On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase

Abstract

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

Graphical Abstract

Figure 1.

Analysis of flanking sequence effects on CpG methylation at HM, OH and UM sites. (A) Flanking sequence preferences of DNMT1 for the −4 to + 4 region. Profiles are based on the observed/expected ratio of nucleotide distribution in the methylated and unmethylated sequence reads. Shown are averages of profiles of individual time points, experiments and repeats and their SEM. N = 21 (including two data sets from Adam et al., 2020) in case of HM, 18 in case of OH and UM. See also Supplemental Figure S6. (B) Box plots of the average reaction rates at all 256 NNCGNN sites. Boxes show the median, 1st and 3rd quartile. Whiskers display the 1.5 IQR distance. Outliers are indicated by dots. X indicates the average. (C) Heatmap of average methylation rates at the NNCGNN sites. (D) Correlation of average methylation rates at NNCGNN sites in different substrates. Shown are Pearson r-values.

Figure 2.

Sequence preferences and DNMT1 specificity. (A) Occurrence of bases in the –2 to +2 positions of the 20 most preferred and most disfavored NNCGNN flanks. Flanks were sorted based on the methylation rates of all 256 HM, OH and UM substrates determined in the exponential fitting of the methylation data. (B) Degree and sequence context of HM/UM, HM/OH and OH/UM ratio preferences of WT DNMT1 based on the NNCGNN methylation rates.

Figure 3.

HM/UM specificity of the DNMT1 methylation reactions with mixed long HM, UM and patterned substrates. (A) Examples of methylation levels (see also Supplemental Figure S9). Yellow shading highlights the hemimethylated sites on the patterned substrate. (B) Average methylation rates of HM and UM substrates and HM as well as UM sites on the patterned substrate. UM, completely unmethylated substrate; HM, substrate only containing hemimethylated CpG sites; patterned substrate is hemimethylated at GCGC and CCGG sites and unmethylated at all other CpG sites.

Figure 4.

HM/UM specificity of the CXXC mutant DNMT1. (A) Compilation of NNCGNN methylation rates determined on HM and UM substrates with single CpG sites in randomized flanking context. Boxes show the median, 1st and 3rd quartile. Whiskers display the 1.5 IQR distance. Outliers are indicated by dots. X indicates the average. (B) Comparison of the methylation rates of WT DNMT1 and CXXC mutant on each NNCGNN substrate. See also Supplemental Figure S11. (C) Fits of the methylation of HM and UM sites on the long patterned DNA substrate by WT DNMT1 or the CXXC mutant.

Figure 5.

DNMT1 UM flanking sequence preferences shape cellular methylation patterns. (A) Compilation of cell lines used in this analysis. WT, 1KO, DKO and TKO data were taken from Li et al., 2015 (34), 5KO, 6KO and 6KO* date were taken from Wang et al., 2020 (35). (B) Correlation of the cellular NNCGNN methylation levels with the DNMT1 HM (D1 HM) and UM (D1 UM) preferences. Shown are Pearson r-values. (C) Correlation of cellular NNCGNN methylation levels of cell lines containing DNMT1 with DNMT1 flanking sequence preferences. (D) Correlation of UM/HM ratios of DNMT1 methylation preferences with genomic methylation levels of cell lines containing DNMT1. Boxes in panels C and D show the median, 1st and 3rd quartile. Whiskers display the 1.5 IQR distance. X indicates the average.