Inhibition of IFN-gamma transcription by site-specific methylation during T helper cell development - PubMed (original) (raw)

Inhibition of IFN-gamma transcription by site-specific methylation during T helper cell development

Brendan Jones et al. EMBO J. 2006.

Abstract

Polarization of naïve CD4 T cells into T helper type 2 (TH2) cells is characterized by expression of IL-4 and silencing of IFN-gamma. Here we show that during TH2 polarization, the DNA methyltransferase Dnmt3a is recruited to the IFN-gamma promoter and correspondingly the promoter undergoes progressive de novo methylation. Notably, the CpG located at the -53 position becomes methylated rapidly and this methylation inhibits ATF2/c-Jun and CREB transcription factor binding in vitro. In vivo, the same factors bind to the unmethylated IFN-gamma promoter in T helper type 1 (TH1) cells but not the methylated IFN-gamma promoter in TH2 cells. Furthermore, methylation at the -53 CpG alone is sufficient to inhibit the IFN-gamma promoter-driven reporter gene expression in a TH1 cell line. These findings suggest that rapid methylation of the evolutionarily conserved -53 CpG by Dnmt3a may suppress IFN-gamma transcription in developing TH2 cells by directly inhibiting transcription factor binding.

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Figures

Figure 1

Figure 1

Methylation changes in the IFN-γ locus during TH1 and TH2 cell polarization. (A) CpG locations in the IFN-γ promoter and the contiguous transcribed region. (B) IFN-γ and IL-4 expression by polarized TH1 and TH2 cells. Day 6-polarized TH1 and TH2 cells were stimulated with PMA and ionomycin for 3 h and cytokine expression was measured. Open histogram, anti-IFN-γ or anti-IL-4 staining; shaded histograms, isotype control. (C and D) Changes in the levels of methylation at different CpGs in the IFN-γ locus during the course of TH1 (C) and TH2 (D) polarization. Polarized TH cells were stimulated every 6 days and, with the exception of day 3, were harvested for methylation assay just before restimulation. The same data are shown in part of Table II.

Figure 2

Figure 2

Inhibition of ATF2/c-Jun and CREB binding by the −53 CpG methylation. (A) EMSA using probes corresponding to the proximal AP1 site of the IFN-γ promoter methylated at the indicated CpGs and nuclear extracts from the AE7 (TH1) cells. (B) EMSA in the presence of competitor oligonucleotides. The assays were carried out with AE7 nuclear extract and labeled, unmethylated AP1 probe in the presence of increasing amount of unlabeled, methylated or unmethylated, or nonspecific (NFAT) competitor oligonucleotides. The first lane was from the incubation without competitor oligonucleotide. The remaining lanes were from incubations with the indicated unlabeled competitor oligonucleotides. (C) EMSA in the presence of specific antibodies. The assays were carried out with nuclear extracts from either AE7 or D10 (TH2) cells and labeled, unmethylated AP1 probe in the absence (No Ab) or presence of the indicated antibodies. The EMSA with labeled, methylated AP1 probe serves as controls. The anti-ATF1 antibody crossreacts with CREB. Data shown are representative from one of the three to five experiments.

Figure 3

Figure 3

Transcription factor binding to the IFN-γ promoter in TH1 and TH2 cells. (A) ChIP assays of the indicated AP1 and CREB/ATF family members binding within the IFN-γ promoter in the AE7 or the D10 cell lines. PCR assays of IFN-γ intron 1 were used to demonstrate the specificity of immunoprecipitation. Representative data from one of two independent experiments is shown. (B) Western blots demonstrating the relative nuclear protein levels of c-Jun, CREB, ATF1/CREB, and ATF2 in AE7 and D10 cells. (C) ChIP assays of Dnmt3a and Dnmt3b binding and tri-methyl H3K9 levels within the IFN-γ promoter in the indicated cell populations.

Figure 4

Figure 4

Inhibition of IFN-γ promoter activity by site-specific methylation. (A) IFN-γ expression by AE7 and D10 cells. IFN-γ expression in AE7 and D10 cells with or without P/I stimulation (for 2 h) was detected by cytokine recapture assay. Stimulated cells were also stained with an isotype control antibody. (B) The 250 bp IFN-γ promoter is capable of driving reporter gene expression in both TH1 and TH2 cells. Data shown are firefly luciferase activities normalized to renilla luciferase activities for γ-luc and pGL3 vectors in transfected TH1 and TH2 cells with or without P/I stimulation. The error bars indicate standard deviations from triplicate samples in each experiment. Representative data from one of the two independent experiments are shown. (C) Effects of CpG methylation on the IFN-γ promoter activity. The γ-luc vector with the indicated methylation(s), or methylated at all CpG sites by mSss I, or deleted of the proximal AP1-binding site, and the pGL3 vector were transfected together with the renilla luciferase vector into AE7 cells. After 6 h, the transfected cells were stimulated with P/I for 2 h and luciferase activities were measured. (+) Indicates a methylated CpG at the indicated position, whereas (−) indicates an unmethylated CpG at the indicated position. The error bars indicate standard deviations from triplicate samples in each experiment. _P_-values were calculated by Student's _t_-test. Representative data from one of the three to five independent experiments are shown.

Figure 5

Figure 5

IFN-γ promoter methylation and transcriptional repression. (A) In naïve CD4 T cells, the promoter is hypomethylated but IFN-γ is not transcribed possibly because activating complexes, such as c-Jun/ATF2, do not bind to the promoter. (B) Activation under TH1 polarizing conditions allows the c-Jun/ATF2 complex to bind to the promoter and contribute to IFN-γ transcription. (C) During activation under TH2 polarizing conditions, rapid methylation of the −53 CpG prevents c-Jun/ATF2 from binding to the promoter, and thus contributes to the suppression of IFN-γ transcription. (D) In long-term polarized TH2 cells, the remaining CpGs in the IFN-γ promoter are methylated, contributing to chromatin remodeling and the permanent suppression of the locus.

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