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DNMT3A is the gene product of the Dnmt3a gene and is an enzyme responsible for de novo DNA methylation. Such function is to be distinguished from maintenance DNA methylation which ensures the fidelity of replication of inherited epigenetic patterns. DNMT3A forms part of the family of DNA methyltransferase enzymes, which consists of the protagonists DNMT1, DNMT3A and DNMT3B.

While de novo DNA methylation modifies the information passed on by the parent to the progeny, it enables key epigenetic modifications essential for processes such as cellular differentiation and embryonic development, transcriptional regulation, heterochromatin formation, X-inactivation, imprinting and genome stability.

As of now, however, the full spectrum of action of DNMT3A remains unknown, and its association with diseases such as cancer remains blurry as shall be discussed below. The following quote is very much illustrative of our current understanding of thi key enzyme. "On a mechanistic level, despite nearly two decades of basic research, we understand only poorly how DNMT3A carries out its duties with regard to DNA methylation and gene expression, and we have no inkling of any DNA methylation-independent functions."

Structure and Phylogenetic Profile
DMT3A is a 130 kDa protein encoded by 23 exons found on chromosome 2p23 in humans. Interestingly, the whole protein considered, there exists a 98% homology between human and murine homologues, thus showing structural conservation across species.

Due to splicing, there exist two main murine RNA isoforms, Dnmt3a1 and Dnmt3a2. These isoforms exist in different cell types, the rationale for such variation being non-determined.

DNMT3A consists of three major protein domains: the Pro-Trp-Trp-Pro (PWWP) domain, the ATRX-DNMT3-DNMT3L (ADD) domain and the catalytic methyltransferase domain. Interestingly, the ADD domain serves as an inhibitor of the methyltransferase domain until DNMT3A binds to the unmodified lysine 4 of histone 3 (H3K4me0) for its de novo methylating activity. This protein thus seems to have an inbuilt control mechanism targeting histones only for methylation. Finally, the methyltransferase domain is highly conserved, even among prokaryotes!

Mechanism of Action and Function
As mentioned in the introduction, DNMT3A is involved in de novo DNA methylation. It is important to know, however, how this happens chemically."Methylation of DNA refers to the addition of a methyl (CH3) group to the C5 position of the pyrimidine ring of cytosines to form 5-methylcytosine (5mC)8, usually in the context of a CpG dinucleotide pair."Indeed, CpG sites are pervasive throughout the genome and exist in two forms, CpG individual sites and CpG islands – clusters of CpG sites. 60-80 % of the individual sites are methylated while the islands are characteristically unmethylated. Such unmethylated sites offer opportunities for epigenetic modifications via de novo methylation. High levels of methylation are associated with gene silencing, a characteristic property of cancerous cells.

It is also important to consider how DNMT3A fits within the epigenetic machinery. We will focus here on its functions relative to its methyltransferase counterparts, DNMT1 and DNMT3B.

Briefly, DNMT1 carries out maintenance DNA methylation while DNMT3A and DNMT3B are believed to carry out both maintenance – correcting the errors of DNMT1 – and de novo DNA methylation. Such belief for the dual function of the DNMT3s originates in a number of findings. For example, DNMT1 knockout human cancer cells were found to retain about 80% of their inherited methylation pattern, thus suggesting maintenance activity by the expressed DNMT3s – assuming no other methyltransferases exist. Further evidence arose from the discovery that DNMT3s show equal affinity for unmethylated and hemimethylated DNA substrates while DNMT1 has a 10-40 fold preference for hemimethylated DNA. This means that the DNMT3s can bind to both forms and hence potentially do both maintenance and de novo modifications.

Yet, de novo methylation is the main recognized activity of DNMT3A, which is essential for processes such as those mentioned in the introductory paragraphs. An interesting fact to note is that genetic imprinting prevents parthenogenesis in mammals, and hence forces sexual reproduction and its multiple consequences on genetics and phylogenesis. DNMT3A is essential for genetic imprinting.

Clinical relevance
The study of this gene in mice has shown its reduced expression in ageing animals causes cognitive long-term memory decline. It is also frequently mutated in cancer, being one of 127 frequently mutated genes identified in the Cancer Genome Atlas project, in a publication involving whole-genome sequencing on 3,281 cancers of various types. In the Cancer Genome Atlas study, DNMT3A mutations were most commonly seen in acute myeloid leukaemia (AML) where they occurred in just over 25% of cases sequenced. The most often occur at the R882 residue of the protein, an amino acid substitution at position 882 from an arginine (R) to another amino acid, and this mutation probably causes loss of function. DNMT3A mutations are associated with poor overall survival, suggesting that they have an important common effect on the potential of AML cells to cause lethal disease.

