Dnmt3a and Dnmt3b are two major de novo DNA methyltransferases essential for embryonic development in mammals. It has been shown that Dnmt3a and Dnmt3b have distinct substrate preferences for certain genomic loci, including major and minor satellite repeats. However, the exact target CpG sites where Dnmt3a and Dnmt3b catalyze DNA methylation remains largely unknown. To identify a CpG site that is specifically methylated by Dnmt3a or Dnmt3b, we screened methylated genomic loci by methylation sensitive restriction fingerprinting using genomic DNA from wild-type, Dnmt3a null, Dnmt3b null, and Dnmt3a-Dnmt3b double null ES cells. Interestingly, one of the CpG sites was preferentially methylated in wild-type and Dnmt3b null ES cells but not in Dnmt3a null or Dnmt3a-Dnmt3b double null ES cells, suggesting that the site-specific methylation was Dnmt3a-dependent. Sequencing results revealed that the isolated CpG site is located within the 1st exon of the G isoform of fibroblast growth factor (Fgf-1.G) on mouse chromosome 18. Exogenous expression of Dnmt3a but not Dnmt3b in the double null ES cells restored DNA methylation of this CpG site. When we examined alternative transcription initiation sites, we determined that another CpG site in the 5'-flanking region of the Fgf-1.A isoform was also methylated specifically by Dnmt3a. Using chimeric constructs between Dnmt3a and Dnmt3b, we further determined that the NH(2)-terminal regulatory domain of Dnmt3a was responsible for establishing its substrate specificity. These results indicate that certain CpG sites within the Fgf-1 gene locus are preferentially methylated by Dnmt3a but not by Dnmt3b. Selective methylation by a specific member of Dnmt3 may therefore play a role in the orchestration of gene expression during embryonic development.
"Lately, high recurrence of Dnmt3a mutations has been reported in acute myeloid leukemia (Ley, et al., 2010; Shah & Licht, 2011). In addition, certain CpG sites within the Fgf-1 gene locus have been proved to be selectively methylated by Dnmt3a in vivo but not by Dnmt3b (Oka, et al., 2006). The mechanism and significance of this activity are still undiscovered. "
"However, the methylation activity of both Dnmt3a and Dnmt3b substantially decreases when the DNA substrate is packaged into nucleosomes, the basic unit of chromatin in eukaryotic cells (7–9), and Dnmt3b was reported to have higher nucleosomal DNA methylation activity than Dnmt3a (8). Moreover, although Dnmt3a and Dnmt3b have overlapped targets in vivo, Dnmt3a is specifically required for the methylation of some single copy genes, such as imprinted genes in germ cells and the Xist gene on the X chromosome (10–12), while Dnmt3b is irreplaceable in the methylation of minor satellite repeats and other different repetitive sequences depending on the cell types (5,11,13,14). Notably, repetitive sequences have increased dramatically in the mammalian genomes (15,16), and these repetitive sequences represent a large part of the genome and contain the large majority (>90%) of 5-methylcytosines (m5C) in the genome (17). "
[Show abstract][Hide abstract] ABSTRACT: Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.
Nucleic Acids Research 10/2010; 38(18):6054-64. DOI:10.1093/nar/gkq456 · 9.11 Impact Factor
"It is an open question how DNA methylation patterns are generated by DNA methyltransferases. For the catalytic domain of Dnmt3a it has been observed that it shows some flanking sequence preferences (33,34) and it favors the methylation of CpG sites in a distance equal to one helical turn of DNA (12,35). Both these properties were shown to influence the DNA methylation in cells, but they cannot explain the generation of a specific DNA methylation pattern during embryogenesis and germ cell development. "
[Show abstract][Hide abstract] ABSTRACT: Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1-19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1-19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.
Nucleic Acids Research 03/2010; 38(13):4246-53. DOI:10.1093/nar/gkq147 · 9.11 Impact Factor
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