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De Novo DNA Methylation Is Required to Restrict T Helper Lineage Plasticity

Thomas, Rajan M., Gamper, Christopher J., Ladle, Brian H., Powell, Jonathan D., Wells, Andrew D.·2012·Journal of Biological Chemistry
DOI 10.1074/jbc.m111.312785
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Abstract

Naïve CD4+ T cells are highly plastic and can differentiate into discrete lineages with unique functions during an immune response. Once differentiated, helper T cells maintain a stable transcriptional memory of their initial lineage choice and resist redifferentiation. During embryogenesis, de novo DNA methylation operates on the hypomethylated genome of the blastocyst to achieve tissue-specific patterns of gene expression. Similarly, the ifn γ promoter is hypomethylated in naïve T cells, but Th2, Th17, and iTreg differentiation is accompanied by substantial de novo DNA methylation at this locus. To determine whether de novo DNA methylation is required to restrict T helper lineage plasticity, we used mice with T cell-specific deletion of the methyltransferase DNMT3a. Induction of lineage-specific cytokines occurred normally in the absence of DNMT3a, however, DNMT3a-deficient Th2, Th17, and iTreg completely failed to methylate the ifn γ promoter. This was accompanied by an increase in the transcriptionally permissive trimethyl H3K4 mark, and a reduction in inhibitory H3K27 methylation at the ifn γ locus. Failed de novo methylation resulted in failed silencing of the ifn γ gene, as DNMT3a-deficient Th2, Th17, and iTreg cells produced significant levels of IFNγ following restimulation in the presence of IL-12. Therefore, DNMT3a-mediated DNA methylation restricts T helper plasticity by establishing an epigenetically silent chromatin structure at regulatory regions of the ifn γ gene. Background: T cells undergo lineage commitment during an immune response and "remember" their lineage choice after the instructive signals cease. Results: T cells lacking a de novo DNA methyltransferase fail to silence the ifn γ gene. Conclusion: DNMT3a opposes T cell trans -differentiation by epigenetically silencing "off-lineage" genes. Significance: The proper control of inflammatory cytokine gene expression is crucial for immune homeostasis.

