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. 2024 May 16;7(1):582.
doi: 10.1038/s42003-024-06252-z.

Specific DNMT3C flanking sequence preferences facilitate methylation of young murine retrotransposons

Leonie Dossmann  1 , Max Emperle  1 , Michael Dukatz  1 , Alex de Mendoza  2 , Pavel Bashtrykov  1 , Albert Jeltsch  3
Affiliations

Specific DNMT3C flanking sequence preferences facilitate methylation of young murine retrotransposons

Leonie Dossmann et al. Commun Biol. .

Abstract

The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline. Using specialized assay systems, we investigate the flanking sequence preferences of DNMT3C and observe characteristic preferences for cytosine at the -2 and -1 flank that are unique among DNMT3 enzymes. We identify two amino acids in the catalytic domain of DNMT3C (C543 and V547) that are responsible for the DNMT3C-specific flanking sequence preferences and evolutionary conserved in muroids. Reanalysis of published data shows that DNMT3C flanking preferences are consistent with genome-wide methylation patterns in mouse ES cells only expressing DNMT3C. Strikingly, we show that CpG sites with the preferred flanking sequences of DNMT3C are enriched in murine retrotransposons that were previously identified as DNMT3C targets. Finally, we demonstrate experimentally that DNMT3C has elevated methylation activity on substrates derived from these biological targets. Our data show that DNMT3C flanking sequence preferences match the sequences of young murine retrotransposons which facilitates their methylation. By this, our data provide mechanistic insights into the molecular co-evolution of repeat elements and (epi)genetic defense systems dedicated to maintain genomic stability in mammals.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structure and purification of DNMT3C catalytic domain proteins.
a Domain composition of different DNMT3 enzymes. The murine DNMT3C protein (Uniprot entry P0DOY1) consists of 740 amino acids. See also Supplementary Fig. 1. b Structure of the heterotetrameric complex of DNMT3B catalytic domain and DNMT3L C-terminal domain. DNMT3B is shown in orange and brown, DNMT3L in grey. Residues differing in DNMT3C and DNMT3B are colored pink in the orange DNMT3B subunit. DNA is shown in blue with the -1 flank base pair in cyan and the -2 flank base pair in yellow. c Purification of WT DNMT3C catalytic domain and mutants. Lane 1: WT DNMT3C, lane 2: C543N/V547A mutant, lane 3: C543N mutant, lane 4: V547A mutant, lane 5: E590K mutant. M: Protein size marker. d Zoom into the DNMT3B/3L structure focusing on the -2 and -1 flank region. Coloring is as described in panel (b). N656 (corresponding to C543 in DNMT3C), A660 (V547 in DNMT3C) and K703 (E590 in DNMT3C) are highlighted. There are no other amino acids differences between DNMT3C and DNMT3B near the DNA at the -2/-1 flank region.
Fig. 2
Fig. 2. Flanking sequence preference analysis of DNMT3 enzymes.
a Schematic picture of the design of the random flank substrates. b-e Combined -4 to +4 flanking profiles of WT DNMT3C, WT DNMT3B, WT DNMT3A and the DNMT3C C543N/V547A mutant. DNMT3A data were combined from,; DNMT3B data were from. Shown are observed/expected (o/e) values for the occurrence of individual bases at each position in the methylated products. DNMT3C-specific C-preference at position -2 and -1 is highlighted by blue arrows, the DNMT3A and DNMT3B characteristic preference for T(-2) is labelled with a red arrow. See also Supplementary Figs. 2 and 3. f Correlation of the combined -8 to +8 flank profiles of the different DNMT3s. Shown are Pearson r values.
Fig. 3
Fig. 3. Comparison of the flanking sequence preferences of DNMT3B, DNMT3C and DNMT3C mutants determined with model substrates using radioactively labelled AdoMet.
