Activity Of Dnmt3a Research Articles

MicroRNA (miRNA)-29b Targets DNMT3A and B and Induces Re-Expression of the Hypermethylated ESR1 and p15 Genes in Acute Myeloid Leukemia (AML).

Aberrant DNA methylation has been shown to play an important role in AML by silencing structurally normal genes important for hematopoiesis. MiRNAs are small non-coding RNAs that down-regulate gene expression. Although aberrant methylation of miRNAs has been reported, it is not known whether miRNAs themselves can modulate DNA methylation. To address this question, we tested the functional role of miRNAs that are predicted to target both DNA methyltransferase 3A and 3B (DNMT3A and B), in AML cells. We cloned the 3′UTR of both DNMT3s into a luciferase reporter and co-transfected K562 cells with scrambled or sense sequences of each of the miRNAs predicted to interact with DNMT3A and B. We found that miR-29b significantly repressed luciferase activity of both DNMT3A and B constructs (42% and 67%, respectively) compared with the controls. Transfection of the pre-miR-29b, but not of the scrambled, resulted in a marked reduction of the DNMT3A and B mRNA levels measured by qRT-PCR in MV4-11 cells (80% and 90%, respectively) and in K562 cells (23% and 67% respectively).We also observed a significant down-regulation of DNMT3s protein by immunoblotting in K562 cells transfected with miR-29b relative to the controls. These results were then validated in AML patients. Both miR-29b and DNMT3B expression levels were measured by qRT-PCR in bone marrow samples obtained from 12 AML patients with primary disease. We found that miR-29b expression was inversely correlated to DNMT3B mRNA levels (Pearson coefficient R=−0.66; P=0.03). Functionally, we showed that the mRNA expression of p15 and ESR1, that are silenced through promoter hypermethylation in AML, were increased in K562 and MV4-11 cells transfected with miR-29b compared with the controls. ESR1 was 1.9 and 1000 fold upregulated in miR-29b-tranfected K562 and MV4-11cells, respectively and p15 was 11.6 fold upregulated in miR-29b-tranfected MV 4–11 cells. Interestingly, we observed that the expression of miR-29b-1, which is silenced in K562 cell lines, was 6.5 fold up-regulated following treatment with 2.5 uM decitabine, a DNA hypomethylating nucleoside compared with untreated controls. Consistent with these results, we also showed 5.6 fold increase in miR-29b-1 expression in post-treatment CD34+ selected bone marrow blasts from AML patients (n=3) who received decitabine (20 mg/m2/day x 10 days) on a clinical protocol (OSU 0336) compared with pretreatment baselines. These results were consistent with the methylation analysis of CpGs up to 30kb upstream from the 5′ encoding regions of the miR-29b-1 precursors in the same patients. Using MassArray (sequenom) technology, we showed that all the CpGs were highly methylated (95 to 100%) in pre-treatment baseline samples and these methylation levels decreased by 20% after decitabine treatment. These preliminary data provide a functional link between miRNAs and aberrant epigenetics by suggesting that:a.miR-29b targets DNMT3A and B;b.miR-29b is silenced in AML cell lines and patient primary blasts and its expression is restored in vitro and in vivo by ypomethylating agents;3) increase of miR-29b levels results in DNMTs down-regulation and re-expression of otherwise hypermethylated genes. Altogether, there data support a rationale for developing novel miRNA-based therapeutic strategies that alone and in combination with other hypomethylating agents may target aberrant epigenetics in AML.

Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (Dnmt3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain. Here we show, with the use of crystallography, that the carboxy-terminal domain of human Dnmt3L interacts with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and Dnmt3L showed further dimerization through Dnmt3a-Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a-Dnmt3L interface or the Dnmt3a-Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA-Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.

Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair

While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.

Distinct DNA Methylation Activity of Dnmt3a and Dnmt3b towards Naked and Nucleosomal DNA

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.

RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.

