Background: The methylation of cytosine residues at CpG sites within the O6-methylguanine-DNA methyltransferase (MGMT) promoter is a key biomarker in glioblastoma therapy. The MGMT promoter (MGMTp) contains multiple guanine-rich sequences capable of folding into G-quadruplexes (G4s), but their relevance for MGMTp methylation is poorly understood. Objectives: Our study explores the impact of potential G-quadruplex-forming sequences (PQS) in the MGMT promoter CpG island on the activity of de novo DNA methyltransferase Dnmt3a. Additionally, we investigate their influence on the accuracy of methylation pattern detection using nanopore sequencing. Methods: Nanopore sequencing was employed to analyze the methylation of 94 clinically significant CpG sites in the human MGMTp using an in vitro de novo methylation system. Circular dichroism spectroscopy was used to identify G4 structures within the MGMTp CpG island. Interactions between the catalytic domain of Dnmt3a and the PQS from the MGMTp were examined by biolayer interferometry. Results: Guanine-rich DNA strands of the PQSs in the MGMTp were hypomethylated, while the complementary cytosine-rich strands were methylated by DNA methyltransferase Dnmt3a with higher efficiency. The accuracy of detecting modified bases in the PQS was significantly lower compared to surrounding sequences. Single-stranded guanine-rich DNA sequences from the MGMTp exhibited strong binding to Dnmt3a-CD, with an affinity approximately 10 times higher than their cytosine-rich complements (K_d = 3 × 10⁻⁸ M and 3 × 10⁻⁷ M, respectively). By binding to Dnmt3a, G4-forming oligonucleotides from MGMTp effectively inhibited the methylation reaction (IC₅₀ 6 × 10⁻⁷ M). Conclusions: The obtained data indicate the role of PQSs in establishing de novo methylation of the MGMT promoter. They also highlight the challenges of sequencing guanine-rich regions and the impact of specific de novo methylation patterns on clinical data interpretation.

DNA methylation, the covalent addition of methyl groups to cytosine (5mC) or adenine (6mA) in DNA, is a fundamental mechanism of epigenetic inheritance conserved from bacteria to humans. Fungi provide a uniquely informative window into the evolutionary logic of methylation systems: spanning more than 1 billion years of diversification, the kingdom encompasses species that have lost cytosine methylation entirely, lineages that use 5mC to silence transposons and drive the irreversible genome-defense process known as repeat-induced point mutation (RIP), and early-diverging lineages in which 6mA has displaced 5mC as the dominant chromatin mark. The methyltransferases underlying these strategies (DIM-2, RID, DNMT1-RFD, DNMT5, and the MT-A70 complex) and the recently characterized demethylases Dmt1 and CcTet are structurally and mechanistically distinct from their mammalian counterparts. Here we review the mechanisms, targets, and biological functions of fungal DNA methyltransferases and demethylases, incorporating cryo-EM structural insights into DIM-2 and DNMT5 catalysis, analyses of DNMT gene loss as a continuous evolutionary process, the antiviral role of DIM-2 in vegetative hyphae, and the emerging model of 6mA as an essential, heritable regulatory mark in early-diverging lineages. By integrating these advances, this review offers the most current and comprehensive account of DNA methylation across fungi.

Background: The methylation of cytosine residues at CpG sites within the O6-methylguanine-DNA methyltransferase (MGMT) promoter is a key biomarker in glioblastoma therapy. Single-molecule nanopore sequencing enables direct detection of methylation patterns across the genome. Our study investigates how potential G-quadruplex-forming sequences (PQS) in the MGMT promoter CpG island influence the activity of de novo DNA methyltransferase Dnmt3a and the accuracy of methylation pattern detection via nanopore sequencing. Methods: Nanopore sequencing was employed to analyze the methylation of 97 clinically significant CpG sites in the human MGMT promoter (MGMTp) using an in vitro de novo methylation system. Circular dichroism spectroscopy was used to identify G-quadruplex (G4) structures within the MGMTp CpG island. Interactions between the catalytic domain of Dnmt3a and the PQS from the MGMTp were examined by biolayer interferometry. Results: Guanine-rich DNA strands of the PQSs in the MGMTp were hypomethylated, while the complementary cytosine-rich strands were methylated by DNA methyltransferase Dnmt3a with higher efficiency. The accuracy of detecting modified bases in the PQS was significantly lower compared to surrounding sequences. Single-stranded guanine-rich DNA sequences from the MGMTp exhibited strong binding to Dnmt3a-CD, with an affinity approximately 10 times higher than their cytosine-rich complements (Kd = 3 × 10⁻⁸ M and 3 × 10⁻⁷ M, respectively). By binding to Dnmt3a, G4-forming oligonucleotides from MGMTp effectively inhibited the methylation reaction (IC50 6·10-7 M). Conclusions: The obtained data indicate the role of PQSs in establishing de novo the methylation of the MGMT promoter and highlight the challenges of sequencing guanine-rich regions using nanopore sequencing as well as impact of specific pattern of de novo methylation in the clinical data interpretation.

DNA methylation, the covalent addition of methyl groups to cytosine (5mC) or adenine (6mA) in DNA, is a fundamental mechanism of epigenetic inheritance conserved from bacteria to humans. Fungi provide a uniquely informative window into the evolutionary logic of methylation systems. Spanning more than 1 billion years of diversification, the kingdom encompasses species that have lost cytosine methylation entirely, lineages that use 5mC to silence transposons and drive the irreversible genome-defense process known as repeat-induced point mutation (RIP), and early-diverging lineages, in which 6mA has emerged as a prominent chromatin mark. The methyltransferases underlying these strategies (DIM-2, RID, DNMT1-RFD, DNMT5, and the MT-A70 complex) and the recently characterized demethylases Dmt1 and CcTet are structurally and mechanistically distinct from their mammalian counterparts. Here we review the mechanisms, targets, and biological functions of fungal DNA methyltransferases and demethylases, incorporating cryo-EM structural insights into DIM-2 and DNMT5 catalysis, analyses of DNMT gene loss as a continuous evolutionary process, the antiviral role of DIM-2 in vegetative hyphae, and the emerging model of 6mA as a heritable regulatory mark in early-diverging lineages. By integrating these advances, this review offers the updated and comprehensive account of DNA methylation across fungi.