Evolutionary and structural insights into DNMTs and TETs: decoding their functional heterogeneity and oncogenic roles in methylation regulation

Abstract

DNA methylation in mammals is dynamically regulated by DNMTs and TETs. Despite their critical roles, comparative structural analyses of these protein families have been relatively scarce. To address the above problems, this study first constructed a phylogenetic tree of DNMT and TET proteins to investigate their evolutionary relationships. Furthermore, the structural exploration revealed that both protein families possess conserved β-sheet structures and exhibit the characteristics of alternating β-sheets in their catalytic domains. Interestingly, DNMTs contain more α helices and fewer loops compared to TETs. Several notable structural changes were discovered, including unique flexibility of the CXXC domain and divergences in DNA binding mechanisms among DNMT1, TET1, and TET3. Additionally, the results showed that a distinctive loop present in DNMT2 may indicate its specialized functional role. This research provides fundamental evolutionary and structural insights into DNMT and TET proteins, emphasizing their significance in tissue-specific distribution and cancer signaling, thereby establishing a foundation for future investigations in the field of epigenetics.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12860-025-00552-w.

Introduction

DNA methylation is a significant epigenetic modification in mammals, the establishment and erasure of which are co-regulated by DNA methyltransferases (DNMTs) and Ten-Eleven Translocation family proteins (TET1/2/3). DNMTs fulfill a critical function in maintaining gene silencing and genomic stability by catalyzing methylation at CpG sites, while TETs initiate active DNA demethylation by oxidizing 5-methylcytosine (5mC), thereby reshaping the epigenetic state and transcriptional program [1, 2].

In mammals, the DNMT family includes DNMT1, DNMT3A, DNMT3B, and the catalytically weaker DNMT2 [3]. Family members exhibit distinct functional specificity and catalyze DNA methylation at CpG sites. DNMT1 maintains methylation patterns during replication, ensuring epigenetic inheritance [4]. DNMT3A and DNMT3B establish de novo methylation during early development and differentiation, aided by the catalytically inactive cofactor DNMT3L [5]. In contrast to conventional DNMTs, which are known to catalyze the specific methylation of DNA cytosine, DNMT2 has been observed to exhibit RNA methylation activity. This activity involves the methylation of the cytosine (C38) at position 38 of tRNA Asp, tRNA Gly, tRNA Val, and tRNA Glu, resulting in the formation of 5-methylcytosine (m5C) [6–8]. DNMT3L was considered a non-catalytically active cofactor that complexes with DNMT3A and DNMT3B and has been shown to enhance their catalytic activity when combined [9]. Instead, the current study suggests that DNMT3L may also be required for de novo DNA methylation in human embryos [9].

Opposing this modification are the ten-eleven translocation (TET) proteins - TET1, TET2, and TET3 - which sequentially oxidize 5-methylcytosine to 5-hydroxymethylcytosine and its derivatives, initiating active DNA demethylation [10, 11]. Both DNMT and TET families are essential for normal development; dysregulation of their activity leads to developmental disorders and tumorigenesis [12]. DNMTs and TETs antagonistically regulate DNA methylation homeostasis, ensuring spatiotemporal control of epigenetic states [13].

TET enzymes catalyze the iterative oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), initiating DNA demethylation via replication-dependent dilution or base excision repair [2, 14]. This dynamic regulation of methylation states orchestrates transcriptional plasticity and cell fate determination. TET1 and TET3 are critical for embryonic development and stem cell differentiation by reshaping epigenetic landscapes and transcriptional programs to direct cell fate. In contrast, TET2 dysfunction is linked to malignancies such as leukemia [15].

Although early studies have basically elucidated the methylation/demethylation mechanism of DNMT and TET [15–20], critical gaps remain in comparative analyses of their evolutionary trajectories, structural divergence, tissue-specificity, and cancer-related dysregulation. A systematic, cross-family comparison is essential to map evolutionary-structural-functional relationships in DNA methylation machinery and identify therapeutic targets against methylation-driven pathologies. We hypothesized that evolutionary constraints and structural innovations underpin functional divergence between DNMTs and TETs.

