Overexpression

Overexpression of DNMTs (DNMT1, DNMT3A, and DNMT3B) in a variety of tumors results in hypermethylation and oncogenic activation [11]. DNMT1 overexpression correlates well with aberrant DNA methylation in solid tumors, thus resulting in lymph node metastasis and poor prognosis in patients [22–24]. Similarly, highly expressed DNMT3A or DNMT3B has been found in a large number of patient specimens, and increased DNMT3A expression is involved in hepatocellular carcinogenesis [25]. Moreover, high expression levels of DNMT3B and CTCF are critical in the epigenetic inactivation of BRCA1 in sporadic breast tumors [26]. Additional studies have suggested that DNMT3B is required for the outgrowth of colonic micro-adenomas [27, 28]. Several studies have provided explanations for the relationship between overexpressed DNMTs and tumorigenesis. Zhao et al. have shown that DNMT1 knockdown has an inhibitory effect on the cell cycle in esophageal squamous cell carcinoma, indicating that increased methylation levels promote cell mitosis [22]. Two groups have demonstrated that DNMT3B overexpression is closely related to CIMP-high in colon cancers [29, 30]. Additional studies performed on cultured primary prostate cells have shown that the overexpression of DNMT3B1 and DNMT3B2, the two subtypes of DNMT3B, leads to an increase in methylation [31].

Mutation

Somatic mutations in DNMTs are the prominent features of many tumors and substantially contribute to malignant transformation [32]. As shown in Table 1, DNMT1 mutations in colon tumors and DNMT3A mutations in hematological malignancies have been observed in the cancer genome. Kanai et al. have shown that DNMT1 inactivation due to mutational changes in colon cancers results in genome-wide alterations of the DNA methylation status [33]. Critical findings on DNMT3A variation have suggested that DNMT3A is frequently mutated in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and adult early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) and is associated with disease aggressiveness and treatment resistance [15, 16, 34–36]. Mice expressing the Dnmt3a Arg882 mutant protein developed chronic myelomonocytic leukemia with thrombocytosis [37]. Moreover, DNMT3A mutations, particularly those in the catalytic domain, substantially decrease enzymatic activity [16, 34]. In DNMT3A-mutated AML samples and relevant mouse models, such loss of function results in the hypomethylation of HOX family genes [16, 37]. Together, these studies suggest that mutated DNMTs disrupt genomic methylation and play significant roles in tumor formation.

Deletion

An in vivo mouse model with embryonically inactive DNMT3A and DNMT3B has shown that the deletion of de novo methyltransferases leads to lethal phenotypes [9]. Recently, the effects of de novo methyltransferase on hematopoiesis have been evaluated through conditional knockout technology. The deletion of Dnmt3a in adult mice induces the proliferation of hematopoietic progenitors [38]. On the basis of this abnormality, researchers then demonstrated that mutated NRAS- or FLT3-ITD-driven malignancy is accelerated by a lack of Dnmt3a [39–42]. Furthermore, the ectopic introduction of c-Kit variants into a Dnmt3a-deficient background produces acute leukemia [43]. Moreover, DNMT3A inactivation leads to the progression of peripheral T cell lymphoma (PTCL) and lung tumors, thus indicating that DNMT3A may act as a tumor-suppressor gene [44, 45]. Studies have also shown that DNMT3B acts as a tumor suppressor in Myc-induced lymphomas and MLL-AF9-driven AML [46]. A lack of maintenance methyltransferase activity is also related to carcinogenesis. Studies have shown that DNMT1 deletion leads to DNA demethylation and that DNMT1 is critical for T-cell lymphoma prevention and maintenance, contributing to aberrant methylation by de novo and maintenance methylation [47]. Therefore, deletion of genes encoding DNMTs also participates in tumor development.

