Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells

Abstract

Background: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair.

Results: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis.

Conclusions: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.

Fig. 1

TET enzymes are important, but TDG is dispensable, for IL-4 production and DNA demethylation of the Il4 locus. A Flowchart of experiments. B Quantification of IL-4 production by Th2 cells after the second cycle of differentiation of naïve T cells from WT vs. TET iTKO, or WT vs. TDG iKO mice. C Schematic representation of the Il4 locus. The locations of all CpGs, the locations of PCR amplicons, and the numbers of CpGs per amplicon are indicated. D Bar graphs show the percentage of (5mC + 5hmC)/total C in 47 CpGs in the Il4 locus with confidence intervals (CIs) in WT vs. TET iTKO cells (upper), or WT vs. TDG iKO cells (lower), as determined by BS-seq. Results for naïve CD4⁺ T cells and Th2 cells after the second cycle of differentiation are shown. Data are representative of two independent experiments. Note that the CNS1 enhancer and the intronic enhancer near the border of exon 1 and intron 1 undergo substantial demethylation during Th2 differentiation. E Bar graphs show the percentages of (5mC + 5hmC)/total C (upper) and (5fC + 5caC)/total C (lower) in 47 CpGs in the Il4 locus in WT vs. TDG iKO cells, as determined by BS-seq (upper) and PB-seq (lower). Results for naïve CD4⁺ T cells (adapted from D) and Th2 cells after a single cycle of differentiation are shown. Data are representative of two independent experiments. TDG deficiency results in clear increases in 5fC/5caC, but no decrease in 5mC+5hmC, at CpGs that undergo TET-dependent demethylation. Statistical significance was calculated using an unpaired two-tailed t test. *P < 0.05

Fig. 2

Summary of experiments showing TET-dependent, TDG-independent demethylation of the Il4 locus during Th2 differentiation. A Heat maps depicting the percentage of (5mC + 5hmC)/total C in 64 CpGs in the Il4 locus in wildtype naïve CD4⁺ T cells and Th2 cells after the first and second cycles of differentiation, as determined by BS-seq. Data show the results of thousands of sequencing reads from two or three independent experiments. The numbers of CpGs in each amplicon are shown above the heat maps. B, C Heat maps depicting the percentage of (5mC + 5hmC)/total C in 64 CpGs in the Il4 locus in WT vs. TET iTKO cells (B), or WT vs. TDG iKO cells (C), as determined by BS-seq. Results for Th2 cells after the first and second cycles of differentiation are shown. The data show the results of thousands of sequencing reads from two independent experiments. D Heat maps depicting the percentage of (5fC + 5caC)/total C in 47 CpGs in the Il4 locus in WT vs. TDG iKO cells, as determined by PB-seq. Tdg gene deletion was achieved using Cre-ERT2 (upper) or CD4Cre (lower). Results for Th2 cells after the first cycle of differentiation are shown

Fig. 3

LPS stimulation of bone marrow-derived macrophages induces 5hmC deposition at enhancers. A Flowchart of experiments. B Number of differentially hydroxymethylated regions (DhmRs) comparing unstimulated vs. LPS-stimulated BMDMs. C LPS induces 5hmC deposition in BMDMs at an intergenic region between the Jdp2 and Batf genes (for genome browser views of the Il1b and Il6 loci, see Fig. S3). D For all the de novo 5hmC peaks in LPS-stimulated BMDMs, Log2 fold change in 5hmC is plotted against Log2 fold change in RNA expression. 5hmC peaks overlapping with latent enhancers (regions that acquire H3K4me1 and H3K27Ac only after stimulation) are shown in red; of these, peaks in the vicinity of the Batf, Ptgs2, Stat1, Alcam, Mdfic, Il1b, and Il6 genes are shown in light blue. E Bar graphs show the RNA expression levels of the Batf, Ptgs2, Stat1, Alcam, Mdfic, Il1b, and Il6 genes in WT vs. TET iTKO (left), or WT vs. TDG iKO (right), as determined by RNA-seq. BMDMs stimulated with (+) or without (−) LPS were used. F Heat maps depicting the percentage of (5mC+5hmC)/total Cs for each CpG in enhancers close to the Batf, Ptgs2, Stat1, Alcam, Mdfic, Il1b, and Il6 genes, as determined by BS-seq. All seven selected latent enhancers show 5hmC deposition upon LPS stimulation (see Fig. S3E), but none undergoes DNA demethylation (loss of 5mC+5hmC)

