An essential role for DNA methyltransferase DNMT3B in cancer cell survival

Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival.     

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.     

Role of DNMT3B in the regulation of early neural and neural crest specifiers

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.     

Role of DNMT3B in the regulation of early neural and neural crest specifiers

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.     

Evolution of the vertebrate DNMT3 gene family: a possible link between existence of DNMT3L and genomic imprinting

DNA methylation plays an essential role in genomic imprinting observed in eutherian mammals and marsupials. In mouse, one of the two de novo DNA methyltransferases, Dnmt3a, and a related protein, Dnmt3L have been shown to be essential for imprint establishment in the parental germline. To gain insights into the evolution of imprinting mechanisms, we have identified and characterized the DNMT3 family genes in other vertebrate species. We cloned cDNAs for chicken DNMT3A and DNMT3B, whose putative protein products shared 81.5% and 48.6% amino acid sequence identity with their mouse orthologues. Using computer-assisted database searches, we also identified DNMT3A and DNMT3B orthologues in fish (fugu and zebrafish) and marsupials (opossum). We found that, while opossums had an orthologue for DNMT3L, chickens and fish did not have this gene. Thus, unlike the other DNMT3 members, DNMT3L was restricted to the species in which imprinting occurs. The acquisition of DNMT3L by a common ancestor of eutherians and marsupials might have been closely related to the evolution of imprinting.     

Down-regulation of DNMT3b in PC3 cells effects locus-specific DNA methylation,and represses cellular growth and migration

Background  

Aberrations in DNA methylation patterns promote changes in gene expression patterns and are invariably associated with neoplasia. DNA methylation is carried out and maintained by several DNA methyltransferases (DNMTs) among which DNMT1 functions as a maintenance methylase while DNMT3a and 3b serve as de novo enzymes. Although DNMT3b has been shown to preferentially target the methylation of DNA sequences residing in pericentric heterochromatin whether it is involved in gene specific methylation remains an open question. To address this issue, we have silenced the expression of DNMT3b in the prostate-derived PC3 cells through RNA interference and subsequently studied the accompanied cellular changes as well as the expression profiles of selected genes.     

Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L

The DNMT3-like protein, DNMT3L, is required for germ line DNA methylation, although it is inactive as a DNA methyltransferase per se. Previous studies have shown that DNMT3L physically associates with the active de novo DNA methyltransferases, DNMT3A and DNMT3B, and stimulates their catalytic activities in a cell culture system. However, the mechanism by which DNMT3L stimulates de novo methylation remains unclear. Here, we have purified the full-length human DNMT3A2 and DNMT3L proteins and determined unique conditions that allow for the proper reconstitution of the stimulation of DNMT3A2 de novo methyltransferase activity by DNMT3L. These conditions include the use of buffers resembling physiological conditions and the preincubation of the two proteins. Under these conditions, maximal stimulation is reached at equimolar amounts of DNMT3L and DNMT3A2 proteins, and the catalytic efficiency of DNMT3A2 is increased up to 20-fold. Biochemical analysis revealed that whereas DNMT3L on its own does not significantly bind to the methyl group donor, S-adenosyl-L-methionine (SAM), it strongly increases the binding of SAM to DNMT3A2. DNA binding, on the contrary, was not appreciably improved. Analysis of DNA methyltransferase complexes in solution using size exclusion chromatography revealed that DNMT3A2 forms large structures of heterogeneous sizes, whereas DNMT3L appears as a monomer. Binding of DNMT3L to DNMT3A2 promotes a dramatic reorganization of DNMT3A2 subunits and leads to the formation of specific complexes with enhanced DNA methyltransferase activity and increased SAM binding.