Global loss of DNA methylation uncovers intronic enhancers in genes showing expression changes

Abstract

Background: Gene expression is epigenetically regulated by a combination of histone modifications and methylation of CpG dinucleotides in promoters. In normal cells, CpG-rich promoters are typically unmethylated, marked with histone modifications such as H3K4me3, and are highly active. During neoplastic transformation, CpG dinucleotides of CG-rich promoters become aberrantly methylated, corresponding with the removal of active histone modifications and transcriptional silencing. Outside of promoter regions, distal enhancers play a major role in the cell type-specific regulation of gene expression. Enhancers, which function by bringing activating complexes to promoters through chromosomal looping, are also modulated by a combination of DNA methylation and histone modifications.

Results: Here we use HCT116 colorectal cancer cells with and without mutations in DNA methyltransferases, the latter of which results in a 95% reduction in global DNA methylation levels. These cells are used to study the relationship between DNA methylation, histone modifications, and gene expression. We find that the loss of DNA methylation is not sufficient to reactivate most of the silenced promoters. In contrast, the removal of DNA methylation results in the activation of a large number of enhancer regions as determined by the acquisition of active histone marks.

Conclusions: Although the transcriptome is largely unaffected by the loss of DNA methylation, we identify two distinct mechanisms resulting in the upregulation of distinct sets of genes. One is a direct result of DNA methylation loss at a set of promoter regions and the other is due to the presence of new intragenic enhancers.

Figure 1

Whole genome bisulfite sequencing comparative analysis of HCT116 and DKO1 cells. (A) Light blue and pink tracks represent the sequencing coverage along a segment of human chromosome 19 in HCT116 and DKO1 cells, respectively. Dark blue and red tracks illustrate the percentage of methyl-C/C in HCT116 and DKO1, respectively. Light-colored lines within the percentage methylation (%mCpG) tracks represent the average percentage methylation in the immediate region. CpG islands are shown above the RefSeq genes as green bars. (B) Box plot illustrating the percentage methylation in promoters, gene bodies, and random regions of the genome; the horizontal line in each bar indicates the median value. For HCT116 cells the median values are 30% (promoters), 84% (gene bodies), and 84% (random regions) and for DKO1 cells the median values are <1% (promoters), 13% (gene bodies), and 9% (random regions). (C) The number of promoters containing varying levels of methylation in HCT116 and DKO1 are shown; the minimum and maximum DNA methylation values for the region between -100 and +700 relative to the start site at the promoters in each group is indicated by the color key.

Figure 2

Overview of ChIP-seq data in HCT116 and DKO1 cells. Shown is a region of chromosome 6 illustrating the reduction in levels of active marks in DKO1 cells compared with HCT116 cells, for RNAP II (blues), H3K4me3 (reds), H3K27ac (greens), and H3K36me3 (oranges). Dark and light colors represent HCT116 and DKO1 cells, respectively. Although peaks are reduced in height at most sites, some genomic locations do have increases in H3K4me3 (blue boxes) or H3K27ac (red boxes) in DKO1 cells.

Figure 3

Active histone modifications and RNA polymerase II are drastically reduced at promoters in DKO1 cells. Shown is the percentage DNA methylation (heat bars) and the density of ChIP-seq tags for H3K4me3 (reds), H3K27ac (greens), and RNAPII (blues) surrounding the transcription start sites of promoters that had less than 50% methylation (left) and promoters that had more than 50% methylation in HCT116 cells (right). Light-colored dashed lines represent the marks in DKO1 cells.

Figure 4

A small set of promoters are de-repressed in DKO1 cells. (A) Plot showing relative DNA methylation across the gene bodies of all RefSeq genes in HCT116 and DKO1 cells. (B) Gene expression differences in HCT116 and DKO1; log2 expression values are plotted for every gene expressed in HCT116 and/or DKO1. The orange line represents a slope of x = y. The green dots represent a group of de-repressed genes with log2(HCT116 normalized expression) <1.5 and log2(DKO1 normalized expression) >2.5.

Figure 5

Identification and characterization of de-repressed genes. (A) Average DNA methylation percentage (heat bar) and densities of ChIP-seq tags for H3K4me3 (reds), H3K27ac (greens), and RNAPII (blues) are plotted relative to the transcription start sites of de-repressed genes in HCT116 and DKO1. Light-colored dashed lines represent the marks in DKO1 cells. (B) Venn diagram displaying the overlap of the promoters of de-repressed genes and H3K4me3 peaks unique to DKO1 cells. (C) Gene Ontology results for the 1,086 de-repressed genes in DKO1 (top) and the subset of those genes whose promoters have DKO1-unique H3K4me3 peaks (bottom).

Figure 6

Identifcation and characterization of de-regulated genes. (A) The 1,640 genes significantly differentially expressed between HCT116 and DKO1 cells are shown in red (P-value <0.05, fold-change >1.2). (B) Gene Ontology results for down-regulated genes; up-regulated genes were not enriched for any Gene Ontology terms. (C) The epigenetic profiles at the promoters of genes up-regulated (right panel) and down-regulated in DKO1 (left panel).

Figure 7

Loss of DNA methylation uncovers thousands of distal H3K27ac sites. (A) Venn diagram comparing distal H3K27ac peaks in HCT116 and DKO1 cells. (B) DNA methylation status of common and unique enhancers. Pie charts show the breakdown of genomic locations for peaks residing within intergenic, intronic, and exonic regions of the genome. Tag density plots show the density of H3K27ac tags relative to the centers of these peaks. (C) Distances from the TSS of genes up-regulated (right panel) or down-regulated (left panel) in DKO1 to the nearest categorized enhancer. Also indicated is the median distance from each category of enhancer to the nearest up-regulated or down-regulated gene.

Figure 8

Genes up-regulated in DKO1 cells have new intragenic enhancers. (A) Graph plotting the number of intragenic enhancers for the 274 genes up-regulated in DKO1 cells. (B) Graph plotting the number of intragenic enhancers for the 1,366 genes down-regulated in DKO1 cells.

Figure 9

New H3K27ac peaks are enriched within the gene bodies of up-regulated genes. (A) DNA methylation profiles and tag density plots for H3K27ac at the promoters of genes containing intronic enhancers (left) and at the locations of intronic enhancers (right). Pie chart shows the percentage of the 310 intergenic enhancers identified within the gene bodies of the up-regulated genes that are intronic or exonic. (B) Genome browser snapshot of the genomic region surrounding the SASH1 gene. The H3K27ac track in green shows an increase in signal within the gene’s intronic regions in DKO1 (light green) relative to HCT116 (dark green).

Figure 10

Schematic illustrating the characterization of gene expression categories. Shown is the breakdown of all expressed genes in HCT116 and DKO1 cells into groups based on their expression in the two cell types. Three categories of genes were identified as de-repressed, up-regulated or down-regulated.