Elizaveta Sergeevna Gromova

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.

Diabetic retinopathy (DR) remains one of the leading causes of vision loss. Its development is driven by complex interconnected pathophysiological mechanisms triggered by chronic hyperglycemia. Traditional diagnostic approaches, which rely on detecting visible microangiopathies, often identify the disease only after significant and progressive retinal damage had already occurred. Consequently, current therapeutic strategies are largely focused on managing advanced, proliferative stages of DR. A major direction in modern ophthalmology is the shift toward identifying and intervening at stages that precede clinical DR manifestations. This review presents current data on the pathogenesis and molecular mechanisms of DR, discusses associated biochemical alterations revealed through multi-omics approaches, and examines methods for preclinical diagnostics, innovative therapeutic strategies, and novel targets for early pathogenetic intervention. Overall, the evidence suggests that that effective treatment options for DR might include multi-target therapeutic approaches extending beyond the blockade of angiogenesis and aimed at addressing metabolic, inflammatory, and oxidative disorders causing neurodegeneration and vascular dysfunction.

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.

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.

S-Adenosyl-L-methionine (SAM) is a central cofactor in cellular methylation, donating methyl groups to a wide range of biological substrates. SAM analogues are promising tools for selective modulation of methyltransferase activity. Here, we investigated phosphorus-containing analogues of SAM and S-adenosyl-L-homocysteine (SAH), focusing on the H-phosphinic SAM analogue ((R,S)-SAM-P_H) with the HO(H)(O)P group replacing the carboxyl group of SAM. We examined the interaction of (R,S)-SAM-P_H with three representative methyltransferases: Dnmt1, responsible for maintenance of DNA methylation; Dnmt3a, which establishes de novo DNA methylation; and catechol-O-methyltransferase (COMT), which methylates protocatechuic aldehyde to yield vanillin and isovanillin. (R,S)-SAM-P_H is a methyl group donor for Dnmt3a and COMT, but not for Dnmt1, despite the high structural similarity of the Dnmt1 and Dnmt3a catalytic domains. These results demonstrate that targeted modification of the carboxyl group of SAM can yield analogues with specific activity towards various methyltransferases. The different recognition of (R,S)-SAM-P_H by Dnmt3a and Dnmt1 highlights its potential as a molecular probe for distinguishing de novo from maintenance DNA methylation. This work enriches our understanding of methyltransferase substrate specificity and provides a new tool for selective modulation of epigenetic processes.

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.

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.

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

Methylation of cytosines at CpG sites in DNA, carried out by de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed by physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduce Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface.