Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.     

Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome

Mammalian DNA methyltransferases methylate cytosine residues within CG dinucleotides. By statistical analysis of published data of the Human Epigenome Project we have determined flanking sequences of up to +/-four base-pairs surrounding the central CG site that are characteristic of high (5'-CTTGCGCAAG-3') and low (5'-TGTTCGGTGG-3') levels of methylation in human genomic DNA. We have investigated the influence of flanking sequence on the catalytic activity of the Dnmt3a and Dnmt3b de novo DNA methyltransferases using a set of synthetic oligonucleotide substrates that covers all possible +/-1 flanks in quantitative terms. Methylation kinetics experiments revealed a >13-fold difference between the preferred (RCGY) and disfavored +/-1 flanking base-pairs (YCGR). In addition, AT-rich flanks are preferred over GC-rich ones. These experimental preferences coincide with the genomic methylation patterns. Therefore, we have expanded our experimental analysis and found a >500-fold difference in the methylation rates of the consensus sequences for high and low levels of methylation in the genome. This result demonstrates a very pronounced flanking sequence preference of Dnmt3a and Dnmt3b. It suggests that the methylation pattern of human DNA is due, in part, to the flanking sequence preferences of the de novo DNA MTases and that flanking sequence preferences could be involved in the origin of CG islands. Furthermore, similar flanking sequence preferences have been found for the stimulation of the immune system by unmethylated CGs, suggesting a co-evolution of DNA MTases and the immune system.     

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

DNA methylation is critical for Arabidopsis embryogenesis and seed viability

DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.     

Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA.

Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo.     

Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements.

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.     

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.     

Longevity of 5-azacytidine-mediated gene expression and re-establishment of silencing in transgenic rice

Epigenetic silencing of a bialaphos resistance (bar) gene in R1 progeny of a transgenic rice line was found to be meiotically stable since selfed (R2) progeny were also susceptible and the bar locus highly methylated. A high proportion of R2 seedlings germinated in the presence of 5-azacytidine (AzaC) were herbicide-resistant and also contained at least one unmethylated copy of the bar gene, further establishing the relationship between silencing and methylation. Restored bar gene expression was typically maintained for 20–50 days, but eventual methylation and silencing of the bar locus underscores the ability of the recipient genome to recognize and inactivate intrusive DNA.     

Plant DNA methyltransferases

DNA methylation is an important modification of DNA that plays a role in genome management and in regulating gene expression during development. Methylation is carried out by DNA methyltransferases which catalyse the transfer of a methyl group to bases within the DNA helix. Plants have at least three classes of cytosine methyltransferase which differ in protein structure and function. The METI family, homologues of the mouse Dnmt1 methyltransferase, most likely function as maintenance methyltransferases, but may also play a role in de novo methylation. The chromomethylases, which are unique to plants, may preferentially methylate DNA in heterochromatin; the remaining class, with similarity to Dnmt3 methyltransferases of mammals, are putative de novo methyltransferases. The various classes of methyltransferase may show differential activity on cytosines in different sequence contexts. Chromomethylases may preferentially methylate cytosines in CpNpG sequences while the Arabidopsis METI methyltransferase shows a preference for cytosines in CpG sequences. Additional proteins, for example DDM1, a member of the SNF2/SWI2 family of chromatin remodelling proteins, are also required for methylation of plant DNA.     

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.     

Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure

Dnmt1 is the predominant DNA methyltransferase (MTase) in mammals. The C-terminal domain of Dnmt1 clearly shares sequence similarity with many prokaryotic 5mC methyltransferases, and had been proposed to be sufficient for catalytic activity. We show here by deletion analysis that the C-terminal domain alone is not sufficient for methylating activity, but that a large part of the N-terminal domain is required in addition. Since this complex structure of Dnmt1 raises issues about its evolutionary origin, we have compared several eukaryotic MTases and have determined the genomic organization of the mouse Dnmt1 gene. The 5' most part of the N-terminal domain is dispensible for enzyme activity, includes the major nuclear import signal and comprises tissue-specific exons. Interestingly, the functional subdivision of Dnmt1 correlates well with the structure of the Dnmt1 gene in terms of intron/exon size distribution as well as sequence conservation. Our results, based on functional, structural and sequence comparison data, suggest that the gene has evolved from the fusion of at least three genes.     

Cooperation between EZH2, NSPc1-mediated histone H2A ubiquitination and Dnmt1 in HOX gene silencing

An intricate interplay between DNA methylation and polycomb-mediated gene silencing has been highlighted recently. Here we provided evidence that Nervous System Polycomb 1 (NSPc1), a BMI1 homologous polycomb protein, plays important roles in promoting H2A ubiquitination and cooperates with DNA methylation in HOX gene silencing. We showed that NSPc1 stimulates H2A ubiquitination in vivo and in vitro through direct interaction with both RING2 and H2A. RT-PCR analysis revealed that loss of NSPc1, EZH2 or DNA methyltransferase 1 (Dnmt1), or inhibition of DNA methylation in HeLa cells de-represses the expression of HOXA7. Chromatin immunoprecipitation (ChIP) assays demonstrated that NSPc1, EZH2 and Dnmt1 bind to the promoter of HOXA7, which is frequently hypermethylated in tumors. Knockdown of NSPc1 results in significant reduction of H2A ubiquitination and DNA demethylation as well as Dnmt1 dissociation in the HOXA7 promoter. Meanwhile Dnmt1 deficiency affects NSPc1 recruitment and H2A ubiquitination, whereas on both cases EZH2-mediated H3K27 trimethylation remains unaffected. When EZH2 was depleted, however, NSPc1 and Dnmt1 enrichment was abolished concomitant with local reduction of H3K27 trimethylation, H2A ubiquitination and DNA methylation. Taken together, our findings indicated that NSPc1-mediated H2A ubiquitination and DNA methylation, both being directed by EZH2, are interdependent in long-term target gene silencing within cancer cells.