The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites

In the cell, Dnmt1 is the major enzyme in maintenance of the pattern of DNA methylation after DNA replication. Evidence suggests that the protein is located at the replication fork, where it could directly modify nascent DNA immediately after replication. To elucidate the potential mechanism of this process, we investigate the processivity of DNA methylation and accuracy of copying an existing pattern of methylation in this study using purified Dnmt1 and hemimethylated substrate DNA. We demonstrate that Dnmt1 methylates a hemimethylated 958-mer substrate in a highly processive reaction. Fully methylated and unmethylated CG sites do not inhibit processive methylation of the DNA. Extending previous work, we show that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation. Completely unmodified DNA is methylated even more slowly due to an allosteric activation of Dnmt1 by methylcytosine-containing DNA. Interestingly, Dnmt1 is not able to methylate hemimethylated CG sites on different strands of the DNA in a processive manner, indicating that Dnmt1 keeps its orientation with respect to the DNA while methylating the CG sites on one strand of the DNA.     

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.     

Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase

DNA methyltransferase 1 (Dnmt1) has a central role in copying the pattern of DNA methylation after replication which is one manifestation of epigenetic inheritance. With oligonculeotide substrates we show that mouse Dnmt1 has a 30- to 40-fold preference for hemimethylated DNA that is almost lost after addition of fully methylated oligonucleotides. Using long hemimethylated DNA substrates that carry defined methylation patterns and bisulfite analysis of the methylation reaction products, we show a 15-fold preference for hemimethylated CG sites. Dnmt1 moves along the DNA in a random walk methylating hemimethylated substrates with high processivity (>50 sites are visited on average which corresponds to linear diffusion over 6000 bp). The frequency of skipping sites is very low (<0.3%) and there is no detectable flanking sequence preference. CGCTC sites tend to terminate the processive methylation of DNA by Dnmt1. Unmethylated DNA is modified non-processively with a preference for methylation at CCGG sites. We simulate the propagation of methylation patterns using a stochastic model with the specificity of Dnmt1 observed here and conclude that either methylation of several sites is required to propagate the methylation information over several cellular generations or additional epigenetic information must be used.     

The DNA Methyltransferase Dnmt1 Directly Interacts with the SET and RING Finger-associated (SRA) Domain of the Multifunctional Protein Uhrf1 to Facilitate Accession of the Catalytic Center to Hemi-methylated DNA

Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.     

The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2′-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.     

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.     

Molecular enzymology of the catalytic domains of the Dnmt3a and Dnmt3b DNA methyltransferases

The C-terminal domains of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b harbor all the conserved motifs characteristic for cytosine-C5 methyltransferases. Whereas the isolated catalytic domain of Dnmt1 is inactive, we show here that the C-terminal domains of Dnmt3a and Dnmt3b are catalytically active. Neither Dnmt3a nor Dnmt3b shows a significant preference for the satellite 2 sequence, although Dnmt3b is required for methylation of these regions in vivo. However, the catalytic domain of Dnmt3a methylates DNA in a distributive reaction, whereas Dnmt3b is processive, which accelerates methylation of macromolecular DNA in vitro. This property could make Dnmt3b a preferred enzyme for methylation at satellite 2 repeats, since they are highly CG-rich. We have also analyzed the catalytic activities of six different mutations found in ICF (immunodeficiency, centromeric instability, and facial abnormalities) patients in the catalytic domain of Dnmt3b. Five of them display catalytic activities reduced by 10-50-fold; one mutant was inactive in our assay (residual activity <1%). These results confirm that a reduced catalytic activity of Dnm3b causes ICF. However, the mutations in general do not completely abrogate catalytic activity. This finding may explain why ICF patients are viable, whereas nmt3b knock-out mice die during embryogenesis.     

The impact of 6-thioguanine incorporation into DNA on the function of DNA methyltransferase Dnmt3a

The incorporation of chemotherapeutic agent 6-thioguanine (^SG) into DNA is a prerequisite for its cytotoxic action. This modification of DNA impedes the activity of enzymes involved in DNA repair and replication. Here, using hemimethylated DNA substrates we demonstrated that DNA methylation by Dnmt3a-CD is reduced if DNA is damaged by the incorporation of ^SG into one or two CpG sites separated by nine base pairs. An increase in the number of ^SG substitutions did not enhance the effect. Dnmt3a-CD binding to either of ^SG-containing DNA substrates was not distorted. Our results suggest that ^SG incorporation into DNA may influence epigenetic regulation via DNA methylation.     

Mammalian DNA methyltransferases show different subnuclear distributions

In mammalian cells, DNA methylation patterns are precisely maintained after DNA replication with defined changes occurring during development. The major DNA methyltransferase (Dnmt1) is associated with nuclear replication sites during S-phase, which is consistent with a role in maintenance methylation. The subcellular distribution of the recently discovered de novo DNA methyltransferases, Dnmt3a and Dnmt3b, was investigated by immunofluorescence and by epitope tagging. We now show that both Dnmt3a and Dnmt3b are distributed throughout the nucleoplasm but are not associated with nuclear DNA replication sites during S-phase. These results suggest that de novo methylation by Dnmt3a and Dnmt3b occurs independently of the replication process and might involve an alternative mechanism for accessing the target DNA. The different subcellular distribution of mammalian DNA methyltransferases might thus contribute to the regulation of DNA methylation.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

Oligomerization and binding of the Dnmt3a DNA methyltransferase to parallel DNA molecules: heterochromatic localization and role of Dnmt3L

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting.     

Novel anticancer drug curaxin CBL0137 impairs DNA methylation by eukaryotic DNA methyltransferase Dnmt3a

Novel DNA intercalating anticancer drug curaxin CBL0137 significantly inhibited in vitro DNA methylation by eukaryotic DNA methyltransferase Dnmt3a catalytic domain (Dnmt3a-CD) at low micromolar concentrations (IC₅₀ 3–9 µM). CBL0137 reduced the binding affinity of Dnmt3a-CD to its DNA target, causing up to four-fold increase in the K_d of the enzyme/DNA complex. Binding of CBL0137 to Dnmt3a-CD was not observed. The observed decrease in methylation activity of Dnmt3a-CD in the presence of CBL0137 can be explained by curaxin’s ability to intercalate into DNA.     

Mechanism of CpG DNA Methyltransferases M.SssI and Dnmt3a Studied by DNA Containing 2-Aminopurine

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase–DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme–substrate complexes were observed.     

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.     

Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.     

Np95 interacts with de novo DNA methyltransferases,Dnmt3a and Dnmt3b,and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.     

Stimulation effect of Dnmt3L on the DNA methylation activity of Dnmt3a2

Quantification of DNA methyltransferases Dnmt3a and Dnmt3a2, and Dnmt3L in isolated male gonocytes in day 16.5 embryos confirmed that not Dnmt3a but Dnmt3a2 and Dnmt3L were the major Dnmt3s. The expression level of Dnmt3L constituted 5- to 10-fold molar excess compared to that of Dnmt3a2. The stimulation property of the DNA methylation activity of Dnmt3a2 with Dnmt3L towards substrate DNA in naked or nucleosomes was similar to that of Dnmt3a. However, the DNA methylation activity of not Dnmt3a but Dnmt3a2 was severely inhibited at the physiological salt concentration. Interestingly, the activity of Dnmt3a2 was significantly detected in the presence of Dnmt3L even at the physiological salt concentration. This indicates that Dnmt3a2 functions only in the presence of Dnmt3L in male gonocytes, and may explain why Dnmt3L is required specifically in mouse gonocytes for DNA methylation.