DNA methyltransferase-3a interacts with p53 and represses p53-mediated gene expression

Genome stability maintenance is regulated by both genetic and epigenetic mechanisms. DNA methylation is the predominant epigenetic mechanism in regulation of gene expression and in suppression of mobile DNA elements from random integration in the genome. The importance of DNA methylation in tumorigenesis has been demonstrated in cancer cells which harbor global genomic DNA hypomethylation and regional hypermethylation at CpG islands of tumor suppressor genes. DNA methylation is mediated by a class of DNA methyltransferases (Dnmts) involved in de novo methylation of genomic DNA and in the maintenance of DNA methylation patterns during replication. Global genomic DNA demethylation induced by 5-Aza-deoxycytidine activates the p53 signaling pathway and induces apoptosis, suggesting that DNA methylation mediated by Dnmts is associated with p53 signaling in maintaining genome stability. In this report, we show that Dnmt3a interacts with p53 directly and represses p53-mediated transactivation of the p21 gene. It was found that trans-repression by Dnmt3a does not require the methyltransferase activity implying that transcriptional repression does not involve promoter silencing through DNA methylation by Dnmt3a. Finally, the activity of Dnmt3a in vivo was demonstrated when this enzyme was overexpressed in a breast cell line in which Dnmt3a repressed p21 upregulation following DNA damage. The results presented in this study provide new understanding of tumor promotion as mediated by Dnmt3a through its interaction with p53, and suppression of the p53-mediated transcription of tumor suppressor genes. Given that the expression of Dnmts is increased in certain cancers, it is likely that increased Dnmts could block the transactivation function of p53 following its induction by chemotherapeutic drugs resulting in chemoresistance. The use of a DNA methyltransferase inhibitor would therefore restore the p53 tumor suppression function and the utilization of such an inhibitor in combination with DNA damage agents might be an effective therapy for certain cancers.

De Novo Methylation of Nucleosomal DNA by the Mammalian Dnmt1 and Dnmt3A DNA Methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.

Mechanism of Stimulation of Catalytic Activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L

Dnmt3L has been identified as a stimulator of the catalytic activity of de novo DNA methyltransferases. It is essential in the development of germ cells in mammals. We show here that Dnmt3L stimulates the catalytic activity of the Dnmt3A and Dnmt3B enzymes by directly binding to their respective catalytic domains via its own C-terminal domain. The catalytic activity of Dnmt3A and -3B was stimulated approximately 15-fold, and Dnmt3L directly binds to DNA but not to S-adenosyl-L-methionine (AdoMet). Complex formation between Dnmt3A and Dnmt3L accelerates DNA binding by Dnmt3A 20-fold and lowers its K(m) for DNA. Interaction of Dnmt3L with Dnmt3A increases the binding of the coenzyme AdoMet to Dnmt3A, and it lowers the K(m) of Dnmt3A for AdoMet. On the basis of our data we propose a model in which the interaction of Dnmt3A with Dnmt3L induces a conformational change of Dnmt3A that opens the active site of the enzyme and promotes binding of DNA and the AdoMet. We demonstrate that the interaction of Dnmt3A and Dnmt3L is transient, and after DNA binding to Dnmt3A, Dnmt3L dissociates from the complex. Following dissociation of Dnmt3L, Dnmt3A adopts a closed conformation leading to slow rates of DNA release. Therefore, Dnmt3L acts as a substrate exchange factor that accelerates DNA and AdoMet binding to de novo DNA methyltransferases.

The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1

BackgroundThough Dnmt1 is considered the primary maintenance methyltransferase and Dnmt3a and Dnmt3b are considered de novo methyltransferases in mammals, these three enzymes may work together in maintaining as well as establishing DNA methylation patterns. It has been proposed that Dnmt1 may carry out de novo methylation at sites in the genome with transient single-stranded regions, such as replication origins, and then spread methylation from these nucleation sites in vivo, even though such activity has not been reported.ResultsIn this study, we show that Dnmt3a does not act on single-stranded substrates in vitro, indicating that Dnmt3a is not likely to initiate DNA methylation at such proposed nucleation sites. Dnmt3a shows similar methylation activity on unmethylated and hemimethylated duplex DNA, though with some substrate preference. Unlike Dnmt1, pre-existing cytosine methylation at CpG sites or non-CpG sites does not stimulate Dnmt3a activity in vitro and in vivo.ConclusionThe fact that Dnmt3a does not act on single stranded DNA and is not stimulated by pre-existing cytosine methylation indicates that the de novo methylation activity of Dnmt3a is quite different from that of Dnmt1. These findings are consistent with a model in which Dnmt3a initiates methylation on one of the DNA strands of duplex DNA, and these hemimethylated sites then stimulate Dnmt1 activity for further methylation.

Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells

Background: Acquired drug resistance is a major obstacle to the successful treatment of neuroblastoma by chemotherapy. Recent studies from our laboratory have demonstrated that drug-induced alterations in DNA methylation play an important role in this process. Methods: The reversal of resistance to cisplatin in murine neuroblastoma (MNB) was induced by inhibition of DNA methyltransferase activity. MNB cells overexpressing DNA methyltransferase activity (Dnmt3a or Dnmt3b) were established by stable co-transfection of wild type MNB cells with plasmids containing Dnmt3a or Dnmt3b cDNA. Cytotoxic response (IC 50), total DNA methyltransferase activity and expression of Dnmt3a or Dnmt3b methyltransferase were determined in Dnmt3a or Dnmt3b transfected MNB cells, respectively. Results: These data demonstrated that total DNA methyltransferase activity was increased to 3-fold above controls ( P < 0.001) in cisplatin resistant MNB cells, 3-fold in Dnmt3a and 4-fold in Dnmt3b transfected MNB cells. Western blot and RT-PCR data confirmed a corresponding increase in Dnmt3a and 3b expression in cisplatin resistant and transfected cells when compared with control MNB cells ( P < 0.001). MNB clones overexpressing Dnmt3a or Dnmt3b proteins were resistant to cisplatin treatment (10 −6 M). Incubation of cisplatin resistant, Dnmt3a or Dnmt3b overexpressing MNB cells with 5′-azacytidine (5′-azaC), a methylation inhibitor (2.5 μM) significantly decreased DNA methyltransferase activity, expression of Dnmt3a and Dnmt3b proteins and mRNA levels of cisplatin resistant, Dnmt3a and Dnmt3b transfected MNB cells. Cytotoxicity studies using the MTT assay demonstrated that the sensitivity of cisplatin resistant, Dnmt3a and Dnmt3b overexpressing MNB cells to cisplatin was increased 10-fold ( P < 0.001) following 5′-azaC treatment. Conclusions: These findings suggest that the overexpression of DNA methyltransferase is associated with a cisplatin resistant phenotype in MNB cells that may or may not be true in animal studies or in the clinical setup. Thus, DNA methylation plays a central role in onset of drug resistance in cisplatin resistant neuroblastoma cells in vitro.

Dimethyl sulfoxide stimulates the catalytic activity of de novo DNA methyltransferase 3a (Dnmt3a) in vitro

Mammalian DNA methyltransferase Dnmt3a is required for de novo methylation of CpG dinucleotides in genomic DNA. While DNA methyltransferase inhibitors have been extensively utilized both in vitro and in vivo, no stimulator of catalytic activity has been identified thus far. Here we show that the methyltransfer activity of Dnmt3a is stimulated by the addition of dimethyl sulfoxide (DMSO) to the reaction solution in vitro. Enzymatic analysis of initial reaction velocity suggests that the DMSO stimulation effect depends on the interaction between DMSO and the reaction substrates (DNA and AdoMet), but not the enzyme itself.

DNMT3L Stimulates the DNA Methylation Activity of Dnmt3a and Dnmt3b through a Direct Interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.

Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription.

The de novo DNA methyltransferase Dnmt3a is one of three mammalian DNA methyltransferases that has been shown to play crucial roles in embryonic development, genomic imprinting and transcriptional silencing. Despite its importance, very little is known about how the enzymatic activity and transcriptional repression functions of Dnmt3a are regulated. Here we show that Dnmt3a interacts with multiple components of the sumoylation machinery, namely the E2 sumo conjugating enzyme Ubc9 and the E3 sumo ligases PIAS1 and PIASxalpha, all of which are involved in conjugating the small ubiquitin-like modifier polypeptide, SUMO-1, to its target proteins. Dnmt3a is modified by SUMO-1 in vivo and in vitro and the region of Dnmt3a responsible for interaction maps to the N-terminal regulatory domain. Functionally, sumoylation of Dnmt3a disrupts its ability to interact with histone deacetylases (HDAC1/2), but not with another interaction partner, Dnmt3b. Conditions that enhance the sumoylation of Dnmt3a in vivo abolish its capacity to repress transcription. These studies reveal a new level of regulation governing Dnmt3a whereby a post-translational modification can dramatically regulate its interaction with specific protein partners and alter its ability to repress transcription.

Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpA sites

We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K m values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 μM, and the k cat values are 0.05 h −1 and 0.07 h −1, respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.

Protein binding protects sites on stable episomes and in the chromosome from de novo methylation.

We have utilized the Escherichia coli lac repressor-operator system to test whether protein binding can interfere with de novo DNA methylation in mammalian cells. We find that a DNA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of Dnmt3a. Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, and it does not affect the binding protein protection of these sites from de novo methylation. This study and previous studies provide a possible mechanism for the observation that an Sp1 site can serve as a cis-acting signal for demethylation and for preventing de novo methylation of the CpG island upstream of the mouse adenine phosphoribosyltransferase (Aprt) gene. These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.