5mC oxidation by Tet2 modulates enhancer activity and timing of transcriptome reprogramming during differentiation

Abstract

In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.

Figure 1. Global loss of 5hmC in Tet2^−/− mouse ES cells

(A–B) DNA methylation profiles of the (A) Tet1 and (B) Tet2 genes in Tet1^−/− and Tet2^−/− ESCs, illustrating the loci targeted for TET deletion. Two biological replicates of bisulfite sequencing are shown. (C) Boxplots of hydroxymethylation abundance for non-overlapping 10-kb bins spanning the mouse genome in WT, Tet1^−/−, and Tet2^−/− mESCs. (D) Bulk quantification of 5hmC in triplicates. Isolated genomic DNA was digested to single nucleosides and quantified using LC-MS/MS. Average profiles of absolute 5hmC abundance at (E) H3K4me3-only promoters, (F) bivalent H3K4me3/H3K27me3 promoters, (G) H3K27me3-only promoters, (H) gene bodies, (I) active H3K4me1/H3K27ac enhancers, (J) poised H3K4me1-only enhancers, and (K) CTCF-bound insulators. (L) Boxplot quantification of 5hmC abundance in E–J. For boxplots, notches indicate median, boxes extend to the 25th and 75th percentiles, and whiskers extend to non-outliers. In bar charts, error bars indicate standard deviation. wrt, with respect to.

Figure 2. Hypermethylation of enhancers in Tet2^−/− cells

Genome-wide DNA methylation profiles were segmented by a hidden Markov model to identify cell-specific DNA methylation. Shown are (A) the relative abundance of DMRs in Tet1^−/− and Tet2^−/− cells and (B) the distribution of 5mC change at DMRs of knockout compared to wild-type cells. (C) The relative enrichment of Tet2^−/− hyper(left)/hypo(right) DMRs (black) and random sites (gray) at genomic elements, normalized to the total coverage of the element type. “P 250bp” indicates promoter defined as transcription start site +/− 250bp, DHS denotes DNase I hypersensitive sites, and LAD denotes lamina-associated domains (Peric-Hupkes et al., 2010). Random consists of 5 random samplings of genomic loci. (D) The relative abundance of Tet2^−/− hyper-DMRs at regulatory elements. Red indicates distal regulatory elements. In bar charts, error bars indicate standard deviation. See also Figure S1 and Table S1.

Figure 3. 5hmC-containing enhancers are hypermethylated in Tet2^−/− cells

(A) (left) Abundance of 5mC and 5hmC at mESC enhancers in WT, Tet1^−/−, and Tet2^−/− cells. (right) Enhancer chromatin state (me1: H3K4me1; ac: H3K27ac) and expression state (RNA: Global Run-On) in WT cells is also indicated. Enhancers are ranked by change in methylation state between Tet2^−/− and WT cells. (B) For enhancers that are hypermethylated in Tet2^−/− cells (red) and those that remain hypomethylated (gray), shown is the enrichment of 5mC in WT and Tet2^−/− cells. (C) Quantification of mESC enhancers that are hypermethylated, hypomethylated, or unchanged in knockout cells compared to wild-type. (D–E) Profiles of average (left) 5mC and (right) 5hmC centered at Tet2^−/− hyper-DMRs at (D) poised enhancers and (E) active enhancers. (F) For enhancers that are hypermethylated in Tet2^−/− cells (red, left) and those that remain hypomethylated (gray, right), shown is the enrichment of 5hmC in wild-type cells. (G) Density plot illustrating the relationship between 5hmC abundance in WT cell and 5mC abundance in Tet2^−/− cells. ρ indicates Spearman rank correlation. (H) Quantification of TET enrichment, using data previously mapped by ChIP-Seq (Chen et al., 2013; Williams et al., 2011), at enhancers active in non-ES cells (white), enhancers hypermethylated in Tet2^−/− cells (red), and enhancers that remain hypomethylated (grey) in Tet2^−/− cells. (I) The relative enrichment of Tet2^−/− hyper-DMRs (black) and random sites (gray) at peaks of 5hmC and 5fC enrichment, at domains of 5fC/5hmC (fhMR) or 5hmC alone (hMR), and at active/poised enhancers. Error bars indicate standard deviation. (J–L) For enhancers that are hypermethylated in Tet2^−/− cells (red, left) and those that remain hypomethylated (gray, right), shown is the enrichment of (J) active chromatin (H3K4me1 and H3K27ac), (K) nascent RNA transcription (by GRO-Seq) in wild-type cells, and (L) transcription factor binding (OCT4, SOX2, NANOG) (Chen et al., 2008; Marson et al., 2008). Boxplot edges indicate the 25th and 75th percentiles, and whiskers indicate non-outlier extremes. See also Figure S2.

