Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase - PubMed (original) (raw)

Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase

Rachna Goyal et al. Nucleic Acids Res. 2006.

Abstract

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.

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Figures

Figure 1

Figure 1

Methylation of unmethylated and hemimethylated oligonucleotides by Dnmt1. Methylation was analyzed by incorporation of radioactive methyl groups into the DNA.In (A) 1 µM of hemimethylated and unmethylated oligonucleotides (CG30hm and CG30um) and 100 nM enzyme were used. In (B) 1 µM fully methylated oligonucleotide (CG30fm) of the same sequence was added.

Figure 2

Figure 2

Methylation of unmethylated and hemimethylated long substrates in different reaction tubes. (A) Methylation of unmethylated substrate 1 by Dnmt1. In (B) substrate 1 was prepared in hemimethylated state at all CG sites and used for methylation by Dnmt1. Methylation was analyzed after bisulfite modification of the DNA, PCR amplification, cloning and sequencing of at least 20 individual clones for each DNA strand. In the figure, some examples of the clones obtained and sequenced are shown. Methylated CG sites are shown black, unmethylated white.

Figure 3

Figure 3

Methylation of unmethylated and hemimethylated substrate in one reaction. (A) Methylation of unmethylated substrate 1 and hemimethylated substrate 2 by Dnmt1 in one reaction tube. Substrate 2 is hemimethylated at all CG sites in this experiment. (B) Methylation of substrate 2 hemimethylated at all CCGG sites. Methylation was analyzed after bisulfite modification of the DNA, PCR amplification, cloning and sequencing of at least 20 individual clones for each DNA strand. In the figure, some examples of the clones obtained and sequenced are shown. Methylated CG sites are shown black, unmethylated white. In (B) the hemimethylated CCGG sites are shaded gray.

Figure 4

Figure 4

(A) Processivity of the methylation of the unmethylated and hemimethylated CG sites by Dnmt1. The figure displays the distribution of lengths of all stretches of methylated sites for the unmethylated (left) and hemimethylated substrate (right). The line in the right panel indicates a fit to the random walk model with a _P_dif of 54. (B) Distribution of the number of termination events of processive methylation of the DNA at the various CG sites. The distribution is roughly fitted by a binomial distribution of the corresponding parameters as indicated by the line, if the two sites 19 and 42 are disregarded. (C) Flanking sequence preference for methylation of unmethylated DNA by Dnmt1.

Figure 5

Figure 5

Simulation of the average methylation level of an unmethylated and hemimethylated population of DNA molecules during several generations. For the simulation an average level of maintenance methylation of 95% and a 20-fold preference for hemimethylated DNA was used. In (A) the average methylation levels of the hemimethylated and unmethylated populations at one CG site are shown, in (B) the discrimination factor (defined as the ratio of the average methylation levels of both populations) is shown if one, three, five or seven CG sites are concerned.

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References

    1. Egger G., Liang G., Aparicio A., Jones P.A. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–463. - PubMed
    1. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 2002;16:6–21. - PubMed
    1. Jeltsch A. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem. 2002;3:274–293. - PubMed
    1. Jones P.A., Takai D. The role of DNA methylation in mammalian epigenetics. Science. 2001;293:1068–1070. - PubMed
    1. Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nature Rev. Genet. 2002;3:662–673. - PubMed

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