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. 2015 Jul;16(7):746-54.
doi: 10.1038/ni.3198. Epub 2015 Jun 1.

Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages

MacLean Sellars  1 , Jun R Huh  2 , Kenneth Day  3 , Priya D Issuree  1 , Carolina Galan  1 , Stephane Gobeil  4 , Devin Absher  3 , Michael R Green  4 , Dan R Littman  1
Affiliations

Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages

MacLean Sellars et al. Nat Immunol. 2015 Jul.

Abstract

During development, progenitor cells with binary potential give rise to daughter cells that have distinct functions. Heritable epigenetic mechanisms then lock in gene-expression programs that define lineage identity. Regulation of the gene encoding the T cell-specific coreceptor CD4 in helper and cytotoxic T cells exemplifies this process, with enhancer- and silencer-regulated establishment of epigenetic memory for stable gene expression and repression, respectively. Using a genetic screen, we identified the DNA-methylation machinery as essential for maintaining silencing of Cd4 in the cytotoxic lineage. Furthermore, we found a requirement for the proximal enhancer in mediating the removal of DNA-methylation marks from Cd4, which allowed stable expression of Cd4 in helper T cells. Our findings suggest that stage-specific methylation and demethylation events in Cd4 regulate its heritable expression in response to the distinct signals that dictate lineage 'choice' during T cell development.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. DNA methylation enzymes are essential for silencing CD4 expression in cytotoxic T cells
Cd4S4-L; Ubc-Cre-ER cytotoxic cells were cultured in vitro and S4 was deleted by adding 400 nM OH-tamoxifen. (a–c) Flow cytometry of CD4 and CD8 expression. (a) Mock or pooled shRNA virus-infected cells (encoding a puromycin resistance gene) were enriched by MACS with anti-CD4 magnetic beads and cultured 4–5 more days in the presence of puromycin (2.5 μg/ml) before analysis. Cd4S4Δ/S4Δ CD4+8+ cells (germline S4 deletion) are shown as a staining control. Representative of 2 independent experiments. (b) Cytotoxic CD4−8+ cells from Dnmt1chip/chip mice were cultured in vitro for 5–6 days after control-ires-gfp or Dnmt1 shRNA-ires-gfp retroviral infection. Staining is on gated GFP+ cells. Representative of 3 independent experiments. (c) 1~1.5 × 106 peripheral CD4−8+ cells from control (Dnmt3aL/L; Dnmt3bL/+; Dnmt1L/+) or Dnmtsreduced (Dnmt3aL/L; Dnmt3bL/+; Dnmt1L/chip; Cd4-Cre+/−) animals were adoptively transferred into Rag2-deficient mice. TCRβ+CD8+ cells from the periphery were analyzed 16 days after transfer. Representative of 3 independent experiments.
Figure 2
Figure 2. Silencer-dependent DMR in the first intron of Cd4
Biological replicates of naïve (Thy1.2+CD44loCD62L+CD25−) WT CD4+, WT CD8+ and Cd4S4Δ/S4Δ CD4+CD8+ cells were isolated from LNs, and their genomic DNA was subjected to CATCH-seq (BAC-mediated enrichment, bisulfite treatment and Illumina sequencing). (a) Percent CpG methylation was calculated at CpGs exhibiting >30x coverage, within ~75 kb of the Cd4 TSS (Chr6:124749635-124906460; mm9), and variance at each CpG across all samples was calculated and graphed on the y-axis versus genomic position on the x-axis. 98–98.5% of targeted CpGs were captured in each sample at >30x coverage; median CpG coverage exceeded 300x. Genes, S4 and E4P are indicated below the graph. (b) A heatmap depicts percent CpG methylation in WT CD4+, WT CD8+ and Cd4S4ΔS4Δ CD4+8+ cells for CpGs from +6200 to −669 relative to the Cd4 TSS (Chr #6:124832027-124838896; mm9). A red line underlines CpGs in the S4 silencer (indicated by the gaps), and a black arrow indicates the Cd4 TSS. (c) CFSE-labeled WT CD4+, WT CD8+ and Cd4S4Δ/S4Δ CD4+CD8+ cells were stimulated in vitro with anti-CD3, anti-CD28 and IL-2, and those cells that had undergone at least 6 divisions after 5 days were sorted for locus enrichment and high-throughput bisulfite sequencing (as above). A heatmap depicts CpG methylation percentage from +6200 to −669 relative to the Cd4 TSS (Chr6:124832027-124838896; mm9). Replicates were derived from two independent experiments.
Figure 3
Figure 3. Cd4 hyper-methylation in immature thymocytes
(a) CATCH-seq was performed on genomic DNA from sorted populations of WT thymocytes: DN3 (Thy1.2+Lin−CD25+CD44−) (n=1), DP (TCRβloCD24+CD69−CD4+CD8+), CD4SP (TCRβhiCD24loCD69loCD4+CD8–−) and CD8SP (TCRβhiCD24loCD69loCD4−CD8+); the SP suffix denotes thymus-derived helper and cytotoxic T cells. A heatmap depicts CpG methylation percentage from +6200bp to −669bp relative to the Cd4 TSS (Chr #6:124832027-124838896; mm9); Biological replicates are noted. (b) BAC-enrichment and bisulfite, high-throughput sequencing was performed on DP cells sorted from Cd4S4Δ/S4Δ mice. Data are displayed as in (a). Replicates were derived from two independent experiments.