Global hypomethylation

DNA hypomethylation of tumor cells is the first process characterized as an epigenetic abnormality [19]. The genome-wide hypomethylation of tumor cells results in a reduction of 5-mC, mainly in gene-coding regions and satellite repeats (Fig. 1). These changes cause mitotic recombination, copy number deletion and chromosomal rearrangement, and even genomic imprinting annihilation. Gaudet et al. have demonstrated that deletion or reduction of DNMT1 leads to substantial genome-wide hypomethylation and chromosomal instability [51]. Through methylated DNA immunoprecipitation (MeDIP)-chip analysis, hypomethylated CpG islands (CGIs) of the HOXB cluster have been found in AML samples with DNMT3A mutations [16]. Although the underlying mechanism governing the effects of genome-wide hypomethylation on the process of tumorigenesis is not fully understood, these limited data have provided alternative insight into a relationship between aberrant DNMTs and global hypomethylation, along with subsequent tumor occurrence [1].

Localized hypermethylation

In normal somatic cells, DNA methylation occurs primarily in dinucleotides containing less CpG, whereas the CpG-enriched region is unmethylated [6, 50]. Throughout malignant transformation, the global methylation level of DNA changes, thus leading to non-CpG island hypomethylation and CGI hypermethylation. As a result, the number of genes that are hypermethylated on their promoters increases. In particular, hypermethylation induces the silencing of several key tumor-suppressor genes (TSGs), which play important roles in tumor progression (Fig. 1). Generally, abnormal CGI hypermethylation is an epigenetic characteristic of tumors, of which hypermethylated TSGs are the most common feature [1, 13].

A great deal of research has been performed to explore the mechanism of aberrant TSG methylation in tumor tissue. As expected, DNMTs have been included in the aforementioned studies. In leukemia, deletions or mutations in DNMTs often disrupt the distribution of 5-mC in the genome [52]. Butcher et al. have shown that in some sporadic breast tumors, hypermethylation of the BRCA1 promoter is partially due to DNMT3B overexpression [26]. Using a conditional Dnmt3a knockout mouse model, researchers have observed a general decrease in hypomethylation in the transcription factor-binding sites of cross-regions (Canyons) [53]. Additionally, canyon-associated genes, including HOX genes, are markedly enriched in DNMT3A mutant AMLs [53].

Tumor-associated DNA methylation generally occurs in the promoter regions of TSGs [13]. However, owing to rapid advancements in methylation sequencing, data increasingly indicate that a large number of non-TSGs are methylated at the early stage of tumor initiation, and methylation changes within the gene body have a substantial effect on the process of transcription. The TCGA network has reported the integrated methylation profiles of AML samples with mutations in DNMT3A, as determined with Human Methylation 450 Bead Chip arrays [54]. This complete epigenomic landscape reveals a large amount of hypermethylated cytosine bases in the gene body and intergenic regions. Similarly, in a Dnmt3a mutant-transduced mouse model, hypermethylation is greater in the intergenic regions, and a cluster of suppressed genes related to lymphocyte development, such as Notch1 and Gata3, are hypermethylated in the gene body regions [37]. Furthermore, Yang et al. have suggested that DNMT3B-dependent gene body methylation enhances transcription and may be a potential therapeutic target in cancer [55].

Interaction with histone modifications

The entire epigenetic profile of the genome shows that active chromatin regions are generally characterized by acetylated histones and unmethylated DNA, whereas methylated histones associated with repressed chromatin and methylated DNA are enriched in suppressed regions [50]. Thus, the two chromatin markers interact in a highly orchestrated manner and are closely linked: DNA methylation helps guide histone modification, and histone modification directs DNA methylation (Fig. 1). For example, DNMT1 is required for the maintenance of H3K9 methylation in human cancer cells [56], and DNMT3A PWWP interacts with H3K36me3 and consequently enhances DNMT3A activity [57]. These effects can be regarded as the outcome of cooperation between histone methyltransferase (HMT) and DNMTs. Indeed, DNMTs form complexes with HMTs and consequently regulate transcription. Both the H3K36 methyltransferase SETD2 and the PWWP domain of DNMT3B are required for the de novo methylation of transcribed genes [58]. Likewise, the ADD domain of DNMT3A recognizes unmodified H3, which is repressed by H3K4 methylation [59]. In undifferentiated human embryonic carcinoma cells, promoter-related DNMTs overlap with different histone modifications [60]. Two groups have demonstrated that DNMT1 improves genomic methylation through enhanced histone modification by EZH2. EZH2 polycomb group protein mediates H3K27 methylation and recruits and directly controls DNA methylation [61, 62]. Thus, the above-mentioned investigations confirm that abnormal DNA methylation in tumor cells is closely related to histone modification. The relationship between DNA methylation and histone modification should provide more comprehensive insights into epigenetic regulation in tumorigenesis.

