Journal: Molecular Oncology

Article Title: Allele‐specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene ( TERT ) associates with TERT expression in multiple cancers

doi: 10.1002/1878-0261.12786

Figure Lengend Snippet: Cells with monoallelic expression of TERT have allelic methylation of the proximal TERT promoter. (A) Extent of allelic methylation across the TERT gene, which is transcribed from right to left. Relative allelic methylation was measured by calculating the difference between the mean and mode values of raw read Bis‐Seq CpG methylation data, where greater levels suggest greater allelic methylation behavior (see Fig. for examples). Positions included contained 3–6 CpGs per read and coverage of ≥ 5 reads per cell line. Error bars represent standard error of the mean. n represents the number of cancer cell lines. * P ≤ 0.05, where statistical analysis was performed using 2‐tailed Student's t ‐test with unequal variance. (B) Bisulfite conversion cloning data from genomic DNA of select CpGs flanking the TERT transcription start site (5:1295138–1295413, spanning 33 CpGs). Each row represents a different clone (or genome copy, or allele) and each circle represents a CpG, with black circles representing a methylated CpG and white circles representing an unmethylated CpG. For chromosomal positions of noted TERT features, see Table .

Article Snippet: For Fig. , raw read Bis‐Seq DNA CpG methylation data from CCLE's Bis‐Seq dataset were obtained by direct request to the Broad Institute's CCLE and analyzed for allelic patterns using stringent cutoff criteria (reads analyzed contained 3–6 CpGs per read, and coverage of ≥ 5 reads per cell line).

Techniques: Expressing, Methylation, CpG Methylation Assay, Cloning

Journal: Molecular Oncology

Article Title: Allele‐specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene ( TERT ) associates with TERT expression in multiple cancers

doi: 10.1002/1878-0261.12786

Figure Lengend Snippet: Decreased TERT promoter methylation associates with histone marks of active transcription and an active exonic SNP. (A) ChIP‐Bis‐Seq of the TERT promoter using an H3ac antibody shows enrichment of unmethylated DNA in the pulled‐down samples (black) relative to the input (gray) in LN‐18 cells. The absence of any bars indicates zero percent methylation. Inclusion criteria for read positions were a greater number of reads in the pull‐down relative to the input and ≥ 10 reads in the pull‐down (mean input coverage was 9 reads; mean pull‐down coverage was 13 reads; P = 0.01 for pull‐down efficiency). (B) Confirmation of long‐range bisulfite conversion PCR enriching for unmethylated or methylated CpGs at the TERT proximal promoter (16 CpGs spanning 5:1295265–1295396; region overlaps with some of the CpGs analyzed in 3A) using unmethylated (gray)‐ or methylated (black)‐specific bisulfite conversion PCR, respectively. PCR products generated a 1448‐bp product including the proximal promoter and the exon 2 SNP analyzed in Panel C. * P ≤ 0.05 (C) Long‐range bisulfite conversion PCR (same PCRs as shown in Panel B) showing representative Sanger sequencing results (upward arrow indicates position of the exon 2 SNP) and graphs of the sequencing results ( n = 2–3 sequenced reactions). ‘Active SNP’ means that the nucleotide at the position of the SNP is the one found in the TERT mRNA transcribed in that cell line. The active SNP was either previously identified in all cell lines or was identified here (Fig. S6). Error bars represent standard error of the mean. * P ≤ 0.01, where statistical analysis was performed using 2‐tailed Student's t ‐test with unequal variance.

Article Snippet: For Fig. , raw read Bis‐Seq DNA CpG methylation data from CCLE's Bis‐Seq dataset were obtained by direct request to the Broad Institute's CCLE and analyzed for allelic patterns using stringent cutoff criteria (reads analyzed contained 3–6 CpGs per read, and coverage of ≥ 5 reads per cell line).

Techniques: Methylation, Generated, Sequencing

Journal: Molecular Oncology

Article Title: Allele‐specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene ( TERT ) associates with TERT expression in multiple cancers

doi: 10.1002/1878-0261.12786

Figure Lengend Snippet: TERT promoter is characterized by conserved upstream hypermethylation and proximal hypomethylation across different cancer tissue types. (A) Bisulfite conversion sequencing (Bis‐Seq) DNA CpG methylation data for 95 positions across the TERT promoter for 23 different cancerous tissues, showing mean values from 833 cancer cell lines ( n represents the number of cell lines per tissue). Colored circles indicate individual CpG sites with statistically significant ( P ≤ 0.005) differences between the tissue and all other tissues. Each graph groups tissues by total mean percent methylated, from most to least methylated (top to bottom, respectively). Each chromosomal position includes data from at least two cell lines for all 23 tissues. (B) Bis‐Seq DNA CpG methylation data for 129 positions across the TERT promoter for 109 cell lines with known allelic expression and activating mutation classifications. Lines had been classified as having wild‐type (WT) monoallelic expression (MAE) of TERT (‘WT MAE’), −124 or −146 C>T activating promoter mutations (‘mutant’), biallelic expression (BAE) of TERT (‘WT BAE’), or alternative lengthening of telomeres (ALT). Each row represents a different cell line. Colors range from red to blue for more to less methylated CpGs, respectively. White represents unavailable data. Each chromosomal position includes data from at least 10 cell lines. (C) Bis‐Seq DNA CpG methylation data for 122 positions across the TERT promoter for 107 cell lines shown in 1B ( n represents the number of cell lines per tissue). Colored circles indicate statistically significant ( P ≤ 0.05) differences in the listed pairwise comparisons, where statistical analysis was performed using 2‐tailed Student's t ‐test with unequal variance. Each chromosomal position includes data from at least two cell lines for all three cell types. See Table for chromosomal positions and Table for cell line data.

Article Snippet: For Fig. , raw read Bis‐Seq DNA CpG methylation data from CCLE's Bis‐Seq dataset were obtained by direct request to the Broad Institute's CCLE and analyzed for allelic patterns using stringent cutoff criteria (reads analyzed contained 3–6 CpGs per read, and coverage of ≥ 5 reads per cell line).

Techniques: Sequencing, CpG Methylation Assay, Methylation, Expressing, Mutagenesis