Fig. 4.

The TREX2 DMR displays gene enhancer characteristics in multiple cell lines. a DNA methylation at individual CpG sites at the TREX2 locus across different cancer cell lines and normal human epidermal keratinocyte (NHEK) specimens, matched with respective mRNA expression values (black bars). EHEB was included as a control with low TREX2 expression. Dark gray: data unavailable. b Correlation of average DNA methylation at TREX2_1 EpiTYPER and TREX2 mRNA expression determined by qRT-PCR in cell lines from a. LS174T was excluded due to reported X-chromosomal aberrations [24]. c 5-Aza-dC treatment in three cell lines and effects on TREX2 DNA methylation and mRNA expression. Bar charts show cell lines after 6 days of treatment with 0.5 μM 5-aza-dC, depicting average TREX2 DNA methylation (upper panel) and TREX2 mRNA expression (lower panel). Average expression ratios of TREX2 versus the housekeeping gene HPRT1 are depicted below each data point for approximate estimation of TREX2 mRNA abundance. d Upper panel: map of the TREX2 gene with ChIP-qPCR amplicons and regions used for luciferase reporter assays. Lower panel: heat map depicting relative luciferase signals in cell lines transfected with TREX2 reporter constructs. Schematic view of reporters is shown to the left. Signals depict mean of quadruplicate measurements normalized to empty vector (pGl4.23). minP/luc2, minimal promoter/luc2 luciferase. e HEK293T cells transfected with CpG-free reporter vectors carrying TREX2 PROM1 and DMR (with minimal promoter) regions. Data show luciferase signal of in vitro CpG-methylated and non-methylated reporter plasmids. EV, empty vector (pCpGfree-promoter-lucia). f ChIP-qPCR assays at the TREX2 locus. Bars show H3K27ac signal at four regions (see d) of the TREX2 gene with mean and standard deviation from four replicates. Dot plot shows correlation of H3K27ac signal at the TREX2 DMR (ChIP region 3) and relative TREX2 mRNA expression in cell lines (n = 6). All data depict mean of three replicates with standard deviation, unless stated otherwise. p values refer to unpaired Student’s t test. For correlations, Pearson coefficient (R) is shown