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FIGURE 1 DNMT3a expression and recruitment to the ifn 𝛄 promoter in polarized T helper subsets. Sorted CD4 + CD62L hi CD44 lo naïve T cells were differentiated in vitro into Th1, Th2, Th17, or iTreg as described under 'Experimental Procedures.' A , DNMT3a protein in whole cell extracts was measured by immunoblotting using polyclonal anti-DNMT3a Ab ( upper panel ), and actin was measured as a loading control ( lower panel ). B , activated CD4 + T cells from wild-type mice ( black histogram) or DNMT3a2loxPxCD4-Cre mice (DNMT3a-ko, gray histogram) were stained with a monoclonal anti-DNMT3a ( solid histograms) or isotype control Ab ( dotted gray histogram) and analyzed by flow cytometry. The mean fluorescence intensity ( MFI ) of each population is depicted. C , DNMT3a levels in wild-type Th1, Th2, Th17, and iTreg were measured using the same approach as in B . D , chromatin extracts from naive, Th1 Th2, Th17, or iTreg cells were immunoprecipitated using polyclonal antiDNMT3a or control Ab, and co-precipitation of the ifn 𝛄 promoter was quantified by quantitative PCR. Specific binding was determined by normalizing the promoter signal in the anti-DNMT3a ChIP samples to the background detected in isotype ChIP controls. The data depicted are representative of two to three separate experiments.
FIGURE 1 DNMT3a expression and recruitment to the ifn 𝛄 promoter in polarized T helper subsets. Sorted CD4 + CD62L hi CD44 lo naïve T cells were differentiated in vitro into Th1, Th2, Th17, or iTreg as described under 'Experimental Procedures.' A , DNMT3a protein in whole cell extracts was measured by immunoblotting using polyclonal anti-DNMT3a Ab ( upper panel ), and actin was measured as a loading control ( lower panel ). B , activated CD4 + T cells from wild-type mice ( black histogram) or DNMT3a2loxPxCD4-Cre mice (DNMT3a-ko, gray histogram) were stained with a monoclonal anti-DNMT3a ( solid histograms) or isotype control Ab ( dotted gray histogram) and analyzed by flow cytometry. The mean fluorescence intensity ( MFI ) of each population is depicted. C , DNMT3a levels in wild-type Th1, Th2, Th17, and iTreg were measured using the same approach as in B . D , chromatin extracts from naive, Th1 Th2, Th17, or iTreg cells were immunoprecipitated using polyclonal antiDNMT3a or control Ab, and co-precipitation of the ifn 𝛄 promoter was quantified by quantitative PCR. Specific binding was determined by normalizing the promoter signal in the anti-DNMT3a ChIP samples to the background detected in isotype ChIP controls. The data depicted are representative of two to three separate experiments.CC-BY · From the PDF
FIGURE 2 DNMT3a-dependent DNA methylation at the ifn 𝛄 promoter in non-Th1 cells. Sorted CD4 + CD62L h iCD44 lo naïve T cells ( A ) from DNMT3a2loxP (wild-type, toppanels ) or DNMT3a2loxPxCD4-Cre(DNMT3ako, bottompanels ) mice weredifferentiatedintoTh1( B ), Th2 ( C ), Th17 ( D ), or iTreg ( E ) in vitro through one or two rounds of stimulation. Genomic DNA was purified and subjected to bisulfite conversion, and the promoter ( left panels ) and intron I enhancer ( right panels ) regions were amplified, cloned, and sequenced. Heat maps depict the aggregate methylation levels ( yellow , 0% to blue , 100%) at each CpG from 25-30 individual clones in one of two separate, representative experiments.
FIGURE 2 DNMT3a-dependent DNA methylation at the ifn 𝛄 promoter in non-Th1 cells. Sorted CD4 + CD62L h iCD44 lo naïve T cells ( A ) from DNMT3a2loxP (wild-type, toppanels ) or DNMT3a2loxPxCD4-Cre(DNMT3ako, bottompanels ) mice weredifferentiatedintoTh1( B ), Th2 ( C ), Th17 ( D ), or iTreg ( E ) in vitro through one or two rounds of stimulation. Genomic DNA was purified and subjected to bisulfite conversion, and the promoter ( left panels ) and intron I enhancer ( right panels ) regions were amplified, cloned, and sequenced. Heat maps depict the aggregate methylation levels ( yellow , 0% to blue , 100%) at each CpG from 25-30 individual clones in one of two separate, representative experiments.CC-BY · From the PDF
FIGURE 3 Histone methylation at the ifn 𝛄 locus in primary, wild-type and dnmt3a -deficient T helper lineages. Naïve CD4 + T cells ( A ) from DNMT3a2loxP (wild-type, dark green and dark red bars ) or DNMT3a2loxPxCD4Cre (DNMT3a ko, light green and light red bars ) mice were differentiated into Th1 ( B ), Th2 ( C ), Th17 ( D ), or iTreg ( E ) in vitro as described under 'Experimental Procedures.' Cells were fixed, and soluble chromatin was prepared, and nucleosomes containing histone H3 trimethylated at lysine 4 ( green bars ) or lysine 27 ( redbars ) was enriched by precipitation with specific Ab as described (34). The presence of the ifn 𝛄 promoter, intronic enhancer, CNS-5, CNS-22,
FIGURE 3 Histone methylation at the ifn 𝛄 locus in primary, wild-type and dnmt3a -deficient T helper lineages. Naïve CD4 + T cells ( A ) from DNMT3a2loxP (wild-type, dark green and dark red bars ) or DNMT3a2loxPxCD4Cre (DNMT3a ko, light green and light red bars ) mice were differentiated into Th1 ( B ), Th2 ( C ), Th17 ( D ), or iTreg ( E ) in vitro as described under 'Experimental Procedures.' Cells were fixed, and soluble chromatin was prepared, and nucleosomes containing histone H3 trimethylated at lysine 4 ( green bars ) or lysine 27 ( redbars ) was enriched by precipitation with specific Ab as described (34). The presence of the ifn 𝛄 promoter, intronic enhancer, CNS-5, CNS-22,CC-BY · From the PDF
