Itative DNA methylation analysis of the DAPK1 gene 59 region (amplicons A ) in untreated and 5-aza-29-deoxycytidine (DAC)-treated Granta-519 cells was performed using the MassARRAY-based MassCleave method. Bars represent quantitative DNA methylation values ( ) at single CpG units. (C) Bisulfite-sequencing of the DAPK1 59 region including the SNP rs13300553 (G/A) used for allelic separation in Granta-519 cells. Sequenced clones carrying A at the respective SNP site (+520) are grouped in the upper panel, the G alleles are displayed in the lower panel. Black boxes represent single-CpG methylation, grey boxes represent unmethylated CpGs, white boxes stand for missing data. Methylation levels are calculated over the area between +58 and +263 in both SC 1 chemical information allele groups. (D) Detection of ASM by separate amplification of either the unmethylated or methylated alleles by methylation-specific PCR on bisulfite-converted genomic DNA. Genotype distribution between the differentially methylated alleles was performed by SNuPE/MALDI-TOF. Untreated Granta-519 (PBS), 7-day treatment with the DNMT inhibitor 5-aza-29-deoxycytidine (DAC), and assessment of ASM after 33 days of withdrawal of DAC (33-day recovery) are shown. The right panel shows the assessment of a CpG Itacitinib web dinucleotide as specificity control (see results section for detailed explanation). doi:10.1371/journal.pone.0055261.gSNP A allele that represents the transcriptionally repressed allele showed 83.0 methylation whereas the G allele was methylated at considerably lower levels (,32.3 ) in the region of interest (Figure 4C). In EHEB cells exhibiting almost monoallelic expression, we found a similar separation in completely unmethylated and (almost) fully methylated alleles at the same region that exhibited ASM in Granta-519 cells (Figure S7A and S7B). However, as the SNP rs13300553 was not heterozygous in this cell line and other informative SNPs could not be detected between position 220 and +600, a clear allelic separation was not possible despite the strong evidence for two distinct allele populations. In JVM-2 cells with perfectly balanced DAPK1 transcription, DNA methylation was entirely absent (Figure S7C). In order to quantitatively confirm the allele-specific promoter methylation (ASM), we designed a methylation-specific genotyping assay basedon the SNuPE method (ASM-SNuPE). This method was used to determine the SNP 15857111 ratio between the amplification of unmethylated and methylated alleles. Unmethylated and methylated amplicons were specifically amplified from bisulfite-treated DNA using PCR with primers specific for unmethylated or methylated template (UMSP/MSP) (Figure 4D). The primer design was based on differentially methylated CpGs as determined by the previous methylation results. We used 24786787 an extension primer as an amplification specificity control to ensure for strict separation of methylated and unmethylated alleles. Quantitative genotyping of the SNP site rs13300553 in Granta-519 showed a strong enrichment of the G allele in the unmethylated fraction, whereas the A genotype almost exclusively appeared in the methylated alleles. DAC treatment increased the appearance of the A allele in the unmethylated fraction, indicating loss of methylation of thisAllele-Specific Expression of DAPK1 in CLLallele. Withdrawal of DAC restored the allele-specific methylation after cultivation for one month. Taken together, these experiments show that in Granta-519 DAPK1 ASE and ASM are functionally related.Dis.Itative DNA methylation analysis of the DAPK1 gene 59 region (amplicons A ) in untreated and 5-aza-29-deoxycytidine (DAC)-treated Granta-519 cells was performed using the MassARRAY-based MassCleave method. Bars represent quantitative DNA methylation values ( ) at single CpG units. (C) Bisulfite-sequencing of the DAPK1 59 region including the SNP rs13300553 (G/A) used for allelic separation in Granta-519 cells. Sequenced clones carrying A at the respective SNP site (+520) are grouped in the upper panel, the G alleles are displayed in the lower panel. Black boxes represent single-CpG methylation, grey boxes represent unmethylated CpGs, white boxes stand for missing data. Methylation levels are calculated over the area between +58 and +263 in both allele groups. (D) Detection of ASM by separate amplification of either the unmethylated or methylated alleles by methylation-specific PCR on bisulfite-converted genomic DNA. Genotype distribution between the differentially methylated alleles was performed by SNuPE/MALDI-TOF. Untreated Granta-519 (PBS), 7-day treatment with the DNMT inhibitor 5-aza-29-deoxycytidine (DAC), and assessment of ASM after 33 days of withdrawal of DAC (33-day recovery) are shown. The right panel shows the assessment of a CpG dinucleotide as specificity control (see results section for detailed explanation). doi:10.1371/journal.pone.0055261.gSNP A allele that represents the transcriptionally repressed allele showed 83.0 methylation whereas the G allele was methylated at considerably lower levels (,32.3 ) in the region of interest (Figure 4C). In EHEB cells exhibiting almost monoallelic expression, we found a similar separation in completely unmethylated and (almost) fully methylated alleles at the same region that exhibited ASM in Granta-519 cells (Figure S7A and S7B). However, as the SNP rs13300553 was not heterozygous in this cell line and other informative SNPs could not be detected between position 220 and +600, a clear allelic separation was not possible despite the strong evidence for two distinct allele populations. In JVM-2 cells with perfectly balanced DAPK1 transcription, DNA methylation was entirely absent (Figure S7C). In order to quantitatively confirm the allele-specific promoter methylation (ASM), we designed a methylation-specific genotyping assay basedon the SNuPE method (ASM-SNuPE). This method was used to determine the SNP 15857111 ratio between the amplification of unmethylated and methylated alleles. Unmethylated and methylated amplicons were specifically amplified from bisulfite-treated DNA using PCR with primers specific for unmethylated or methylated template (UMSP/MSP) (Figure 4D). The primer design was based on differentially methylated CpGs as determined by the previous methylation results. We used 24786787 an extension primer as an amplification specificity control to ensure for strict separation of methylated and unmethylated alleles. Quantitative genotyping of the SNP site rs13300553 in Granta-519 showed a strong enrichment of the G allele in the unmethylated fraction, whereas the A genotype almost exclusively appeared in the methylated alleles. DAC treatment increased the appearance of the A allele in the unmethylated fraction, indicating loss of methylation of thisAllele-Specific Expression of DAPK1 in CLLallele. Withdrawal of DAC restored the allele-specific methylation after cultivation for one month. Taken together, these experiments show that in Granta-519 DAPK1 ASE and ASM are functionally related.Dis.
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