Figure 4. Enhancer loss of acetylation and reduced gene expression

(A) Partitioning of active enhancers hypermethylated in Tet2^−/− ESCs into those that (top) lose H3K27ac and (bottom) retain H3K27ac. The middle column indicates difference in H3K27ac, and the right columns indicate DNA methylation abundance. (B) UCSC Genome Browser snapshots of Tet2^−/− hyper-DMRs that lose H3K27ac (highlighted yellow) near an enhancer of the differentially expressed (bottom) Lefty1 gene. (C) The enrichment of different enhancer groups near genes differentially repressed in Tet2^−/− cells. (D) The distribution of expression change for genes physically interacting with enhancers that (red) lose H3K27ac and (green) retain H3K27ac. The ratio of down-regulated to up-regulated genes is indicated. (E) Difference in enrichment of active chromatin (H3K4me1, left; H3K27ac, right) between Tet2^−/− and WT cells, as a function of hypermethylation. Average percentage change in ChIP enrichment is indicated. Boxplot edges indicate the 25th and 75th percentiles, and whiskers indicate non-outlier extremes. See also Figure S3 and Table S2.

Figure 5. Delayed gene induction of Tet2^−/− cells during NPC differentiation

(A) Dendrogram summarizing RNA-Seq experiments during differentiation of mES cells to NPCs. Red indicates Tet2^−/− branches; bold black indicates WT branches. (B–C) Expression of (B) neuronal markers and (C) Tet genes during NPC differentiation. (D) Examples of genes exhibiting delayed gene induction in Tet2^−/− specifically at differentiation day 3. Error bars indicate standard deviation. (E) The total number of differentially expressed genes in Tet2^−/− (black) and WT (white) cells, as compared to undifferentiated ES cells. Shown in red are those genes commonly differentially expressed in these two cells. (F) Of the genes induced in WT cells at day 3 (left) or day 6 (right) during differentiation towards NPCs, shown are the number of genes repressed (blue) or induced (red) in Tet2^−/− cells compared to WT. (G) Expression of genes in Tet2^−/− relative to WT differentiated cells for delayed induction genes (top) and all other genes (bottom). Genes in (F) that exhibited differential expression in d0 or d6 were removed. (H) Ontology terms enriched for delayed induction genes. See also Figure S4.

Figure 6. Differential enhancer methylation contributes to delayed gene induction

(A) Distribution of change in 5mC for DMRs identified in Tet2^−/− cells compared to WT cells 3 days after mESC differentiation towards NPCs. (B) Relative enrichment of Tet2^−/− hyper DMRs from (A) (black) and random sites (gray) at genomic elements, normalized to the total coverage of the element type. Error bars indicate standard deviation. (C) Density heatmap representing the methylation state of hypermethylated enhancers before (x axis) and after (y axis) differentiation in WT (left) and Tet2^−/− (right) cells. (D) Heatmap representing the epigenetic state of d3 Tet2^−/− hypermethylated enhancers. (E) Genome browser snapshots of DNA methylation and chromatin state at d3 Tet2^−/− hypermethylated enhancers (yellow). (F) Boxplots quantifying mCG (top) and quantile-normalized H3K27ac (bottom) at enhancers before (right) and after (left) differentiation. Day 3 Tet2^−/− specific hypermethylated enhancers are labeled as “Tet2^−/− > WT”, and other active enhancers as “remainder”. Boxplot edges indicate the 25th and 75th percentiles, and whiskers indicate non-outlier extremes. (G) The number of enhancers within TADs containing delayed induction genes is indicated in red, compared to the distribution of 5000 random gene sets. Enhancers are defined as: (top) the set of all WT day 3 active enhancers, (middle) the subset that is hypermethylated in Tet2^−/− cells at day 3, and (bottom) the subset with WT specific H3K27ac at day 3.

Figure 7. Model of enhancer hypermethylation in Tet2^−/− cells

Transcription factors bind to DNA and therefore occlude hypermethylation by DNMTs. Enhancers with high TF occupancy (left) are more resistant to DNMTs, and therefore remain hypomethylated in Tet2^−/− cells. However, enhancers with low TF occupancy (right) are prone to hypermethylation, which is balanced by the action of Tet2. Loss of Tet2 causes re-methylation.