FIGURE 4 Cytokine production by primary, wild-type, and dnmt3a -deficient T helper cells. Sorted CD4 + CD62L hi CD44 lo naïve T cells from DNMT3a2loxP (WT, top ) or DNMT3a2loxPxCD4-Cre (ko, bottom ) mice were differentiated into Th1 ( A and E ), Th2 ( B , F , and G ), Th17 ( C and H ), or iTreg ( D ) in vitro . Cultures were restimulated with PMA/ionomycin, and IFN 𝛄 , IL-4, IL-17, and Foxp3 expression were measured by intracellular staining at 4 h ( A-D ) or by ELISA at 24 h ( E-H ). Results are representative of two to four separate experiments.
FIGURE 4 Cytokine production by primary, wild-type, and dnmt3a -deficient T helper cells. Sorted CD4 + CD62L hi CD44 lo naïve T cells from DNMT3a2loxP (WT, top ) or DNMT3a2loxPxCD4-Cre (ko, bottom ) mice were differentiated into Th1 ( A and E ), Th2 ( B , F , and G ), Th17 ( C and H ), or iTreg ( D ) in vitro . Cultures were restimulated with PMA/ionomycin, and IFN 𝛄 , IL-4, IL-17, and Foxp3 expression were measured by intracellular staining at 4 h ( A-D ) or by ELISA at 24 h ( E-H ). Results are representative of two to four separate experiments.CC-BY · From the PDF
FIGURE 5 Histone methylation at the ifn 𝛄 locus following secondary Th1 polarization of wild-type and dnmt3a -deficient T helper lineages. Naïve CD4 + T cells from DNMT3a2loxP (wild-type, dark green and dark red bars ) or DNMT3a2loxPxCD4-Cre (DNMT3a ko, light green and light red bars ) mice were differentiated into Th1 ( A ), Th2, or Th17 cells as described. Established Th2 ( B and D , Th2 3 Th1) and Th17 (C and E, Th17 3 Th1) lines were then restimulated in the presence of IL-12 for an additional week, and histone methylation ( B and C , H3K4me3, green bars ; H3K27me3, red bars ) at the ifn 𝛄 locus was assessed as described in Fig. 3, and DNA methylation at the promoter and intronic enhancer ( D and E ) was measured as described in Fig. 2. Statistically significant differences between wild-type and dnmt3a -ko in A-C were determined by paired t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Results are representative of three separate experiments.
FIGURE 5 Histone methylation at the ifn 𝛄 locus following secondary Th1 polarization of wild-type and dnmt3a -deficient T helper lineages. Naïve CD4 + T cells from DNMT3a2loxP (wild-type, dark green and dark red bars ) or DNMT3a2loxPxCD4-Cre (DNMT3a ko, light green and light red bars ) mice were differentiated into Th1 ( A ), Th2, or Th17 cells as described. Established Th2 ( B and D , Th2 3 Th1) and Th17 (C and E, Th17 3 Th1) lines were then restimulated in the presence of IL-12 for an additional week, and histone methylation ( B and C , H3K4me3, green bars ; H3K27me3, red bars ) at the ifn 𝛄 locus was assessed as described in Fig. 3, and DNA methylation at the promoter and intronic enhancer ( D and E ) was measured as described in Fig. 2. Statistically significant differences between wild-type and dnmt3a -ko in A-C were determined by paired t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Results are representative of three separate experiments.CC-BY · From the PDF
FIGURE 6 IFN 𝛄 production by established, wild-type, and dnmt3a -deficient T helper lineages following repolarization to Th1. Naïve CD4 + T cells from DNMT3a2loxP (WT, open symbols ) or DNMT3a2loxPxCD4-Cre (ko, filled symbols ) mice were differentiated under Th2 ( A ), iTreg ( B ), or Th17 ( C ) conditions for 2 weeks. These established lines were then cultured in the presence of IL-12 for an additional week, and cytokine production was assessed as described by intracellular staining ( A-C ) and ELISA ( D-F ). Intracellular staining results are representative of four separate experiments, and ELISA results depict two mice per group and are representative of two separate experiments.
FIGURE 6 IFN 𝛄 production by established, wild-type, and dnmt3a -deficient T helper lineages following repolarization to Th1. Naïve CD4 + T cells from DNMT3a2loxP (WT, open symbols ) or DNMT3a2loxPxCD4-Cre (ko, filled symbols ) mice were differentiated under Th2 ( A ), iTreg ( B ), or Th17 ( C ) conditions for 2 weeks. These established lines were then cultured in the presence of IL-12 for an additional week, and cytokine production was assessed as described by intracellular staining ( A-C ) and ELISA ( D-F ). Intracellular staining results are representative of four separate experiments, and ELISA results depict two mice per group and are representative of two separate experiments.CC-BY · From the PDF

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Metadata

Authors
Thomas, R. M., Gamper, C. J., Ladle, B. H., Powell, J. D., & Wells, A. D.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2012-06
Volume
287
Issue
27
Pages
22900-22909
ISSN
0021-9258
DOI
10.1074/jbc.m111.312785
Cited by
79
References
34
Language
en
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