We performed phylogenetic and structural analyses of DNMT and TET protein families to resolve outstanding questions regarding their evolutionary relationships and functional divergence. Cross-species sequence alignment enabled phylogenetic reconstruction, revealing distinct evolutionary trajectories between these enzyme families. Comparative structural modeling using PyMOL identified significant variations in secondary structure composition and divergent spatial organization of catalytic domains. Particularly, the C-terminal regulatory domains exhibited family-specific folding patterns that correlate with differential substrate recognition capabilities. This integrated analysis provides mechanistic insights into how structural diversification of these epigenetic regulators underlies their specialized roles in DNA modification processes, with implications for understanding their dysregulation in carcinogenesis.

Materials and methods

Protein sequence data collection

From the UniProt database (https://www.uniprot.org/), Protein sequences of DNMT and TET family members from seven species (human, mouse, cow, sheep, pig, horse and dog) were obtained. The selection of seven species was based on two criteria: (1) they are relatively well-annotated mammalian species in UniProt, with relatively complete genome and protein annotations; and (2) they represent phylogenetic diversity within placental mammals, which is sufficient for comparative inference. Protein accession numbers were carefully checked to ensure sequence accuracy. Protein sequence numbers used herein are provided in the Supporting Table 1.

Table 1.

Protein names and PDB IDs

Protein	PDB ID
DNMT1	3PTA
DNMT2	1G55
DNMT3A	5YX2
DNMT3B	6PA7
DNMT3L	2PV0
TET1	6ASD
TET2	4NM6
TET3	4Z3C

Phylogenetic tree construction

The full-length protein sequences of DNMT and TET members were analyzed. Using the Clustal Omega program (https://www.clustal.org/omega/), multiple sequence alignments were performed. Then, using the MEGA 11 software (https://www.megasoftware.net/), build a phylogenetic tree based on maximum likelihood estimation (MLE). The bootstrap value was set to 500 for the robustness of the tree. Self-spread analysis was performed with 500 replicates to assess the reliability of the tree topology. The tree is presented as an unrooted tree. According to Tamura et al. [21], it is a common and reliable method of phylogenetic tree construction that can effectively reflect the evolutionary relationships between different proteins based on sequence similarity.

Protein structure analysis

We retrieved the 3D structures of human DNMT and TET proteins from the Protein Data Bank (https://www.rcsb.org/) and the UniProt database (https://www.uniprot.org/) Structures determined by X-ray crystallography or high-resolution NMR spectroscopy were preferred. For proteins lacking experimental structures, we used the SWISS-MODEL (https://swissmodel.expasy.org/) to build homology models based on known structural templates. AlphaFold predicted data is also used (https://alphafold.com/)

We visualized and analyzed the protein structures using the PyMOL 2.6.0 software. Using the software’s built-in function, we explored the differences in flexibility by comparing the B-factor values of amino acid residues in the CXXC domain. The B factor provides valuable information about the flexibility and mobility of protein regions, contributing to the understanding of the functional characteristics of different domains. We calculated the percentage of secondary structure for TET and DNMT proteins. The formula for each protein individually is: number of residues forming a specific secondary structure for that protein/total number of residues in that structure. The formula for protein families is: total number of residues forming a specific secondary structure for that protein family/total number of residues in all structures of that protein family. The PDB IDs of the structures used in this article are shown in the following Table 1.

B-factor analysis

B-factor is a measure used to describe the thermal motion or position uncertainty of atoms in the crystal structure of proteins or other molecules [22]. It reflects the vibration amplitude or mobility of atoms in the crystal structure: Low B-factor indicates that the thermal motion of atoms is very small, which usually occurs in relatively rigid regions of the structure. High B-factor indicates that the mobility of atoms is large, which is common in flexible regions of proteins. In PyMOL, by mapping the B-factor to the protein structure, it is possible to intuitively show which regions are more flexible. Using the A->preset->B-factor Putty-L-B-factor command in PyMOL software allows readers to view B-factor values in different structures and to display them in a gradient of blue, green, yellow, and red to compare flexibility differences.

Tissue specificity analysis

In this study, tissue-specific expression data of human DNMT and TET proteins were collected from the consensus dataset of the Human Protein Expression Atlas (https://www.proteinatlas.org/). Expression values were curated and summarized for multiple organs, and bar plots were generated to visualize distribution patterns across tissues. The tissue-specific analysis graph was drawn using the ggplot2 package in the R language.

