Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity

Abstract

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4(+) T cells (Tconv) and CD4(+)CD25(+) regulatory T cells (Treg), cell types whose differentiation and function are known to be controlled by epigenetic mechanisms. Using a novel approach that is based on the separation of a genome into methylated and unmethylated fractions, we examined the extent of lineage-specific DNA methylation across whole gene loci. More than 100 differentially methylated regions (DMRs) were identified that are present mainly in cell type-specific genes (e.g., FOXP3, IL2RA, CTLA4, CD40LG, and IFNG) and show differential patterns of histone H3 lysine 4 methylation. Interestingly, the majority of DMRs were located at promoter-distal sites, and many of these areas harbor DNA methylation-dependent enhancer activity in reporter gene assays. Thus, our study provides a comprehensive, locus-wide analysis of lineage-specific methylation patterns in Treg and Tconv cells, links cell type-specific DNA methylation with histone methylation and regulatory function, and identifies a number of cell type-specific, CpG methylation-sensitive enhancers in immunologically relevant genes.

Figure 1.

Locus-wide identification of DMRs using the MCIp “mirror image” approach. (A) Schematic outline of the MCIp fragmentation and hybridization strategy. The fragmented genomes of Tconv and Treg cells are separated into unmethylated (CpG) and methylated (mCpG) pools. Each pool is directly labeled using fluorescent dyes, and each pool of one cell type is compared with the corresponding pool of the other cell type on a locus-wide microarray. Microarray images are compared to identify regions that show a reciprocal hybridization behavior. (B) Representative scatter plots of CpG- and mCpG-pool hybridizations are shown. Probes with reciprocal signal intensity ratios indicate the presence of DMRs. (Red) Treg cells; (blue) Tconv cells. (C) Exemplary validation of microarray results using mass spectrometry. The intron 1 region of LRRC32 is enriched in the unmethylated (CpG, red line) and depleted in the methylated (mCpG, black line) pools of Treg cells. A large hypomethylation score (defined as the difference product of log₁₀ signal intensity ratios of both hybridizations) indicates differential methylation (bottom left panel). The same region was analyzed by MALDI-TOF MS (Epityper), and results are shown as a heat map (the scale ranges from white [no methylation] to dark blue [100% methylation]).

Figure 2.

Chromatin modification and CTCF binding patterns across the CTLA4 gene locus. Shown are the following tracks (from top to bottom): mammalian Consensus (Cons, black); repetitive regions as identified by the RepeatMasker program (black); ChIP-chip tracks for CTCF (blue); monomethylated (pale green), dimethylated (green), and trimethylated (dark green) lysine 4 of histone H3; and the CpG index (indicating the methylation density 300 bp up- or downstream of each microarray probe) as well as hypomethylation scores (red) for both cell types. Several amplicons were designed for MALDI-TOF MS analysis of bisulfite-treated DNA as indicated below the tracks. Methylation levels of individual CpGs in the indicated cell types are shown color-coded as described in the Fig. 1 legend.

Figure 3.

Chromatin modification and CTCF binding patterns across the IL2RA gene locus. Tracks and heat maps are shown as described in the legend of Fig. 2.

Figure 4.

Correlation of DNA demethylation and H3K4 methylation status. (A–C) Probe signal ratios of Tconv and Treg cells are plotted against each other for ChIP-chip experiments of monomethylated (A), dimethylated (B), and trimethylated (C) lysine 4 of histone H3. Probes that appear along the diagonal indicate similar H3K4 methylation levels, whereas probes above or below the diagonal indicate higher methylation levels in Tconv or Treg cells, respectively. Probes in DMRs: (blue) unmethylated in Tconv; (red) unmethylated in Treg; (gray) all other probes. (D) The two pie charts illustrate the relationship of associated H3K4 methylation and DMRs hypomethylated in Tconv (left) or Treg (right). The H3K4 methylation status was classified as follows: DMRs with increased H3K4 trimethylation in Treg or Tconv cells (Treg Tri or Tconv Tri, respectively); DMRs with increased H3K4 mono- or dimethylation, but no trimethylation, in Treg or Tconv cells (Treg or Tconv Mono/Di respectively); DMRs with H3K4 methylation present but no difference between T cell subsets (Not diff.); and DMRs with no detectable H3K4 methylation (No H3K4me). The numbers of DMRs in each subclass are shown next to each piece of pie. Circled numbers indicate subclasses where hypomethylation in one cell type correlates with an increased level of H3K4 methylation in the same cell type.

Figure 5.

CpG methylation-dependent enhancer activity of selected DMRs. Several DMRs were cloned upstream of a basic EF1-promoter into the CpG-free luciferase vector pCpGL-P. The indicated plasmids were in vitro SssI-methylated (mCpG) or unmethylated (CpG) and transiently transfected into Jurkat T cells that were left untreated (A), or were stimulated with PMA and ionomycin (B) or PHA (C) after transfection. Luciferase activity was normalized against the activity of a cotransfected Renilla construct, and mean values ± SD are shown relative to the unmethylated pCpGL-P. (*) Significant difference between methylated and unmethylated plasmids (P < 0.05, paired Student's t-test).