Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

Abstract

CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.CpG methylation is considered to be important for development, X chromosome inactivation, genomic imprinting, and suppression of transposons in mammals. The critical role played by the establishment and maintenance of DNA methylation has been demonstrated by the findings that homozygous disruption of any one of the three known methyltransferases in mice leads to lethality (23,28). It has also been shown that some methyl-CpG binding proteins associate with histone deacetylase and repress transcription (for a review, see reference 6). The importance of this has been exemplified by the recent identification that a human disease, Rett syndrome, is caused by mutations in one of the methyl-CpG binding proteins, MeCP2 (1). Recently, changes in DNA methylation patterns have also been implicated in carcinogenesis (for a review, see reference 18).Three vertebrate genes with known methyltransferase function have been identified, namely, Dnmt1, Dnmt3a, and Dnmt3b (4,27). Dnmt1 has been considered as a maintenance methyltransferase mostly based on its association with the replication foci (22) and its preference for hemimethylated DNA (29,30,37). It has been reported that Dnmt1 is capable of methylating fully unmethylated DNA as well as non-CpG sites in vitro (30,37). Dnmt1 has also been described to have no site preference other than CpGs (37). In Dnmt1 knockout mice, there is still a considerable a...

Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells

“…DNMT1 may therefore not be the only enzyme required to maintain methylation of CpG-rich regions, as both DNMT3a and -3b3 were still largely present after drug treatment. As indicated by the hemimethylation data, DNMT3a and -3b, which are presumably targeted to CpG-rich regions, may function to randomly methylate hemimethylated molecules generated by zebularine (26), whereas, in CpG-poor regions, there are more hemimethylated molecules than fully methylated molecules throughout the period of zebularine treatment, suggesting that perhaps these regions are basically maintained by DNMT1 alone.…”

CpG Sites Preferentially Methylated by Dnmt3a in Vivo

“…In addition, Hsieh (10) found that the cloned EBNA region of the Epstein-Barr virus genome on the episome was a better substrate for Dnmt3a than the control luciferase coding region, suggesting that there is a substrate preference for the enzyme regardless of its chromosomal location or structure. In fact, it has been demonstrated that purified Dnmt3a protein has a DNA sequence preference when either a plasmid or an oligonucleotide is used as the methylation substrate in vitro (11,12).In the present study, we attempted to identify the genomic DNA loci that are preferentially methylated by Dnmt3a or Dnmt3b in vivo. This was initially accomplished using methylation-sensitive restriction fingerprinting (MSRF), 2 which has been successfully utilized for the identification of differentially methylated genes in cancer cells (13) or during ES cell differentiation (14).…”

“…In addition, Hsieh (10) found that the cloned EBNA region of the Epstein-Barr virus genome on the episome was a better substrate for Dnmt3a than the control luciferase coding region, suggesting that there is a substrate preference for the enzyme regardless of its chromosomal location or structure. In fact, it has been demonstrated that purified Dnmt3a protein has a DNA sequence preference when either a plasmid or an oligonucleotide is used as the methylation substrate in vitro (11,12).…”

DNA Methylation Profile: A Composer-, Conductor-, and Player-Orchestrated Mammalian Genome Consisting of Genes and Transposable Genetic Elements

“…In vitro experiments indicated the differences in the preferred target sequences of these enzymes [70,71]. An inhibitor of Dnmt1, 5-Aza-deoxycytidine induces global demethylation of the genome as expected; however, locus-specific demethylation and methylation is observed [72].…”