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Nucleic Acids Res
2010 Jul 01;3813:4246-53. doi: 10.1093/nar/gkq147.
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Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail.
Zhang Y
,
Jurkowska R
,
Soeroes S
,
Rajavelu A
,
Dhayalan A
,
Bock I
,
Rathert P
,
Brandt O
,
Reinhardt R
,
Fischle W
,
Jeltsch A
.
???displayArticle.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.
Figure 1. Binding of the Dnmt3a and Dnmt3b ADD domains to peptide arrays and native histones. (A) Binding of Dnmt3a ADD domain to peptide arrays comprising 384 different peptides. The enlargement shows the binding to the H3 1–19 peptides. Peptides containing H3K4me2 or me3, H3T3P, H3S10P or H3T11P are shaded green, red and blue, respectively. The positions of the unmodified H3 1–19 as well as the peptides di- and trimethylated at K4 are annotated. (B) Design of the CelluSpots histone tail peptide arrays. For a detailed annotation of all spots cf. Supplementary Data S1. (C) Binding of Dnmt3b ADD domain to peptide arrays comprising 384 different peptides. (D) Binding of ADD domains to native histones isolated from human cells. Histones were separated by polyacrylamide gel electrophoresis and blotted to Nitrocellulose membrane. The membrane was stained with Ponceau S (PS). Then, membranes were incubated with GST tagged ADD domains, washed and ADD binding detected with anti-GST antibody staining (AS).
Figure 2. Analysis of reconstituted oligonucleosomes. (A) Native agarose gel (0.5%, 0.2× TBE) of free DNA or oligonucleosomes after reconstitution on 12 × 200 × 601 sequence containing uniformly the indicated H3 species. Gel was stained post-running with ethidium bromide (M, molecular size markers). (B) Reconstituted oligonucleosomes containing the indicated H3 species were run on SDS PAGE gel and analyzed by western blotting using the indicated antibodies. A separately run gel was stained with Coomassie blue. (C) DNA used for reconstitution or assembled oligonucleosomes containing different H3 species were digested with MNase for varying times. Samples were run on agarose gels (1.5%, 1× TBE) and stained with ethidium bromide (M, molecular size markers). (D) Scanning force microscopy images of reconstituted oligonucleosomes. At a 1.1 : 1 octamer : DNA ratio the number of visible nucleosomes on one DNA molecule typically ranged from 10 to 12.
Figure 3. DNA methylation of unmodified chromatin and naked DNA by full-length Dnmt3a2, full-length Dnmt3a2/Dnmt3L complex and Dnmt3a-C. DNA methylation analysis was performed for both strands of DNA in chromatin substrates and DNA. Results shown are the average of three independent experiments. Altogether about 250 clones were sequenced for each strand and each data point. (A) Methylation levels of single CpG sites in the upper and lower strand of the DNA. (B) DNA sequence of the 601 monomeric unit (200 bp). The grey color shades the sequences of the forward and reverse primers. The red-color-labeled CpG sites were analyzed in this study. The nucleosome positioning sequence (147 bp) is underlined.
Figure 4. DNA methylation levels of single CpG sites in chromatin with H3K4me3 or H3K9me3 modifications by full-length Dnmt3a2, full-length Dnmt3a2/Dnmt3L and Dnmt3a-C. Results shown are the averages of three independent experiments. Altogether about 250 clones were sequenced for each strand and each data point.
Figure 5. Methylation of DNA on modified oligonucleosomes. (A) Methylation of the first three CpG sites by full-length Dnmt3a2, full-length Dnmt3a2/Dnmt3L, but not Dnmt3a-C is stimulated by the absence of H3K4me3 (data taken from Figures 3 and 4). For each comparison, the methylation levels were normalized to H3K4me3, which showed the lowest methylation. Results shown are the average of three independent experiments; error bars indicate the standard deviations. (B) Schematic picture showing how the H3 tail interaction of Dnmt3a’s ADD domain (orange) anchors the catalytic domain (red) to methylate the linker DNA regions of nucleosomes.
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