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In the 1970s, it was first discovered that the addition of a methyl group at the fifth position of the cytosine in a CpG dinucleotide could inactivate the expression of genes. Therefore, DNA methylation driven by DNA methyltransferase (DNMT) enzymes gained momentum as the most important epigenetic factor. Subsequently, the regulatory effect of histone acetylases and deacetylases added another level of complexity through regulating the acetylation status at the lysine tails at the histone core of the nucleosomes. Furthermore, it was uncovered that these enzymes were also able to bind and regulate DNMTs and other proteins having a DNA methylation-binding domain. The recognition that histone proteins can undergo methylation, phosphorylation and other modifications in addition to acetylation/deacetylation led to the discovery of histone demethylases, histone methyltransferases, and their numerous interactions with components of nucleosomes, histones, and DNA itself, as well as many protein complexes having nucleosome-remodeling activities.
DNA methylation is a significant process involved in developmental patterning, chromatin modification, and imprinting. Aberrant methylation has long been recognized to have a role in disease processes such as cancer. Methylation involves the addition of a methyl group (CH3) to specific cytosines base found in the DNA sequence to produce 5-methylcytosine (5mC) (Figure 1).
Figure 1. DNA methylation by DNA methyltransferase enzymes.
All DNA methyltransferases (DNMTs) use a similar catalytic mechanism that is characterized by the formation of a covalent reaction intermediate between the enzyme and the substrate base. Nevertheless, distinct catalytic motifs in DNMTs are used to generate 4mC, 5mC and 6mA. The catalytic motifs of cytosine‑5 DNMTs are highly conserved and can be used to identify these enzymes in DNA sequences. In fact, the DNMT family was established through the discovery that the catalytic motifs of the first identified mammalian cytosine‑5 DNMT, DNMT1, are highly conserved in bacterial cytosine‑5 DNMTs. Since then, a number of additional eukaryotic genes that contain the ten catalytic signature motifs present in cytosine‑5 DNMTs have been identified; this identification allowed the reconstruction of the evolutionary relationships between these enzymes and their subclassification.
An overview of DNMT family
The human genome encodes five DNMTs: DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. DNMT1, DNMT3A and DNMT3B are canonical cytosine‑5 DNMTs that catalyze the addition of methylation marks to genomic DNA. By comparison, DNMT2 and DNMT3L are non-canonical family members, because they do not possess catalytic DNMT activity. Nevertheless, both proteins have clear sequence conservation with canonical DNMT enzymes and represent evolutionary adaptations of original DNMT genes.
Animal DNMT enzymes are usually structured into an N‑terminal regulatory domain and a C‑terminal catalytic domain (Figure 2). An exception is DNMT2, which is composed of exclusively of the catalytic domain. The N‑terminal domain of DNMT1 contains several smaller conserved subdomains that mediate molecular interactions, such as the DNMT1‑associated protein 1 (DMAP1) binding domain, which is crucial for the interaction of DNMT1 with the transcriptional repressor DMAP1 and with the histone deacetylase HDAC2. The replication foci targeting sequence (RFTS) is necessary for targeting DNMT1 to replication foci and for the replication-dependent maintenance of DNA methylation.
Figure 2. Conserved domains of animal DNMT family members are shown in different colors.
DNMTs in human disease
DNMTs are key regulators of gene transcription, and their roles in carcinogenesis have been the subject of considerable interest. It has been reported that the expression levels of DNMTs are elevated in cancers of the breast, colon, prostate, liver, and in leukemia. Because inhibition of DNMTs is correlated with reduction of tumorigenicity and increased expression of tumor suppressor genes, DNMTs are attractive targets for the development of anticancer agents. Besides cancer, many other diseases have been associated with epigenetic alterations, particularly those influenced by the environment. DNA methylation has shown importance in Alzheimer’s disease and psychiatric diseases such as depression, schizophrenia, and bipolar disorder. DNA methylation is also involved in autoimmune diseases and genetic disorders.
In tumorigenesis, methylation in the promoter regions of some genes-such as tumor suppressor genes (TSGs) involved in cellular cycle (e.g., retinoblastoma protein (RB), cyclin-dependent kinase (CDK) inhibitors), maintenance of genome integrity (e.g., TP53, breast cancer 1 (BRCA1), O6-methylguanine DNA methyltransferase (06-MGMT), mutL homolog 1 (hMLH1)), apoptosis (e.g., caspase 8, death-associated protein kinase (DAPK)), migration process (e.g., E-cadherin (CDH1), metalloproteinase inhibitor 3 (TIMP-3)), and those involved in the response to growth factors (estrogen receptor (ER), phosphatase and tensin homolog (PTEN)) leads to their silencing. Concurrently, low levels of gene body methylation participate in genome instability. Thus, inhibition of DNA methylation is an interesting approach in cancer treatment. So far, several strategies to inhibit DNA methylation have been developed (Figure 3).
Figure 3. Schematic representation of different DNMT-inhibition approaches.
Even though a number of DNMT inhibitors other than cytidine analogues have been developed, their effects are not satisfactory and they do not seem to be able to replace azacytidine and decitabine, as yet. Azacytidine and decitabine, in spite of their limitations, are still used for combination epigenetic therapy. Therefore, attempts to discover and to develop novel compounds targeting DNMTs should be continued. It is important to find more selective and less toxic DNMT inhibitors that will be effective especially in patients with solid tumors.