Figure 4
Figure 4. E4P is required for Cd4 locus hypo-methylation in the T-helper lineage
(a) CATCH-seq was performed on naïve CD4+ cells (Thy1.2+CD25−CD44loCD62L+CD4+) from Cd4E4PΔ/E4PΔ; mice. A heatmap depicts percentage CpG methylation in biological replicates from +6200bp to −669bp relative to the Cd4 TSS (Chr #6:124832027-124838896; mm9). Data from WT CD4 cells in Figure 2b are shown for comparison. (b) CFSE-labeled naïve CD4+ Cd4E4PΔ/E4PΔ cells were stimulated in vitro with anti-CD3, anti-CD28 and IL-2 for 5 days. CD4+ (CD4+ → CD4+) and CD4− (CD4+ → CD4−) that had undergone at least 6 divisions were sorted for CATCH-seq. Data are displayed as in (a). Results from 2 independent experiments are displayed.
Figure 5
Figure 5. Reduction in Dnmt1 activity rescues CD4 expression in Cd4E4PΔ/E4PΔ helper T cells
CFSE-loaded naïve CD4+ T cells from the indicated strains of mice were stimulated in vitro for 72 h with anti-CD3 and anti-CD28 antibodies. (a) Top, post-sort, pre-stimulation CD4 and CD8 expression; bottom, CD4 expression and CFSE dilution after 72 h. Percentages of CD4+ cells are indicated. (b) Graph of CD4 MFI after 72 h activation of Cd4E4PΔ/E4PΔDnmt1L/ChipCd4-cre+ (red) and Cd4E4PΔ/E4PΔDnmt1L/LCd4-Cre− CD4+ T cells (blue) (from CD4+ gates in (a)). Dnmt1 knockout cells were not included as they exhibited growth defects (i.e. lower forward scatter), precluding MFI comparison (data not shown). (c) Percent CD4+ cells from each cell division of samples in (a), as tracked using dilution of CFSE fluorescence. Cells were from mice of the genotypes Cd4E4PΔ/E4PΔDnmt1L/LCd4-Cre− (blue); Cd4E4PΔ/E4PΔDnmt1L/ChipCd4-cre+ (red); and Cd4E4PΔ/E4PΔDnmt1L/LCd4-Cre+ CD4+ (black). Results are representative of at least 4 independent experiments.
Figure 6
Figure 6. Cd4 locus demethylation occurs late in helper lineage differentiation and is largely independent of Zbtb7b
(a) A Cd4 intron 1 amplicon from the indicated sorted cells in (b–d) was subjected to bisulfite sequencing: (b) GFP−, GFPmid or GFPhi CD4+CD8lo (also: CD69+HSAhiTCRβ+) and GFP+ CD4SP (CD69−HSAloTCRb+) thymocytes from Zbtb7bGFP/+ mice, (c) GFPhi CD4+CD8lo and GFP+ CD8SP thymocytes from Zbtb7bGFP/GFP mice. (d) WT CD8SP thymocytes (CD69−HSAloTCRb+). Filled circles indicate methylated CpGs and empty circles indicate unmethylated CpGs. Biological replicates from independent experiments are shown for (b–d). (e) CATCH-seq was performed on GFP+ (MHCII selected) and GFP− (MHCI selected) T cells from the lymph nodes of Zbtb7bGFP/GFP mice (n=1). The heatmap depicts the percentage CpG methylation from +6200bp to −669bp relative to the Cd4 TSS (Chr #6:124832027-124838896; mm9). Data from WT CD4 cells in Figure 2b, are shown for comparison. (f) Zbtb7bGFP/GFP GFP+ and GFP− lymph node CD8+ cells were stimulated in vitro for 3d with anti-CD3 and anti-CD28, and then analyzed for CD4, CD8 and GFP expression; results are representative of 2 independent experiments.
Figure 7
Figure 7. Cd4 TSS-proximal hydroxymethylation in MHCII-selected CD4+CD8lo cells
(a) Genomic DNA from DP, naïve CD4+ and MHCII-selected CD4+CD8lo cells (CD69+HSA+CD4+CD8loGFP+ cells from Zbtb7bGFP/+ mice) was incubated with Uridine Diphosphate Glucose (UDG), with or without β-glucosyltransferase (βGT), before digestion with MspI. T4-βGT transfers UDG specifically to 5hmC residues, blocking MspI digestion of CCGG motifs. MspI digestion (at Chr6:124838036; +191bp relative to the Cd4 TSS) was assessed by qPCR comparison to undigested DNA. As a loading control, values were normalized to an adjacent amplicon without a MspI site. The mean and SD of 3 (CD4+) or 4 (DP and CD4+CD8lo) independent biological samples analyzed in two independent experiments are shown; P values (unpaired student’s t-test) below 0.05 (*) and 0.01 (**) are noted. (b–c) Genomic DNA from DP and CD4+CD8lo cells was subjected to bisulfite amplicon sequencing (BS) or KRuO4-oxidation followed by bisulfite amplicon sequencing (OxBS). (b) Pie charts represent the mean percentage of the indicated cytosine modifications at three CpGs located from +1407 to +1487 relative to the Cd4 TSS (CpG 1 = Chr #6:124836820; CpG 2 = Chr #6:124836779; CpG 3 = Chr #6:124836740), in DP (n=2) and CD4+CD8lo cells (n=2); Data were combined from 2 independent experiments: 4–13 amplicons (BS) or 11–17 amplicons (OxBS) were analyzed for each sample. (c) The mean and SD of the indicated modifications at the 3 CpGs in (b) were graphed for DP and CD4+CD8lo cells (circles and triangles represent the measurements for individual DP and CD4+CD8lo CpGs, respectively). 5hmC levels were calculated by subtracting the percentage “C” after OxBS treatment (5mC only) from the percentage “C” after BS treatment (5mC + 5hmC). P values (paired student’s t-test) below 0.05 (*) and 0.01 (**) are noted.
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