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Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells

Nature Immunology volume 4, pages 1183–1190 (2003)Cite this article

Abstract

How T cells regulate interleukin 4 (IL-4) expression is not completely understood. We show here that single-positive thymocytes express IL-4, but attenuate GATA-3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. Type 2 polarization blocks Dnmt1 recruitment, enhances histone H3 Lys4 methylation (indicative of accessible chromatin) and initiates DNA demethylation of the locus. Dnmt1−/− CD4 and CD8 T cells derepress IL-4 expression considerably, demethylate DNA and increase H3 Lys4 methylation without affecting GATA-3 expression, demonstrating that Dnmt1 and DNA methylation are essential for proper Il4 regulation. These results indicate that Dnmts, DNA and histone methylation, and transcription factors 'collaborate' to determine appropriate Il4 expression patterns.

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Figure 1: Silencing of Il4 expression in CD8 T cells begins in the thymus.
Figure 2: Il4 expression is attenuated in CD4 T cells and abolished in CD8 T cells as they mature from single-positive thymocytes.
Figure 3: DNA methylation of and recruitment of Dnmts to the Il4-Il13 locus as single-positive thymocytes mature into naive T cells.
Figure 4: IL-4 signaling excludes Dnmt1 from and initiates demethylation of the Il4-Il13 locus.
Figure 5: IL-4 signaling increases Il4 and Gata3 expression and H3 Lys4 methylation in both CD4 and CD8 T cells.
Figure 6: Dnmt1−/− T cells substantially increase Il4 expression and show profound DNA demethylation and increased H3 Lys4 methylation at the Il4-Il13 locus.

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Acknowledgements

We thank A. Kelso, H. Harowicz, B. Fallen, K. Allen and F. Lewis for their support and technical assistance, and all members of the Wilson laboratory for discussions. This work was supported by awards from the National Institutes of Health (GM20865, HD18184 and HD39454) and the March of Dimes.

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  1. Mercedes Pérez-Melgosa

    Present address: Unidad de Diagnostico y Terapia Molecular, Hospital Universitario Vall d'Hebron, Barcelona, Spain, 08035

Authors and Affiliations

  1. Department of Immunology, University of Washington, Seattle, 98195, Washington, USA

    Karen W Makar, Mercedes Pérez-Melgosa, Maria Shnyreva, William M Weaver & Christopher B Wilson

  2. Amgen, 51 University Street, Seattle, 98101, Washington, USA

    David R Fitzpatrick

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Supplementary information

Supplementary Figure 1.

DNA methylation of individual CpG sites within the Il4/Il13 locus of resting SP thymocytes, resting naïve T cells and fully polarized TH1 and TH2 cell lines. DNA methylation analysis is shown for individual CpGs within the CNS-1, Il4 promoter and intronic enhancer of resting (t0) or activated (d3) SP CD4 or SP CD8 thymocytes, naïve CD4 or CD8 T cells, and naïve CD8 T cells activated under type 2 polarized conditions for three days. Included for comparison is DNA methylation analysis of the long-term TH1 cell line, AE7, and TH2 cell line, D10.G4.1; AE7 shows overall DNA methylation content similar to resting naïve CD4 and CD8 T cells, whereas D10.G4.1 demonstrates the loss of DNA methylation in the Il4/Il13 locus of fully polarized TH2 cells. Results are the percent of methylated cytosines and represent the average of 17-55 sequenced alleles from 2-5 independent experiments for each primary cell type and >20 alleles for cell lines AE7 and D10.G4.1. (PDF 76 kb)

Supplementary Figure 2.

Representative raw data from Dnmt1 ChIP experiments. (a) NIH3T3 cell extracts (lane 1) or extracts immunoprecipitated with an (NH4)2SO4 concentrated (lane 2) or an affinity purified (lanes 3 and 4) preparation of the polyclonal anti-murine Dnmt1 antibody generated in this study were separated by SDS-PAGE and then analyzed by western blot. A different antibody to murine Dnmt1 kindly provided by R. Jaenisch (HM334, Tucker, K.L. et al., PNAS USA 93:12920-12925 (1996)) was used to detect Dnmt1 in combination with HRP-conjugated goat anti-rabbit IgG and the ECL detection system (Amersham). The upper band noted by the arrow is murine Dnmt1and the dark lower band in lanes 2-4 is due to the presence of rabbit Ig in the immunoprecipitated samples. (b) Real-time PCR raw data are shown to demonstrate the specificity and level of detection of the Dnmt1 ChIP assay. The top panel represents a positive sample and shows the amplification plot (PCR cycle number versus fluorescence signal) for chromatin immunoprecipitated with Dnmt1 antisera or pre-immune sera. The Ct value (the PCR cycle at which fluorescence rises above a minimal threshold) for each sample is given. A lower Ct value indicates a larger amount of starting material, and the difference in Ct between pre-immune and immune sera reflects specific ChIP. PCR products were a single band of the appropriate size as shown in the inset photograph of an agarose gel, and were confirmed by melt curve analysis (data not shown). The second panel shows the amplification plot and Ct values for a negative sample from the same experiment. The third panel shows the amplification plot for duplicate cDNA standards spanning four logs in concentration. The bottom graph is log [DNA] versus Ct value from the standard curve. The line equation generated by the standards is used to calculate the concentration of DNA in each ChIP sample. The specific IP DNA values reported in the manuscript are calculated by the following equation: ([DNA]specific IP – [DNA] non-specific IP) / [DNA]input. The [DNA]input is an aliquot of pre-IP sample of sheared chromatin and is used to normalize a sample to the amount of chromatin added to each ChIP. Analysis was performed by real-time PCR using a DNA Engine Opticon (MJ Research). (c) Real-time PCR data from Dnmt1 ChIP of activated wildtype (WT) and Dnmt1-/- cells. The amplification plots, Ct values and standard curve are shown. The Ct value for Dnmt1-/- cells is comparable with the non-specific (pre-immune) IP from WT cells. (PDF 78 kb)

Supplementary Figure 3.

Representative raw data from Dnmt3b ChIP experiments. (a) Real-time PCR raw data are shown as in Supplementary Figure 2b to demonstrate the specificity and level of detection of the Dnmt3b ChIP assay. The top panel represents a positive sample chromatin immunoprecipitated with anti-Dnmt3b or mouse IgG (mIgG). The second panel shows a negative sample from the same experiment. The third panel and bottom graph show duplicate cDNA standards spanning three logs in concentration, indicating that Ct values above 33 are not accurately quantitative. For control samples with Ct values below the range of the standard curve (such as in the top panel), the value of the lowest point on the standard curve is used to determine the specific IP DNA. (b) Real-time PCR raw data from one sample analyzed at two different regions, showing a positive Dnmt3b ChIP at the CNS-1 region, and a negative Dnmt3b ChIP at the Il4 promoter. The bottom panels show the standard curves for each PCR reaction. (PDF 43 kb)

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Makar, K., Pérez-Melgosa, M., Shnyreva, M. et al. Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells. Nat Immunol 4, 1183–1190 (2003). https://doi.org/10.1038/ni1004

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