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. 2011 Sep 1;39(16):6956-69.
doi: 10.1093/nar/gkr263. Epub 2011 May 27.

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Max Felle  1 , Helen Hoffmeister, Julia Rothammer, Andreas Fuchs, Josef H Exler, Gernot Längst
Affiliations

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Max Felle et al. Nucleic Acids Res. .

Abstract

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.

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Figures

Figure 1.
Figure 1.
Dnmt3a and Dnmt3b2 have distinct substrate binding properties. (A) His-tagged Dnmt3a and Dnmt3b2 were purified using Ni-NTA columns (lanes 1 and 3), followed by cation exchange chromatography (SP-Sepharose; lanes 2 and 4). Proteins were subjected to SDS–PAGE and Coomassie Blue staining. Sizes of the molecular weight marker are indicated. (B) Dnmt3a and Dnmt3b2 (His Dnmt; lanes 1 and 2) isolated only by His-tag-affinity purification, or by His-tag- and subsequent cation exchange purification (SP Dnmt; lanes 3 and 4) were analysed for co-purifying DNA contaminations. Five micrograms of each protein preparation was incubated with RNaseA and proteinaseK. The remaining DNA was purified and analysed by agarose gel electrophoresis and staining with ethidium bromide. Molecular weight marker (M) and sizes are indicated. (C) DNA fragments containing the NPS1 sequence located either in the centre of the DNA or close to the DNA border were partially assembled into mononucleosomes using the salt dialysis method. The sizes of the linker DNA next to the NPS1 sequences are indicated. (D) Different nucleosomal templates were mixed in 1:1 ratio (lanes 1 and 5) and incubated with a 3.5- to 15-fold molar excess of Dnmt3a and Dnmt3b2 (lanes 2–4 and 6–8). Reactions were analysed by native polyacrylamide gel electrophoresis next to a molecular weight marker (M).
Figure 2.
Figure 2.
Nucleosomes are major obstacles for de novo DNA methylation in vitro. (A) DNA templates containing the NPS2 sequence either with linker DNA containing CpG sites (C91-NPS2-C104, 12 CpG sites in linker, lanes 1 and 2), without linker DNA (NPS2, 15 CpG sites, lanes 3 and 4) or with linker DNA depleted of CpG sites (N78-NPS2-N79, lanes 5 and 6) were fully reconstituted into mononucleosomes. Nucleosomes were analysed by native PAGE next to a molecular weight marker (M). Black triangles (filled inverted triangle) indicate CpG sites. (B) Free DNA and nucleosomes described in (A) were subjected to in vitro DNA methylation reactions with the indicated DNA methyltransferases. The incorporation of the 3H-methyl was quantified and the methylation efficiency (given as percentage relative to free DNA) is plotted. Black triangles indicate CpG sites and the oval indicates the position of the nucleosome. (C) The ratios of the methylation efficiencies of DNA compared to nucleosomes (DNA/NUC) are given for the indicated nucleosomal templates. Black triangles indicate CpG sites and the oval indicates the position of the nucleosome.
Figure 3.
Figure 3.
De novo DNA methylation is limited to the entry/exit sites of the nucleosome. (A) DNA methylation reactions of the de novo DNA methyltransferases and M.SssI with the free DNA or the nucleosomal DNA fragment C91-NPS2-C104 were subjected to bisulfite conversion. The methylation frequency for each CpG site is plotted against the CpG sites of the DNA sequence. Analysis of the free DNA (light grey) and the nucleosomal template (dark grey) for the (+) strand and the (−) strand is shown. The position of the nucleosome is indicated. On average 20–40 sequences for each strand were sequenced. (B) The ratio of methylation events within the NPS2 sequence (black box, filled inverted triangle arrows indicated CpG sites) of DNA and nucleosomes as an average of the (+) and the (−) strand was calculated for C91-NPS2-C104 from the bisulfite experiments, and for NPS2 from the radioactive assay. The DNA/NUC ratios are indicated in the figure.
Figure 4.
Figure 4.
Chromatin remodelling stimulates de novo DNA methylation. (A) Chromatin remodelling reactions were performed in the presence of WT Snf2H (lanes 3–5) or mutant Snf2H K211R (lanes 6 and 7) using a 0.7–1.5 molar ratio (+, ++) relative to the nucleosomal N78-NPS2-N79 DNA. Reactions were supplied with ATP or ATPγS as indicated and stopped by the addition of competitor DNA. Nucleosome positions were analysed by native PAGE. Arrows (filled inverted triangle) indicate CpG sites on the DNA fragment. Black triangles indicate CpG sites and the oval indicates the position of the nucleosome. (B) Remodelling reactions were performed as indicated using either nucleosomal (dark grey bars) or free DNA (light grey bars). DNA methylation with the indicated enzymes was performed for 10 min. The incorporation of 3H-methyl groups was measured and the relative methylation activity (normalized to free DNA) of free DNA and nucleosomes was plotted. Black bars mark the reactions on the remodelled substrates. Black triangles indicate CpG sites and the oval indicates the position of the nucleosome. (C) Competitive nucleosome-Dnmt3a EMSA. Either the remodelled nucleosomal template or an equimolar mixture of remodelled and non-remodelled nucleosomes were incubated with a 3.5- to 15-fold molar excess of Dnmt3a (lanes 3–5 and 7–9). Reactions were analysed by native polyacrylamide gel electrophoresis next to a molecular weight marker (lane 1). Black triangles indicate CpG sites and the oval indicates the position of the nucleosome.
Figure 5.
Figure 5.
Nucleosomal DNA is devoid of CpG methylation in vivo. (A) Chromatin from HeLa cells was partially hydrolysed with MNase (lanes 1 and 2) and the purified DNA was analysed by agarose gel electrophoresis. The nucleosome-sized DNA fragments are indicated on the right. Mono-, di- and tri-nucleosomal (1 n, 2 n, 3 n) DNA was isolated and analysed (lanes 3–5). Molecular weight marker (M) and DNA sizes are indicated. (B) DNA methylation levels for mononucleosomal (white bars) and trinucleosomal (black bars) DNA using increasing DNA amounts, was determined with a meCpG-sensitive ELISA assay (Sigma). A comparative DNA methylation analysis for 100 ng of different nucleosomal DNAs (1 n, 2 n, 3 n), methylated control DNA (ctrl mDNA), non-methylated and methylated NPS2 DNA (mNPS2) is shown. (C) Micellar capillary electrophoresis of nucleosides (∼4 µg/µl) from purified and hydrolysed mononucleosomal DNA (left) and genomic HeLa DNA (right). The absorbance (AU) is plotted against the migration time (t; min). The peaks for 2′-deoxycytidine (C), 5-methyl 2′-deoxycytidine (5-meC) and an impurity (asteriks) are indicated. (D) The average (n = 10) of the 5-meC content of mononucleosomal (light grey) and Hela genomic DNA (dark grey) was calculated as percentage of (Area 5-meC/(Area C + Area 5-meC). Statistical significance of the data was calculated using the paired t-test. The calculated 5-meC content of mononucleosomal DNA (light grey, 147 bp) relative to the 5-meC content of linker DNA (dark grey, 41 bp) is given in the graph on the right.
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