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. 2012 Jun;22(6):1128-38.
doi: 10.1101/gr.133728.111. Epub 2012 Mar 30.

Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk

Arie B Brinkman  1 , Hongcang Gu, Stefanie J J Bartels, Yingying Zhang, Filomena Matarese, Femke Simmer, Hendrik Marks, Christoph Bock, Andreas Gnirke, Alexander Meissner, Hendrik G Stunnenberg
Affiliations

Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk

Arie B Brinkman et al. Genome Res. 2012 Jun.

Abstract

Cross-talk between DNA methylation and histone modifications drives the establishment of composite epigenetic signatures and is traditionally studied using correlative rather than direct approaches. Here, we present sequential ChIP-bisulfite-sequencing (ChIP-BS-seq) as an approach to quantitatively assess DNA methylation patterns associated with chromatin modifications or chromatin-associated factors directly. A chromatin-immunoprecipitation (ChIP)-capturing step is used to obtain a restricted representation of the genome occupied by the epigenetic feature of interest, for which a single-base resolution DNA methylation map is then generated. When applied to H3 lysine 27 trimethylation (H3K27me3), we found that H3K27me3 and DNA methylation are compatible throughout most of the genome, except for CpG islands, where these two marks are mutually exclusive. Further ChIP-BS-seq-based analysis in Dnmt triple-knockout (TKO) embryonic stem cells revealed that total loss of CpG methylation is associated with alteration of H3K27me3 levels throughout the genome: H3K27me3 in localized peaks is decreased while broad local enrichments (BLOCs) of H3K27me3 are formed. At an even broader scale, these BLOCs correspond to regions of high DNA methylation in wild-type ES cells, suggesting that DNA methylation prevents H3K27me3 deposition locally and at a megabase scale. Our strategy provides a unique way of investigating global interdependencies between DNA methylation and other chromatin features.

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Figures

Figure 1.
Figure 1.
Integration of capturing methods and bisulfite-deep sequencing: ChIP-BS-seq and MethylCap-BS-seq. (A) Schematic outline of the ChIP-BS-seq and MethylCap-BS-seq procedures. Capturing of genomic regions of interest is achieved by the MethylCap procedure or ChIP with an antibody of interest. Captured DNA is processed as indicated. Shown intermediate products and final PCR fragments indicate the fate of unmethylated as well as methylated cytosines throughout the procedure. (B) Examples of conventional MethylCap-seq and MethylCap-BS-seq data of normal (N) and tumor (T) colon tissues. For each covered CpG, percentage methylation as derived from the MethylCap-BS-seq data is indicated by color (yellow, 0%; blue, 100%). (Green) CpG islands and a CpG density profile (CpG/bp). (C) Average profiles of DNA methylation and coverage in MethylCap peaks of normal colon tissue, as determined by MethylCap-BS-seq. (Blue) Percentage DNA methylation; (magenta) CpG coverage; (brown) read density. (D) MethylCap-BS-seq analysis of differentially methylated regions from normal/tumor colon tissue. Regions that gain DNA methylation in tumor tissue show increased CpG coverage (x-axis) and read-density (y-axis). Color-code depicts absolute changes in percent methylation of these regions, as determined by bisulfite sequencing. (Blue) increase; (yellow) decrease.
Figure 2.
Figure 2.
H3K27me3-enriched CpG island promoters are devoid of DNA methylation. (A,B) Examples of H3K27me3 ChIP-BS-seq data of HCT116 cells. H3K27me3 ChIP-BS-seq profiles are shown, as well as the derived DNA methylation data per covered CpG and per 200-bp window. Percentage methylation is color-coded as in Figure 1B. (C) Histograms showing the distribution of mean methylation in 300-bp windows throughout H3K27me3-enriched regions, as derived from H3K27me3 ChIP-BS-seq results. Windows were categorized according to functional genomic elements (intergenic, intron, exon, non-CpG island promoter, or CpG island promoter). (D) Average profiles of DNA methylation (blue), H3K27me3 (red), and CpG density (green) in regions flanking H3K27me3-enriched CpG islands, as determined from H3K27me3 ChIP-BS-seq data.
Figure 3.
Figure 3.
H3K27me3 is locally depleted at hypermethylated CpG islands. (A) Examples of the genome-wide H3K27me3 ChIP-seq and MethylCap-seq data, demonstrating hypermethylated CpG islands within H3K27me3 BLOCs, and concomitant local depletion of H3K27me3. H3K27me3-enriched BLOCs and MethylCap peaks are indicated as red and blue rectangles, respectively. (B) Density maps of MethylCap-seq and H3K27me3 ChIP-seq read densities in 20-kb regions surrounding MethylCap peaks that reside in H3K27me3 BLOCs and overlap with CpG islands.
Figure 4.
Figure 4.
Localized peaks of H3K27me3 in wild-type mES cells over CpG islands. (A) Screenshots of localized H3K27me3 peaks (red). Per covered CpG, percentage methylation as derived from the H3K27me3-BS-seq data is indicated in color. (B,C) Average profiles of DNA methylation (blue), H3K27me3 (red), and CpG density (green) in H3K27me3 peaks over CpG islands, as determined from H3K27me3-ChIP-BS-seq. (D) Histograms showing the distribution of methylation in 300-bp windows through H3K27me3 peaks, as inferred from H3K27me3 ChIP-BS-seq. Windows were categorized according to CpG density. (E) H3K27me3 changes in CpG islands that were either hypermethylated (>90%, left) or hypomethylated (<10%, right) in wild-type mES cells. Read counts shown are from conventional H3K27me3 ChIP.
Figure 5.
Figure 5.
Changes in H3K27me3 patterns upon loss of DNA methylation in Dnmt triple-knockout (TKO) mES cells. (A) Examples of H3K27me3 BLOCs appearing in TKO cells, and concomitant loss of H3K27me3 in localized peaks. Per covered CpG, percentage methylation as derived from H3K27me3-ChIP-BS-seq is indicated in color. (B) Scatterplot of H3K27me3 changes in peaks (x-axis) vs. BLOCs (y-axis). H3K27me3 peaks were matched against BLOCs in which they reside. I–IV indicate the four quadrants of the plot, as determined by log2 fold changes deviating from zero. (C) Histograms of percent DNA methylation in the BLOCs of each quadrant of B, as deduced from H3K27me3-ChIP-BS-seq in wild-type mES cells. (D) Density maps of H3K27me3 through BLOC transition regions detected in TKO mES cells. Average profiles are shown on top.
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