Single-Molecule Nanopore Sequencing of the TERT Core Promoter CpG Island Reveals PQS-Associated Strand Asymmetry of De Novo Methylation and Its Modulation by Photo-Oxidation
- 1Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia
- 2Belozersky Research Institute of Physical and Chemical Biology, Moscow State University, 119991 Moscow, Russia
Abstract
DNA methylation of the human telomerase reverse transcriptase (hTERT) promoter contributes to telomerase regulation in cancer. We examined how strand identity and sequence context relate to methylation by the Dnmt3a catalytic domain (Dnmt3a-CD). Strand-resolved nanopore sequencing compared four promoter variants (wild type, C228T, C250T, and C228T/C250T) in amplicons and plasmids. Amplicons were also photo-oxidized with methylene blue before methylation. Methylation was higher on the cytosine-rich than on the guanine-rich strand. Defined putative quadruplex-forming sequence (PQS) cores showed reduced strand asymmetry and lower site-to-site variability; grouping sites by high G4Hunter scores did not reproduce this pattern. Variants and plasmid substrates largely retained the spatial profile. Photo-oxidized amplicons showed lower measured 5mC, particularly on the guanine-rich strand, while broadly retaining the profile. Photo-oxidation reduced sequencing quality. Bisulfite analysis of the C-rich strand of Raji DNA revealed a PQS-associated hypomethylation trough in both Dnmt3a-CD-treated DNA and an untreated aliquot. Together, these results highlight the importance of PQS sequence context for hTERT promoter methylation and the challenges of accurately sequencing guanine-rich, oxidation-prone DNA.
Related
- Genome-Wide DNA Methylation Sequencing Identifies Methylation-Associated Genes Related to Porcine Skeletal Muscle Fiber Diversity
- Protocol for genome-wide differential methylation using long-read DNA sequencing in the cortex of a mouse model of Alzheimer's disease.
- Arabidopsis nanopore direct RNA sequencing data for mutants in splicing, polyadenylation, and m6A methylation.
- RiboMeth-Sequencing Reveals Selective Ribosomal RNA 2′-O-Methylation Changes in the Dorsolateral Prefrontal Cortex of Subjects With Major Depressive Disorder
- Decoding common and complex genotypes of facioscapulohumeral muscular dystrophy using methylation profiling and long-read sequencing.
BibTeX
@article{sergeev2026singlemolecule,
title = {{Single-Molecule Nanopore Sequencing of the TERT Core Promoter CpG Island Reveals PQS-Associated Strand Asymmetry of De Novo Methylation and Its Modulation by Photo-Oxidation}},
author = {Alexander Sergeev and Adelya Genatullina and Viktoria Snyga and Mayya Monakhova and Elena Kubareva and Elizaveta Gromova and Maria Zvereva},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209129},
url = {https://www.mdpi.com/1422-0067/27/20/9129},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Single-Molecule Nanopore Sequencing of the TERT Core Promoter CpG Island Reveals PQS-Associated Strand Asymmetry of De Novo Methylation and Its Modulation by Photo-Oxidation
AU - Alexander Sergeev
AU - Adelya Genatullina
AU - Viktoria Snyga
AU - Mayya Monakhova
AU - Elena Kubareva
AU - Elizaveta Gromova
AU - Maria Zvereva
PY - 2026
DA - 2026/10/15
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209129
UR - https://www.mdpi.com/1422-0067/27/20/9129
AB - DNA methylation of the human telomerase reverse transcriptase (hTERT) promoter contributes to telomerase regulation in cancer. We examined how strand identity and sequence context relate to methylation by the Dnmt3a catalytic domain (Dnmt3a-CD). Strand-resolved nanopore sequencing compared four promoter variants (wild type, C228T, C250T, and C228T/C250T) in amplicons and plasmids. Amplicons were also photo-oxidized with methylene blue before methylation. Methylation was higher on the cytosine-rich than on the guanine-rich strand. Defined putative quadruplex-forming sequence (PQS) cores showed reduced strand asymmetry and lower site-to-site variability; grouping sites by high G4Hunter scores did not reproduce this pattern. Variants and plasmid substrates largely retained the spatial profile. Photo-oxidized amplicons showed lower measured 5mC, particularly on the guanine-rich strand, while broadly retaining the profile. Photo-oxidation reduced sequencing quality. Bisulfite analysis of the C-rich strand of Raji DNA revealed a PQS-associated hypomethylation trough in both Dnmt3a-CD-treated DNA and an untreated aliquot. Together, these results highlight the importance of PQS sequence context for hTERT promoter methylation and the challenges of accurately sequencing guanine-rich, oxidation-prone DNA.
SN - 1422-0067
ER -