Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells
Background: <i>TERT</i> promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to <i...
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| Format: | Article |
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MDPI AG
2022-08-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/14/16/4018 |
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| author | Seungjae Lee Ti-Cheng Chang Patrick Schreiner Yiping Fan Neeraj Agarwal Charles Owens Reinhard Dummer John M. Kirkwood Raymond L. Barnhill Dan Theodorescu Gang Wu Armita Bahrami |
| author_facet | Seungjae Lee Ti-Cheng Chang Patrick Schreiner Yiping Fan Neeraj Agarwal Charles Owens Reinhard Dummer John M. Kirkwood Raymond L. Barnhill Dan Theodorescu Gang Wu Armita Bahrami |
| author_sort | Seungjae Lee |
| collection | DOAJ |
| description | Background: <i>TERT</i> promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to <i>TERT</i> activating genetic alterations, as predicted for functionally redundant mechanisms. To annotate the altered patterns of <i>TERT</i> promoter methylation and their relationship with gene expression, we applied a Pacific Biosciences-based, long-read, bisulfite-sequencing technology and compared the differences in the methylation marks between wild-type and mutant cancers in an allele-specific manner. Results: We cataloged <i>TERT</i> genetic alterations (i.e., promoter point mutations or structural variations), allele-specific promoter methylation patterns, and allele-specific expression levels in a cohort of 54 cancer cell lines. In heterozygous mutant cell lines, the mutant alleles were significantly less methylated than their silent, mutation-free alleles (<i>p</i> < 0.05). In wild-type cell lines, by contrast, both epialleles were equally methylated to high levels at the <i>TERT</i> distal promoter, but differentially methylated in the proximal regions. ChIP analysis showed that epialleles with the hypomethylated proximal and core promoter were enriched in the active histone mark H3K4me2/3, whereas epialleles that were methylated in those regions were enriched in the repressive histone mark H3K27me3. Decitabine therapy induced biallelic expression in the wild-type cancer cells, whereas the mutant cell lines were unaffected. Conclusions: Long-read bisulfite sequencing analysis revealed differences in the methylation profiles and responses to demethylating agents between <i>TERT</i> wild-type and genetically altered cancer cell lines. The causal relation between <i>TERT</i> promoter methylation and gene expression remains to be established. |
| first_indexed | 2024-03-09T04:38:29Z |
| format | Article |
| id | doaj.art-cce0a52b8eb0422db4daf8a1f405b2b1 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-09T04:38:29Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-cce0a52b8eb0422db4daf8a1f405b2b12023-12-03T13:25:31ZengMDPI AGCancers2072-66942022-08-011416401810.3390/cancers14164018Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer CellsSeungjae Lee0Ti-Cheng Chang1Patrick Schreiner2Yiping Fan3Neeraj Agarwal4Charles Owens5Reinhard Dummer6John M. Kirkwood7Raymond L. Barnhill8Dan Theodorescu9Gang Wu10Armita Bahrami11Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USACenter for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN 38015, USACenter for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN 38015, USACenter for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN 38015, USACedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA 90048, USADepartment of Surgery, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USADepartment of Dermatology, University Hospital Zurich, 8091 Zurich, SwitzerlandDepartment of Pathology, University of Pittsburgh Cancer Center, Pittsburgh, PA 15232, USADepartment of Translational Research, Institut Curie, 75248 Paris, FranceCedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA 90048, USADepartment of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USADepartment of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30307, USABackground: <i>TERT</i> promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to <i>TERT</i> activating genetic alterations, as predicted for functionally redundant mechanisms. To annotate the altered patterns of <i>TERT</i> promoter methylation and their relationship with gene expression, we applied a Pacific Biosciences-based, long-read, bisulfite-sequencing technology and compared the differences in the methylation marks between wild-type and mutant cancers in an allele-specific manner. Results: We cataloged <i>TERT</i> genetic alterations (i.e., promoter point mutations or structural variations), allele-specific promoter methylation patterns, and allele-specific expression levels in a cohort of 54 cancer cell lines. In heterozygous mutant cell lines, the mutant alleles were significantly less methylated than their silent, mutation-free alleles (<i>p</i> < 0.05). In wild-type cell lines, by contrast, both epialleles were equally methylated to high levels at the <i>TERT</i> distal promoter, but differentially methylated in the proximal regions. ChIP analysis showed that epialleles with the hypomethylated proximal and core promoter were enriched in the active histone mark H3K4me2/3, whereas epialleles that were methylated in those regions were enriched in the repressive histone mark H3K27me3. Decitabine therapy induced biallelic expression in the wild-type cancer cells, whereas the mutant cell lines were unaffected. Conclusions: Long-read bisulfite sequencing analysis revealed differences in the methylation profiles and responses to demethylating agents between <i>TERT</i> wild-type and genetically altered cancer cell lines. The causal relation between <i>TERT</i> promoter methylation and gene expression remains to be established.https://www.mdpi.com/2072-6694/14/16/4018<i>TERT</i> promotermethylationbisulfite sequencingallele-specific methylationproximal and distal promoters |
| spellingShingle | Seungjae Lee Ti-Cheng Chang Patrick Schreiner Yiping Fan Neeraj Agarwal Charles Owens Reinhard Dummer John M. Kirkwood Raymond L. Barnhill Dan Theodorescu Gang Wu Armita Bahrami Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells Cancers <i>TERT</i> promoter methylation bisulfite sequencing allele-specific methylation proximal and distal promoters |
| title | Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells |
| title_full | Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells |
| title_fullStr | Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells |
| title_full_unstemmed | Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells |
| title_short | Targeted Long-Read Bisulfite Sequencing Identifies Differences in the <i>TERT</i> Promoter Methylation Profiles between <i>TERT</i> Wild-Type and <i>TERT</i> Mutant Cancer Cells |
| title_sort | targeted long read bisulfite sequencing identifies differences in the i tert i promoter methylation profiles between i tert i wild type and i tert i mutant cancer cells |
| topic | <i>TERT</i> promoter methylation bisulfite sequencing allele-specific methylation proximal and distal promoters |
| url | https://www.mdpi.com/2072-6694/14/16/4018 |
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