Cancer signaling pathway analysis

Information regarding the roles of DNMT and TET proteins within cancer signaling pathways was gathered through an extensive literature review. The primary sources included PubMed (https://pubmed.ncbi.nlm.nih.gov/) and the Cancer Genome Atlas (https://cancergenome.nih.gov/), in addition to a search for pertinent research articles across other databases. The collected data were systematically organized and analyzed to elucidate the underlying mechanisms through which these proteins modulate cancer progression via diverse signaling pathways. In 2011, Hanahan and Weinberg presented a comprehensive overview of cancer hallmarks and the pivotal signaling pathways implicated, which served as a guiding framework for comprehending the functions of DNMT and TET proteins in the context of cancer [23].

Results

Evolutionary differentiation and structural characteristics of the DNMT and TET families in mammals

DNMT mediates DNA methylation in mammals. TET mediates demethylation mainly by converting 5mC into 5hmC, 5fC, and 5caC (Fig. 1A). Phylogenetic analysis of DNMT and TET families across seven mammalian species reveals distinct evolutionary trajectories (Fig. 1B). Separate evolutionary trees of mammalian TET and DNMT proteins were constructed (Fig. S1), and additional trees focusing on human TET and DNMT proteins were also generated (Fig. S2). From the evolutionary tree, it can be seen that DNMT1 and DNMT2 proteins show a close phylogenetic relationship compared to DNMT3A, DNMT3B and DNMT3L. DNMT1 is primarily involved in the maintenance of DNA methylation during replication, while DNMT3A and DNMT3B are implicated in de novo methylation processes. The divergent domain architectures of these proteins—characterized by DNMT1 possessing zinc-finger-CxxC-domain (CXXC), replication focus targeting sequence (RFTS), and bromo adjacent homology domain (BAH) domains, and DNMT3A/B containing Proline-Tryptophan-Tryptophan-Proline (PWWP) and ATRX-DNMT3-DNMT3L domain (ADD) domains—underscore their functional specialization (Fig. 1C). Similarly, TET1 and TET3 exhibit closer evolutionary kinship than TET2, implying conserved regulatory roles across species.

Fig. 1.

The epigenetic modifications and structural features of DNMT and TET proteins. (A) Chemical reactions depicting the conversion of cytosine bases in DNA by DNMTs and TETs, showing the formation of 5mC, 5hmC, 5fC, and 5caC. (B) Circular phylogenetic tree illustrating the evolutionary relationships among different DNMT and TET proteins across various species, with colored ranges indicating different protein types. The tree was constructed using the MLE method (Bootstrap = 500) (C) Domain architecture diagrams of DNMT and TET proteins, detailing the lengths (in amino acids) and functional domains such as CXXC, PWWP, DMAP1-binding, BAH, ADD, CRD, RFTS, Catalytic domain, Low complexity insert, and DSBH. (D) Structural models of DNMT and TET protein, which are predicted by AlphaFold, highlighting the catalytic domain and other structural elements with different colors for better visualization

All of the DNMTs utilize S-adenosylmethionine (SAM) as a methyl donor and employ a base-flipping mechanism to reposition the target base into the catalytic pocket of the enzyme [24]. Consequently, the C-terminal catalytic domain is crucial for DNMT functionality (Fig. 1D). The TET protein functions as a dioxygenase dependent on iron (II) and α-ketoglutaric acid (Fe (II)/α-KG). Its carboxy-terminal core catalytic domain is characterized by a double-stranded β-helix (DSBH) domain and a cysteine-rich domain [25]. The DSBH domain facilitates the clustering of Fe (II), α-KG, and 5mC together for the oxidation process, while the cysteine-rich domains encase the DSBH core to stabilize the overall structure and TET-DNA interactions [15] (Fig. 1D).

Conserved catalytic structures and DNA-interacting features of DNMT and TET proteins

Notwithstanding their divergent catalytic roles, the DNMT and TET protein families engage with DNA, thereby enabling an investigation into structural similarities via conserved sequences within their protein domains.