Fig. 4

GATA3 is partly responsible for Il4 locus demethylation. A Genome browser view of the 5′ half of the Il4 gene. Track 1: CpGs are shown as short vertical blue lines. CpGs belonging to different amplicons are indicated by colored horizontal bars as in Fig. 1C [i.e., magenta bar for promoter CpGs, yellow bar for exon 1, green bar for intron 1-exon 2, etc.]. Tracks 2 and 3: GATA3 ChIP-seq profiles (GSE28292) at two different scales (0–20 and 0–7) in Th2 cells. Track 4: Positions of known GATA binding motifs (TATC and GATA). Tracks 5–7: ChIP-seq profiles for STAT6 (GSE22104), BATF (GSE85172), and IRF4 (GSE85172) in Th2 cells. Tracks 8 and 9: 5hmC profiles in Th2 and naïve CD4⁺ T cells respectively, determined by CMS-IP. Note the presence of 5hmC at regions occupied by transcription factors. The rightmost 5hmC/CMS-IP peak does not correspond to a region with GATA3, STAT6, BATF, or IRF4 occupancy. B Number of differentially hydroxymethylated regions (DhmRs) in naïve CD4⁺ T cells compared to Th2 cells. C Motif enrichment analysis of DhmRs that are enriched in Th2 cells compared to naïve T cells. The y axis indicates fold enrichment versus background, circle size indicates the percentage of regions containing the respective motif, and the color indicates the significance (-Log10 (p-value)). D Flow cytometry plots showing IL-4 and IFN-γ production by Th2 cells that were transduced with retroviral vectors encoding control or Gata3 sgRNA. Data are representative of two independent experiments. E Gata3 transcripts were quantified by qRT-PCR and normalized to Gapdh and then to the level of empty vector control. F A bar graph depicting the percentage of (5mC + 5hmC)/total C in 47 CpGs in the Il4 locus in cells transduced/electroporated with control vs. Gata3 sgRNA, as determined by BS-seq. Data are representative of two independent experiments

Fig. 5

The Il4 locus is concordantly demethylated during Th2 cell differentiation. A Genome browser view of part of the Il4 locus, with amplicons indicated as in Fig. 1C. B Positions of CpGs in the first amplicon of Il4 CNS1 (CpGs 1–8), and the amplicons for Exon 1 (CpGs 22–28) and Intron 1-Exon 2 (29–37). C Methylation profiles for CNS1 (CpGs 1–8), Exon 1, and Intron 1-Exon 2 amplicons in Th2 cells after the first (upper) and second (lower) cycles of differentiation. Each row represents one read. Black indicates methylation (5mC + 5hmC) and white indicates the presence of unmodified C, 5fC, or 5caC at the indicated CpG. Note that demethylation occurs progressively between the first and second cycles of differentiation at all CpGs in the first amplicon of Il4 CNS1 (CpGs 1–8), with the most extensive demethylation at CpGs 3–8; similarly, demethylation occurs progressively at the GATA3-binding intronic enhancer, with the most extensive demethylation occurring at CpGs 27–29 near the peak of GATA3 occupancy (see Fig. 4A). D Matrix showing odds ratio of any two CpGs as a measure of concordant modification in Th2 cells after the first (upper) and second (lower) cycles of differentiation. The brighter the red color, the more similar the methylation status of the CpGs being compared. The highest levels of concordant demethylation are observed at CpGs 3–8 of Il4 CNS1, CpGs 25–28 of exon 1, and CpGs 29–31 of intron 1-exon 2. E For all possible pairs of CpGs, the odds ratio for a pair of CpGs is plotted against the distance between that pair of CpGs. Note that the odds Ratio (concordant methylation status) is higher the closer the CpGs

Fig. 6

TDG-deficient mice show normal hematopoiesis. A Flow cytometry plots and B quantification of CD4 and CD8 in the thymus (upper) and spleen (lower) in WT (n = 3) and TDG-KO (n = 3) mice, in which CD4Cre-driven gene deletion was achieved. C CD4 single positive (SP) cells were isolated from the thymus. Tdg transcripts were quantified by qRT-PCR and normalized to Gapdh and then to the level of WT control. Data from three independent experiments with three technical replicates each. D Flowchart of experiments. E Flow cytometry plots and F quantification of CD11b, Gr-1, B220, and TCR β chain in cells in the peripheral blood of WT (n = 2) and TDG iKO (n = 2) mice. Statistical significance was calculated using an unpaired two-tailed t test