a) Schematic representation of the DAPK1 transcriptional start site (TSS, “+1”) and exon 1 (black box). A CpG island (CGI) around the transcriptional start site (TSS) is displayed as black bar. Two amplicons (A and B) covering the DAPK1 promoter and exon 1 region were quantitatively analyzed by the MassCleave assay (−267 to +336 bases relative to TSS). The region analyzed by pyrosequencing is labeled PyroSeq (−265 to −153 bases relative to TSS). The amplicon generated by the methylation-specific PCR is indicated by the label MSP.
b) Quantitative DNA methylation results are displayed as heatmap, columns represent single CpG units, rows represent separate samples. The top panel (AML) in red shows 204 AML samples, 31 healthy controls (N) are displayed in different shades of green (dark green, CD34+ selected hematopoietic progenitor cells [CD34+]; medium green, buffy coat cells [BC]; bright green, peripheral blood granulocytes [GRAN]). Nine AML cell lines of different maturation stages (HL-60, OCI-AML3, KG-1a, Kasumi-1, NB-4, U-937, HEL, MEG-01, and K-562) are shown in black (CL) and 61 CLL samples serving as methylated reference are depicted in blue (CLL). Bright green encodes for low methylation levels, dark blue for high methylation levels, grey indicates missing data.
c) Heatmap visualizing quantitative DNA methylation results as assessed at the region indicated in figure 1a by pyrosequencing. The top panel (AML) in red represents 42 AML samples, 42 MDS samples (MDS) are displayed in bright red. The color coding is identical to figure 1b.
d) The upper panel shows a plot of expected versus observed DNA methylation (^mC) for the amplicon A at the DAPK1 locus performed on a 6-point standard (0, 20, 40, 60, 80 and 100% in vitro methylated genomic DNA). Light grey dots represent all single CpG units of amplicon A for a given methylation level. The equation describes the best fitted curve to the DNA methylation standard. The lower panel represents a control plot of expected versus observed DNA methylation after mathematical correction. The equation represents the respective fitted curve which is identical with the ideal relation between expected and observed levels after correction.
e) Summarizing distribution of average DNA methylation (^mC) values across investigated regions as indicated in figure 1a. The upper panel shows MassCleave-derived quantitative DNA methylation data for AML sampls (AML), healthy controls (CD34+, BC and GRAN), AML cell lines (CL) and CLL samples (CLL). The lower panel depicts pyrosequencing derived average methylation levels for AML and MDS.