In our study of human genes that encode DNMT and TET proteins, we have identified an evolutionarily conserved sequence within the DNMT family (Fig. 2A). We labelled the critical binding positions of this conserved region. The 1226 C mutation of DNMT1 to A is inactive [26]. Its corresponding position, C710 of DNMT3A, is important for exerting methylation [27]. G1256-T1273 and G1288-D1312 of TET2 can form a shallow groove that interacts with DNA, and in addition, the H-D/E-H binding site in the TET family, which is essential for binding ferrous ions, was labelled by us [25]. The three-dimensional structure of DNMT3A exhibits a conserved region characterized by six β-sheet structures interconnected by α-helices (Fig. 2B). Similarly, the TET C-terminal catalytic domain features ten conserved and five semi-conserved β-sheets, including DSBH domain elements (Fig. 2B). These structures form rigid planar architectures stabilized by interchain hydrogen bonds, conferring structural stability.

Fig. 2.

Structure and sequence analysis of DNMT and TET proteins. (A) Sequence comparison of DNMT and TET proteins with conserved residues highlighted in yellow (relatively conserved) and red (more conserved). Purple box: critical region for substrate binding. Sky blue dots: DNMT key residues. Dark blue dots: Residues of TET-bound Fe ions. Brown arrows: β-sheet, light blue columns: α-helix. The source of this figure is shown in the Uniprot number of the human in the supporting data. (B) Domain schematic diagrams of DNMT and TET proteins, with arrows indicating β-sheet structures. Pale green: β-sheet structure of DNMT. Pale pink: β-sheet structure of TET. Structures shown are derived from AlphaFold predictions. The AlphaFold predicted structures of DNMT3A and TET2 are used in the structural presentation here. (C) Surface representations of DNMT1, DNMT2, DNMT3A, TET1, TET2, and TET3 proteins, colored by electrostatic potential (blue for positive, red for negative). The charge value range is displayed magnified. All the protein structures used above are predicted by AlphaFold

Consistent with previous studies, the present results suggest that the conserved β-strands of DNMT and TET proteins are essential for the integrity and activity of their catalytic domains [25]. Variant residues are critical for substrate coordination and catalytic activity. Structural modeling suggests β-sheets mediate DNA interactions through base/phosphate-group contacts via dedicated residues, while adjacent α-helices modulate structural polarity and flexibility without compromising β-sheet-derived stability.

Moreover, both protein families demonstrate a significant presence of basic amino acid residues within their catalytic regions (Fig. 2C). This positively charged residue-rich environment likely facilitates interactions with negatively charged DNA, enhancing substrate binding and catalytic efficacy. In summary, the conserved structural architecture of DNMT and TET catalytic domains underpins their functional versatility and efficacy in engaging with DNA.

Analysis of structural differences between DNMTs and TETs

TETs exhibit higher loop content and structural flexibility compared to DNMTs, reflecting distinct epigenetic roles

Using AlphaFold-predicted data, we calculated the secondary structure percentages of the TET and DNMT families. The calculations were based on full-length protein structures, including catalytic domains and intrinsic disordered regions (IDRs). Quantitative secondary structure analysis showed that TET families exhibit more loops than DNMT families (Fig. 3A). We attribute the high loop proportion to the presence of extensive IDRs and flexible linkers. Additionally, we separately calculated the secondary structure percentages of individual members of TET and DNMT in experimental data (partially for catalytic domains) and predicted data (Supporting Data 1 and 2). Furthermore, secondary structure percentages of TET and DNMT families from experimental data were calculated. Considering that DNMT2 may play a less prominent role in vivo, we also computed the mean values for DNMT1/3A/3B/3L only (Supporting Data 3). In all the above results, TET consistently showed a higher proportion of loop structures than DNMT, leading us to speculate that high loop content might be a structural feature of TET.

Fig. 3.