a Multiple sequence alignment of DNMT3A/B/C orthologues in muroidea and humans, highlighting the region around mouse DNMT3C positions C543 and V547. Amino acid color depicts conservation. b Species tree of the selected muroidea species found to contain bona fide DNMT3C orthologues, with topology and divergence time adapted from Steppan and Schenk (2017). c Methylation rates of two designed substrates to be preferred by DNMT3B and DNMT3C. Methylation rates were determined for WT DNMT3B, WT DNMT3C, and DNMT3C mutants C543N, V547A, E590K, and the C543N/V547A double mutant. Shown are averages of three experiments, error bars represent the SD, data points are indicated by circles. The -4 to +4 part of the sequence of the substrates is indicated.
Fig. 4
Fig. 4. Detailed analysis of NNCGNN flanking sequence preferences of DNMT3C.
a Comparison of the NNCGNN methylation rates of WT DNMT3C, DNMT3C C543N/V547A (NA) double mutant and WT DNMT3B. Examples of the rate fittings are shown in Supplementary Fig. 5. Shown are the 10% most preferred/disfavored sites. DNMT3B data were taken from. b Enrichment and depletion of bases at the -2 to +2 flank of the 10% most preferred and most disfavored NNCGNN sequences. DNMT3C-specific effects at positions -2 and -1 are highlighted by arrows.
Fig. 5
Fig. 5. Correlation of DNMT3C flanking sequence preferences and genomic methylation.
a Correlation of the NNCGNN flanking profiles of different DNMTs with the genomic methylation of 6KO cells. DNMT data were taken from: DNMT1, DNMT3A,; DNMT3B. Shown are Pearson r values. See also Supplementary Fig. 8 and Supplementary Table 4. b Correlation of DNMT3C flanking sequence preferences and genomic methylation in o/e ratios for all 256 NNCGNN sequences. The boxes show the median, 1st and 3rd quartile. Whiskers display the 1.5 IQR distance. X indicates the average. c Enrichment and depletion of bases at the -2 to +2 flank of the 10% most highly/lowly methylated NNCGNN sequences in 6KO cells. DNMT3C-specific effects at position -2 and -1 are highlighted by arrows. d Methylation levels of NNCG sites in 6KO cells expressed as o/e. The DNMT3C-specific CC preference is highlighted by an arrow. e Local correlation analysis of methylation levels and DNMT3C NNCGNN preferences of consecutive CpG sites in chromosome 1 of 6KO. R-values were determined in a 22 CpG site window, positive r-values are shown in blue, negative ones in orange. Examples of local correlations are shown in Supplementary Fig. 9.
Fig. 6
Fig. 6. Enrichment of CCCG or CGGG sequences in mouse repeat elements that are DNMT3C targets.
a Image of the L1MdA and IAPEz-int elements showing the frequencies of CpG sites, CCCG + CGGG sites and fraction of CpG sites in CCCG or CGGG context, all expressed as o/e frequencies averaged over 50 bps. “Expected” values were determined using the C and G content of the entry. The annotated ORFs of the elements are indicated. Note the clustering of CCCG/CGGG sites in the promoter of L1MdA and at several regions in the body of IAPEz. b Frequency of CG sites in a CCCG or CGGG context in mouse repeat elements. The figure shows data for three LINE elements and three ERVK elements that are validated DNMT3C targets. As a control, two ERVK elements are shown, which are not methylated by DNMT3C. In addition, data are shown for examples of one murine ERV1 and ERVL element that both are also not targeted by DNMT3C and for one human ERV2 element. For the L1 elements, the promoter regions were used for the analysis, in all other cases the entire sequence of the element was used. See Supplementary Table 5 and Supplementary Fig. 10 for more information. The p-value was determined by a two-flanked T-test assuming unequal variance. c Methylation rates of two designed repeat substrates with sequences taken from known DNMT3C targets. Shown are averages of three experiments, error bars represent the SD. Data points are indicated by circles. P-values were determined by a two-sided T-test assuming equal variance, n.s. not significant. See also Supplementary Fig. 4.
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