DNMT3A structure and function

As a member of the DNMT family, DNMT3A possesses the characteristic peptide structure: its catalytic domain directly binds to S-adenosyl-L-methionine (SAM) and DNA strands, and the N-terminus regulation domain is primarily involved in nuclear localization and protein interactions, in which the PWWP domain interacts with methyl lysine histones, and the PHD domain recognizes unmethylated histones. These functions serve as a signal of the histone transfer effect, thus ensuring diverse epigenetic modification [5]. Specifically, DNMT3A forms a butterfly-shaped tetramer (DNMT3L-DNMT3A-DNMT3A-DNMT3L) in the C-terminus with DNMT3L, thus changing the conformation of DNMT3A and facilitating its catalytic activity. The N-terminus of DNMT3A also operates as a transcriptional repressor. The regulatory domain recruits nucleoproteins into the complex and performs histone modifications, chromatin remodeling and gene transcription. A range of partners is known to interact with DNMT3A, including histone methyltransferases, histone deacetylases, and various transcription factors, even enzymes in the DNMT family [50]. DNMTs are bound to each other, and de novo methyltransferase said in coordination during methylation maintenance [66]. Studies have shown that H3K9 methyltransferases, such as SUV39H1 and SETDB1, can directly bind to the PHD domain of DNMT3A and improve each other’s catalytic activity, thus indicating that different epigenetic modifications can enhance chromatin inhibition by cooperating together [67].

DNMT3A mutation in leukemia

Clinically, in patients with DNMT3A mutations, the number of leukocytes present at diagnosis is relatively higher, and survival is comparatively shorter [15, 16]. To date, numerous functional experiments have provided a better understanding of the effects of DNMT3A mutation on leukemia pathogenesis (Fig. 2). For instance, DNMT3A mutation is an early event in the initiation of hematopoietic disorders and is one of several causative factors for the establishment of founder clones and the transformation of hematopoietic stem cells (HSCs) to pre-leukemic stem cells (Pre-LSCs) [68]. In addition, DNMT3A mutants harbor dominant-negative effects, such as those exhibited by DNMT3A R882H (R878H in mouse) mutated protein against wild-type DNMT3A [69, 70]. DNMT3A mutations also disrupt hematopoiesis. Researchers have used a bone marrow transplantation mouse model to determine that the function of Dnmt3a mutants in blood cell production is aberrant [37]. Additional studies have shown that mutated DNMT3A disrupts normal hematopoiesis and promotes the transformation to malignant cells, in combination with other epigenetic regulators [71]. In Dnmt3a-mutated models, a double-hit is essential for clonal expansion. In vivo experiments suggest that the mutation or deletion of Dnmt3a induces the development of leukemia by cooperating with oncogenic factors, such as RAS mutation, c-Kit variation, or FLT3-ITD abnormalities [39–43, 72]. DNMT3A mutations may also play an important role in tumor metastasis. For example, DNMT3A mutant leukemia cells may undergo leukemic extramedullary infiltration in NOD/SCID mice, a result partially linked to high expression levels of TWIST1, an epithelial-mesenchymal transition (EMT) inducer [73]. In summary, DNMT3A mutation exerts a great influence in hematological malignancy. A variety of small molecule compounds targeting relevant epigenetic disruptions have been developed and applied in the treatment of leukemia, which also provide a comprehensive innovation for the study of pathogenesis and targeted therapy of solid tumors.