Structural insights into DNMT and TET proteins and their interactions with DNA. (A) Cartoon depictions of DNMT and TET proteins, where helices are distinctly colored in cyan, sheets in orange, and loops in light gray. Accompanying pie charts precisely illustrate the proportion of each secondary structure type within DNMT and TET proteins, providing a clear visual summary of their structural composition. (B) Surface models of DNMT1(PDB:3PTA) and DNMT3A (PDB:5YX2) proteins bound to DNA with a 180° rotation view of TET2 protein-DNA complex (PDB:4NM6), where helices are distinctly colored in bright orange, strands in pale cyan, and loops in white. (C) A comparative analysis of the CXXC structures of DNMT1 (PDB:3PTA), TET1 (PDB:6ASD), and TET3 (PDB:4Z3C) is presented. The B-factors of each CXXC were analyzed to observe the differences between the CXXCs from the perspective of protein flexibility. In the B-factor putty representation, the color gradient from blue-green-yellow-red indicates increasing flexibility, with blue representing the most rigid regions and red indicating the most flexible. (D) Key structures of DNMT2, TET1/2/3 are shown. Orange: regions 170–234 of DNMT2. then Self Purple: loop region of TET1/2/3. The protein structures used above are predicted by AlphaFold

The structure of a protein determines its function, and its three-dimensional conformation directly affects its specific binding and biological activity with substrates, ligands, or other molecules. TET protein catalyzes the oxidation of 5mC, which involves multiple reaction intermediates and depends on conformational flexibility, provided by its cyclic structures. Conversely, DNMT proteins maintain stable DNA methylation, necessitating strong binding to DNA, which correlates with a lower loop ratio. Notably, TET proteins also have low-complexity insertion regions that may contribute to their higher loop structure.

Varying evolutionary pressures have shaped the structural adaptations of these protein families. TET proteins likely underwent extensive changes to meet the complexities of DNA demethylation and epigenetic regulation, resulting in an increased loop structure proportion. Conversely, DNMT proteins have exhibited a relatively conserved function to maintain essential functions in DNA methylation across species, resulting in a stable loop ratio.

Structural analyses of protein-DNA complexes indicate that DNMT structures feature multiple helical components within their DNA-binding regions. In TET proteins, the DNA-bound region consists mainly of loop and β-fold structures, while the area behind the DNA-binding site contains helical structures (Fig. 3B). This variation highlights the distinctions in secondary structural components. DNMT proteins necessitate stable binding at CpG sites, rendering helical structures essential for effective methylation. In contrast, the flexibility provided by TET’s loop and β-fold structures allows for adaptation during the oxidative demethylation process. Nevertheless, the helical structures behind the DNA-binding site in TET proteins provide a level of stability that is critical for their diverse interactions.

Distinct conformational flexibility of CXXC domains suggests distinct DNA binding mechanisms between DNMTs and TET proteins

The analysis presented indicates that DNMT1, TET1, and TET3 all contain the CXXC domain. However, there are significant distinctions among these proteins [28, 29].

Subsequent structural analysis of the CXXC-DNA complexes revealed variations in both structural morphology and differences in conformational rigidity among the CXXC domains of different proteins. Specifically, B factor analysis revealed that the CXXC domain of DNMT1 exhibited uniform flexibility distribution, while the CXXC domain of TET1 and TET3 exhibited significant flexibility differences across different regions. These disparities in flexibility may have ramifications for their interactions with DNA and other factors (Fig. 3C). These results suggest that the three proteins differ in structural rigidity and conformational plasticity. However, it can be noted that the B factor itself is not directly indicative of DNA binding affinity, unless it is based on structural data of the complex. Consequently, this study merely postulates that alterations in the B factor may potentially impact the binding mode with DNA or other factors. However, discrepancies in binding affinity require experimental validation to ascertain their precise nature.

The CXXC domain of DNMT1 may exhibit relatively low conformational flexibility, which may support more stable DNA interactions. The CXXC domain of TET1 is relatively flexible and lacks overt DNA binding activity. However, this flexibility allows TET1 to be recruited to specific chromatin regions through interactions with other proteins (such as NANOG). Although the CXXC domain of TET1 does not directly bind DNA, its presence is essential for the biological function of TET1 [30]. The CXXC domain of TET3 exhibits remarkable conformational stability, attributable to its elevated rigidity. This property facilitates its precise anchoring to the gene promoter region, ensuring sustained residence and efficient execution of demethylation reactions [31].

