverse correlation between protein expression and the methylation profile (Spear- man = 20.52 and 20.33 for UCHL1 and THBS1 respectively). Confirmation of candidate gene promoter methylation in fresh-frozen melanoma tumors Simultaneously, in the Epityper assay, we included 30 fresh- frozen melanoma tumor samples and found that 13%, 15% and 30% of them respectively were methylated for COL1A2, THBS1 and TNFRSF10D (Table 1). On average, the COL1A2 and TNFRSF10D promoters appeared .10–fold more methylated in melanoma cell lines compared to melanocytes, and 6-fold higher in the fresh tumors (Figure S4). THBS1 promoter methylation levels were 3.5 and 2 times higher in melanoma cell lines and tumors respectively. The UCHL1 promoter appeared to be 8-fold more methylated in melanoma cell lines compared to melanocytes, and 4-fold more methylated in the 30 fresh tumors compared to the same control. Since mRNA levels were not assessed in the tumors, we are unable to correlate the proportion of methylation with expression in these samples. The observed methylation rates in tumors are lower than in the melanoma cell lines, as expected due to stromal PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26121