DNMT3A in Hematological Malignancies
This section is dedicated to DNMT3A and its association with acute myeloid leukemia (AML). The review article used here is highly recommended for further reading and understanding.

It is judicious to start with the following quote, which gives an idea of the prevalence of DNMT3A-associated malignancies. "'Mutations in DNMT3A have now been found in most types of haematological malignancy with varying frequency... the overwhelming prevalence of DNMT3A mutations across a range of diseases demonstrates the special role of this gene in preventing malignancy.'"Therefore, DNMT3A – in its mutated form – is involved in hematological malignancies. Unfortunately, as illustrated by the quote in the introductory paragraph, the exact role of this enzyme remains an elusive piece of knowledge that needs to be elucidated.

However, an intriguing clue about this protein’s mechanism of influence in malignancies has been found and is absolutely worth discussing here.

As mentioned earlier, DNMT3A and the methyltransferase family are involved in cell differentiation, which requires the silencing of certain genes while specific other ones are expressed. De novo methylation is most likely involved, although there still is no certainty there either. Therefore, inactivating mutations in DNMT3A may hinder such differentiation and favor self-replication instead to make clones. Indeed, ‘In Dnmt3a-/- mice, many genes associated with HSC self-renewal increase in expression and some fail to be appropriately repressed during differentiation.’. As mentioned in the review, this suggests abrogation of differentiation in hematopoietic stem cells (HSCs) and an increase in self-renewal cell-division instead. Indeed, it was found that differentiation was partially rescued if Dnmt3a-/- HSCs experienced an additional Ctnb1 knockdown – Ctnb1 codes for β-catenin, which participates in self-renewal cell division.

Based on such evidence, therefore, it may be suspected that HSCs bearing Dnmt3a mutations will show accumulation over time relative to wild-type HSCs, which fully enter differentiation and lose the ability self-renew. The issue with such an argument is that it assumes the mutants can evade detection by defenses such as the immune machinery while forming a pre-leukemic lesion. Indeed, ‘Two independent studies found that human HSCs purified from patients with AML could harbour DNMT3A mutations in the absence of other common leukaemia-associated mutations.’ Whether such lesions enable the generation of further mutations for overt leukemia is a question that remains unanswered presently. However, another more intricate consequence is possible.

A genomic study involving 42,000 healthy – no hematological malignancies – individuals revealed that hematopoiesis often became clonally derived with time. For instance, it was discovered that ‘5-10% of 70-year-olds derived almost all of their peripheral blood cells from a single HSC.’  This Darwinian nature of the bone marrow could, therefore, be exploited by Dnmt3amut HSCs over time so that all cells eventually emanate from such mutants or one of them as seen in some individuals.

The reason why this could be relevant to leukemia and more specifically to AML is that, should leukemic mutations arise in Dnmt3amut.cells, their proliferation would be facilitated, thus leading to full blown leukemia. Evidently, this excludes the other genetic consequences that that DNMT3Amut has on the genome.

To illustrate such relevance, one can use a recent (2015) bone marrow transplant genomic study. In this study, a brother donated bone marrow (BM) to his brother, and they both eventually – only a few months later – developed AML. The donor probably was on the verge of developing the malignancy and he passed it on to the brother through his donated BM cells. This resembles an infectious disease, and, in some sense, it is. The relevance of this study here is the remarkable presence of Dnmt3amut HSCs in the donor BM. Besides, after treatment and complete remission (CM), it was found that Dnmt3mut-only cells populated the bone marrow in both brothers. Unfortunately, soon after treatment, relapse occurred and the Dnmt3amut load corresponded to the prevalence of mutations typically associated with AML, as if Dnmt3amut cells were the origin of malignant cells. If that is the case, Dnmt3amut cells could be regarded as acting as cancer stem cells (CSCs) that are resistant to treatment. This suggests a possible role of the DNMT3A mutant protein in the generation of further genetic lesions contributing to the development of AML. That, however, still needs to be clarified via further studies.

In essence, DNMT3Amut seems to give a selective advantage to HSCs possessing it, so their accumulation over time and association with AML-linked mutations lead to the development of AML.

Interactions
DNMT3A has been shown to interact with:


 * DNMT1,
 * DNMT3B,
 * HDAC1,
 * Myc,
 * PIAS1,
 * PIAS2,
 * SUV39H1,
 * UBE2I and
 * ZNF238.

Model organisms
Model organisms have been used in the study of DNMT3A function. A conditional knockout mouse line called Dnmt3atm1a(KOMP)Wtsi was generated at the Wellcome Trust Sanger Institute. Male and female animals underwent a standardized phenotypic screen to determine the effects of deletion. Additional screens performed: - In-depth immunological phenotyping