Structural domain divergence within the DNMT family suggests functional specialization and substrate preferences

DNMT1 possesses a conserved C-terminal catalytic domain alongside a distinctive N-terminal regulatory domain, which encompasses functional subdomains such as DNMT1 binding, PCNA binding, NLS, RFTS, CXXC, and two BAH domains [32]. In contrast, DNMT3A and DNMT3B play essential roles in de novo methylation in mammals, with DNMT3L enhancing their activity as a cofactor. Both DNMT3A and DNMT3B share structural similarities, consisting, from N to C terminus, of a variable region for context-dependent regulation, a PWWP domain for initial DNA anchoring, cysteine-enriched regions for stability, and a C-terminal catalytic region [33]. Notably, DNMT3A exhibits a preference for specific CpG sites, while DNMT3B is more active at non-CpG sites, particularly in regions associated with ICF syndrome, reflecting structural differences.

In contrast, DNMT2 possesses only a C-terminal catalytic domain. Structural analysis identified a loop region (residues 170–234) in the catalytic domain of DNMT2 (Fig. 3D). This structural feature may contribute to the unique substrate recognition mechanism of DNMT2, potentially enabling its specialized function in tRNA methylation (Fig. 3D), corresponding to amino acid residues 170–234. To provide a more comprehensive reference within the above context, we further present structural comparisons of the corresponding regions among selected members of both the DNMT and TET families (Fig. S3). This loop is hypothesized to be potentially involved in tRNA binding for methylation, suggesting a distinct substrate-binding mechanism.

Distinct features of structural analysis of CXXC domains in the TET family

Both TET1 and TET3 contain the CXXC domain, which can bind to non-methylated CpG dinucleotides [34, 35]. The DNA-binding ability of the CXXC domain is closely related to its structural rigidity and electrostatic surface properties. In contrast, TET2 lacks an N-terminal CXXC DNA binding domain, but its function can indirectly regulate DNA binding specificity through the IDAX (also known as CXXC4) protein. This unique structural feature confers flexibility to TET2 in regulating chromatin states, enabling it to dynamically control gene expression by oxidizing 5mC [36]. IDAX expression leads to caspase activation and TET2 protein degradation, a process dependent on DNA binding by the IDAX CXXC domain [37].

Additionally, the low-complexity insertion regions among TET1, TET2, and TET3 consist of unconserved amino acid sequences that predominantly form loop structures. Structural comparison indicates variability in the dimensions of these regions (Fig. 3D), which significantly affects the conformation of the protein. Located within the core catalytic cavity, these insertion regions may contain multiple modification sites that could influence catalytic activity and potentially regulate the conformation of TET proteins. Prior studies have highlighted the crucial role of low-complexity insertion regions in the functional integrity of TET proteins, particularly in mechanisms that reduce the risk of overoxidation [38–40].

DNMT and TET family proteins exhibit distinct tissue-specific expression patterns

The tissue-specific expression of DNMT and TET family members is regulated by a combination of transcriptional, epigenetic, and post-transcriptional mechanisms [2, 41]. We mapped their tissue-specific expression profiles (Fig. 4). As shown in Fig. 4, DNMT1 is highly expressed in most tissues, especially in the liver and kidneys. DNMT1-mediated epigenetic regulation is considered essential for postnatal liver growth and regeneration and has also been proposed as a therapeutic target for alleviating diabetic nephropathy [42].

Fig. 4.

Tissue-specific expression profiles of DNMT and TET proteins. Bar charts presenting the expression levels of DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L, TET1, TET2, and TET3 across different tissues. Each bar corresponds to the expression level of a specific protein in a given tissue

DNMT3A and DNMT3B are predominantly active in germ cells and early embryonic development. Their expression underscores an essential role in establishing DNA methylation patterns. Aberrant expression of these proteins has been linked to various cancers, such as colorectal cancer and acute myeloid leukemia. Similarly, DNMT3L is highly expressed in germ cells and, while lacking methyltransferase activity, enhances the function of DNMT3A and DNMT3B through interactions.

Conversely, TET1, TET2, and TET3 are key players in DNA demethylation. TET2 exhibits heightened expression in the hematopoietic system, with its functional impairment has been linked to the development of leukemia. TET3 is notably expressed in the nervous system, indicating its potential role in neurodevelopment and function. These tissue-specific expression patterns underscore the functional specialization of each protein within the DNMT and TET families.

