Uantified by SNuPE/ MALDI-TOF. Representative spectra demonstrate an imbalanced allelic mRNA

Uantified by SNuPE/ MALDI-TOF. Representative spectra demonstrate an imbalanced allelic mRNA expression compared to allelically balanced gDNA. Molecular weight range 5390?500 Da is displayed for rs1056719 (G/A) in Granta-519 and JVM-2 cells and 6985?045 Da for rs3818584 (T/C) in EHEB. Peak height/ signal intensity correlates with allele abundance. (C) qPCR-based DAPK1 mRNA quantification upon treatment with the DNMT inhibitor (5-aza-29deoxycytidine, DAC) in Granta-519. The values are relative to the respective mock control (PBS). (D) Allelic ratios of SNP rs1056719 in Granta-519 cells measured by SNuPE/MALDI-TOF assay upon DAC treatment. doi:10.1371/journal.pone.0055261.gfrom 5.5 kb upstream of the TSS to the end of exon 2 of the DAPK1 gene. We identified heterozygosity for eight known SNPs (rs11141848, rs10746814, 23115181 rs1035262, rs13296984, rs1035261, rs1035260, rs1964911, and rs13300553) indicating that these cells carried no deletions around the promoter region. No further genetic variation between the two alleles could be detected. Thus, we speculated that epigenetic aberrations might contribute to DAPK1 ASE in Granta-519. To functionally test the impact of promoter methylation patterns on DAPK1 transcription balance, we treated Granta-519 cells with different concentrations (1.0 and 1.5 mM) of the DNA methyltransferase inhibitor 5-aza-29-deoxycytidine (DAC) for 7 days. We hypothesized that inhibition of DNA methylation might lead to potential reactivation of the repressed allele. A twofold upregulation of DAPK1 transcription was observed by qPCR (Figure 3C). This finding is in concordance with a reactivation of the silent allele contributing to overall expression. Simultaneously this upregulation was accompanied by a reconstitution of the allelic balance, as illustrated by the equal size of the G and A peak heights at the SNP site rs1056719 in cDNA and shown by mass spectrometryand conventional cDNA sequencing (Figure 3D, Figure S6A, B). The mock-treated control (PBS) retained imbalanced mRNA expression, and expression did not increase. Importantly, after withdrawal of DAC and one month of continued culturing, DAPK1 ASE reappeared with an identical reduction in cDNA of the A allele relative to the G allele compared to untreated Granta519 cells (Figure S6C). To directly prove that allele-specific promoter methylation was associated with DAPK1 ASE in Granta-519 23115181 rs1035262, rs13296984, rs1035261, rs1035260, rs1964911, and rs13300553) indicating that these cells carried no deletions around the promoter region. No further genetic variation between the two alleles could be detected. Thus, we speculated that epigenetic aberrations might contribute to DAPK1 ASE in Granta-519. To functionally test the impact of promoter methylation patterns on DAPK1 transcription balance, we treated Granta-519 cells with different concentrations (1.0 and 1.5 mM) of the DNA methyltransferase inhibitor 5-aza-29-deoxycytidine (DAC) for 7 days. We hypothesized that inhibition of DNA methylation might lead to potential reactivation of the repressed allele. A twofold upregulation of DAPK1 transcription was observed by qPCR (Figure 3C). This finding is in concordance with a reactivation of the silent allele contributing to overall expression. Simultaneously this upregulation was accompanied by a reconstitution of the allelic balance, as illustrated by the equal size of the G and A peak heights at the SNP site rs1056719 in cDNA and shown by mass spectrometryand conventional cDNA sequencing (Figure 3D, Figure S6A, B). The mock-treated control (PBS) retained imbalanced mRNA expression, and expression did not increase. Importantly, after withdrawal of DAC and one month of continued culturing, DAPK1 ASE reappeared with an identical reduction in cDNA of the A allele relative to the G allele compared to untreated Granta519 cells (Figure S6C). To directly prove that allele-specific promoter methylation was associated with DAPK1 ASE in Granta-519 15857111 cells, we quantitatively assessed DNA methylation in the DAPK1 59 upstream region. While MEC-1 cells not expressing DAPK1 exhibited almost complete DNA methylation from 200 bp upstream of TSS to exon 2 (Figure S4), a region of restricted methylation could be detected in ASE-positive Granta-519 at the end of exon 1 (Figure 4A and 4B). Quantitative assessment showed approximately 50 DNA methylation around exon 1, while most of the downstream region starting from intron 1 was unmethylated. Utilizing an informative SNP (rs13300553) in close vicinity to the 50 methylated region, we performed bisulfite sequencing to investigate allele-specific methylation patterns. The rsAllele-Specific Expression of DAPK1 in CLLFigure 4. Allele-specific DNA methylation (ASM) contributes to ASE of DAPK1 in Granta-519 cells. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first 2 exons of DAPK1. Nucleotide positions are given relative to DAPK1 transcriptional start site (TSS). Dashed lines represent positions of the investigated regions/amplicons. (B) Quant.