It is noteworthy that there is a close relationship between protein structure and tissue-specific expression. To illustrate this point, consider the examples of TET2 and DNMT3A. TET2 is distinguished by the absence of the CXXC domain, a consequence of a chromosomal inversion, which is encoded by the adjacent gene IDAX. TET2 is expressed at high levels in hematopoietic stem cells and their progenitor cells, and its expression gradually decreases during differentiation. Studies have demonstrated that the structural features of TET2, including the catalytic domain, directly influence its function, regulating the self-renewal, lineage-specific differentiation, and terminal maturation of hematopoietic stem cells [43]. In 2019, the ADD domain and the PWWP domain of DNMT3A were found to recognize histone modifications H3K4me0 and H3K36me2/3, respectively [44]. This structure-dependent histone interaction confers DNMT3A with tissue-specific localization capabilities. For example, in certain tissues, specific histone modification states recruit DNMT3A to perform DNA methylation in specific genomic regions, thereby influencing the spatiotemporal specificity of gene expression [45]. Future research endeavors must delve deeper into the intricate interplay between protein structure, signaling pathways, and disease mechanisms.

The role of DNMT and TET proteins in multiple signaling pathways affects the occurrence of cancer

Cancer signaling pathway analysis (Fig. 5) underscores the involvement of the DNMT and TET protein families in a range of malignancies, such as liver, colorectal, cervical, ovarian, lung, gastric, and breast cancer. The regulation of these proteins is predominantly mediated by the Wnt/β-catenin, PI3K-AKT, AMPK, NF-κB/Hippo, and Notch1/c-Myc signaling pathways.

Fig. 5.

Analysis of DNMT and TET protein regulatory pathways. Network diagram depicting the associations between DNMT and TET proteins and various cancers. All the protein structures used above are predicted by AlphaFold. Proteins are visualized by their structural representations, and cancers are illustrated as organ icons

DNMT1 affects the self-renewal and maintenance of liver cancer stem cells (CSCs) by regulating the methylation status of BEX 1 in liver cancer, making it a potential therapeutic target in hepatocellular carcinoma (HB) and CSC-HCC [46]. In colorectal cancer, the interaction of DNMT1 with β-catenin regulates the Wnt signaling pathway, affecting tumor genesis and progression [47]. Moreover, DNMT1 affects the expression of matrix metalloproteinase 14 (MMP 14) and Hepatic Nuclear Factor 1 Alpha (HNF1A) in cervical cancer by miR-484, and subsequently regulates the malignant characteristics of tumors [48].

DNMT3A In lung cancer, through miR-708-5p inhibition, reducing DNA methylation level and upregulating the expression of tumor suppressor gene CDH1, thus inhibiting Wnt/β-catenin signaling pathway and impairing the stemness properties of non-small cell lung cancer (NSCLC) cells [49]. In ovarian cancer, DNMT3A expression is inhibited by the combination of curcumin and DAC treatment, affecting the Wnt/β-catenin signaling pathway and alleviating ovarian cancer development [50].

TET1 influences the epithelial-mesenchymal transition (EMT) and cancer stem cells properties. It suppresses tumor metastasis and self-renewal [51]. In NPC, TET1 expression is downregulated, restoring the Wnt antagonists DACT 2 and SFRP 2 expression and inhibiting the Wnt/β-catenin signaling pathway by demethylation [52]. TET2 affects the PI3K / AKT / mTOR signaling pathway in breast cancer by regulating miR-660-5p, and promotes tumor progression [53]. TET3 promotes tumor proliferation, migration, and invasion in thyroid cancer by modulating the AMPK pathway [54]. Among these signaling pathways, the Wnt pathway emerges as the primary signaling pathway associated with DNMT and TET in the context of cancer.

Interestingly, DNMT1 and DNMT3A are often overexpressed in tumors, promoting aberrant methylation of tumor-related genes [55, 56]. TET proteins, especially TET2 often are downregulated in solid tumors [57, 58]. Its internal mechanism still needs further in-depth study. Additionally, Protein structural mutations have been demonstrated to induce aberrant regulation of signaling pathways, thereby precipitating uncontrolled cellular behavior and fostering cancer development. For instance, mutations in the catalytic domain of TET2 result in the loss of its catalytic activity, leading to elevated DNA methylation in the hematopoietic system. This, in turn, inhibits the expression of DNA repair genes, such as Breast Cancer 1 (BRCA1), thereby exacerbating genomic instability and ultimately driving the development of hematological tumors [59, 60]. Mutations in the PWWP domain of DNMT3A disrupt its ability to recognize H3K36me2/3 modifications, thereby interfering with DNA methylation patterns. In the context of cancer, such mutations have been observed to reduce the methylation levels of regulatory elements, such as enhancers, thereby activating stem cell-related genes and promoting tumorigenesis [61, 62].

Discussion

This study addresses the lack of an integrated analysis of DNMT and TET families by systematically comparing their evolutionary divergence, structural domains, regulatory mechanisms, and tissue-specific expression patterns. Phylogenetic analysis showed that DNMT1/2 in the DNMT family were closely related in terms of maintaining methylation function, and DNMT3A/B formed an independent branch responsible for de novo methylation; TET1/3 in the TET family were more closely related in terms of evolution. Structural analysis showed that both contained a conserved β-sheet catalytic core, but DNMT had more α-helices (required for stable binding) and TET contained more ring structures (demethylation flexibility). Functional specificity was manifested as follows: DNMT1 contained an N-terminal regulatory domain, DNMT3A/B had substrate selectivity, and DNMT2 specifically participated in tRNA methylation; TET2 lacked a CXXC domain but could interact and regulate DNA, and TET members contained different low-complexity regulatory regions. Tissue expression profiles showed that DNMT1 was continuously expressed in highly proliferative tissues such as liver/kidney, DNMT3A/B was active in reproductive/embryonic tissues, TET2 was enriched in the hematopoietic system, and TET3 was highly expressed in the nervous system. In terms of carcinogenic mechanisms, the two families jointly participate in the occurrence and development of various cancers through epigenetic regulatory networks, and their abnormal expressions can serve as potential biomarkers and therapeutic targets.

In short, previous studies have made great progress in the study of DNMT and TET. This article provides a more integrated and multidimensional perspective on the study of DNMT and TET from the perspectives of structure, tissue-specific expression, and disease occurrence. A key limitation of this study is that it relies on a protein prediction structural model that lacks experimental structure. Fine interaction details need to be further verified using experimental methods such as cryo-EM, such as the unique loop of DNMT2. Similarly, although B-factor values help assess domain flexibility, they also need further experimental verification. In future studies, cryo-electron microscopy technology can be further used to experimentally verify key structural features, and combined with single-cell multi-omics methods, the expression patterns of DNMT and TET proteins and their regulatory mechanisms can be deeply explored at the spatiotemporal level. Comparative studies of DNMT and TET in cancer will also promote the development of targeted drugs.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

DNMTs

DNA methyltransferases

TETs

TEN-eleven translocation family proteins

C38

Cytosine-38

5mC

5-methylcytosine

5hmC

5-hydroxymethylcytosine

5fC

5-formylcytosine

5caC

5-carboxycytosine

Fe (II)/α-KG

Dioxygenase dependent on iron (II) / α-ketoglutaricacid

DSBH

Double-stranded β-helix

AARS

Aminoacyl tRNA synthetase

CSCs

Cancer stem cells

HB

Hepatocellular carcinoma

NSCLC

Non-small cell lung cancer

EMT

Epithelial-mesenchymal transition

MMP 14

Matrix metalloproteinase 14

HNF1A

Hepatic Nuclear Factor 1 Alpha

HCC

Hepatocellular carcinoma

CXXC

Zinc-finger-CxxC-domain

RFTS

Replication focus targeting sequence

PWWP

Proline-Tryptophan-Tryptophan-Proline

ADD

ATRX - DNMT3 - DNMT3L domain

BAH

Bromo adjacent homology domain

SAM

S-adenosylmethionine

IDRs

Intrinsically disordered regions

BRCA1

Breast Cancer 1

Author contributions

S.C. and Y.Z. conceptualization; S.Y. visualization, formal analysis, writing - original draft; X.L. and L.B. writing - review & editing; J.C. and J.L. visualization.

Funding

This research was funded by the National Natural Scientific Foundation of China (62171241), the Group Project of Developing Inner Mongolia through Talents (2025TEL25), the Natural Science Foundation of Inner Mongolia Autonomous Region of China (2025QN03183) and the Central Guidance Fund for Local Science and Technology Development (2024ZY0168).

Data availability

Data provided in supporting materials.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.