Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May 29:13:16.
doi: 10.1186/1471-2199-13-16.

Plasticity of DNA methylation in mouse T cell activation and differentiation

Yan Li  1 , Guobing Chen, Lina Ma, Stephen J Ohms, Chao Sun, M Frances Shannon, Jun Y Fan
Affiliations

Plasticity of DNA methylation in mouse T cell activation and differentiation

Yan Li et al. BMC Mol Biol. .

Abstract

Background: Circulating CD4+ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4+ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results: Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion: We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Global DNA methylation and chromosomal accessibility in CD4+ T cells. A. - C. Genomic DNA isolated from total CD4+ T cells taken from the spleens of 2 week or 16 week old C57BL/6 mice that were either unstimulated (NS) or stimulated with PMA/I for 4 hours (A), FACS-sorted naive T cells from both age groups (B), or FACS-sorted naive T cells and nTreg cells or differentiated Th1, Th2, Th17, and iTreg cells (C) were digested with McrBC for 6 hours at 37°C and the DNA fragments were resolved on a 1% agarose gel. Uncut genomic DNA is also shown. D. & E. Nuclei isolated from CD4+ T cells of 2 weeks (D) and 16 weeks (E) old C57BL/6 mice were digested with various concentrations of MNase (10 U to 120 U) at 37°C for 5 min, prior to loading onto a 1% agarose gel for separation. The DNA was visualised by ethidium bromide staining in all experiments. The 1 kb plus ladder is indicated by the letter L. Every experiment was repeated at least 3 times
Figure 2
Figure 2
DNA methylation changes at the promoters of Il2 and Csf2 during T cell activation and development. A. Schematic representation of the promoter regions of the Il2 and Csf2 genes, showing the nucleotide distances upstream from the transcription start sites (dark arrows), the locations of the CpG dinucleotides (vertical arrows) and NF-kB binding elements for reference. PCR primers that covered one or multiple CpG dinucleotides were designed for each inducible gene for the MeDIP assay and are shown as thick horizontal lines and denoted by capital letters. B. - F. The MeDIP assay, using a 5-methylcytosine-specific monoclonal antibody (Epiteck), and real-time PCR, was used to determine the DNA methylation levels of individual CpG dinucleotides located at the promoter regions of Il2 and Csf2 in CD4+ T cells. B. Basal DNA methylation levels at the promoter regions of Il2 and Csf2 in unstimulated CD4+ T cells (NS) isolated from both age groups of mice were examined; the data are shown as means and SEM. C. - F. The DNA methylation levels of the Il2 and Csf2 genes in CD4+ T cells upon PMA/I stimulation for 0, 0.5, 2 and 4 hours. The data were normalized to Gapdh, and were then normalized to the unstimulated samples, and are shown as the mean and SEM of three independent experiments. Every experiment was repeated at least 3 times
Figure 3
Figure 3
Chromatin accessibility at the promoters of the Il2 and Csf2 gene promoters. A - C. Nuclei isolated from unstimulated and stimulated CD4+ T cells were digested with MNase (60 U) at 37°C for 5 min, and chromosomal accessibility at the promoter regions of Il2 and Csf2 in unstimulated CD4+ T cells (NS) isolated from both age groups of mice (A), or at Il2 (B) and Csf2 (C) following T cell activation by PMA/I was determined by real-time PCR. Primer set A at the promoters of both cytokines, and Il2 set F located 2-kb upstream of Il2 TSS or Csf2 set B were located as shown in Figure 2A. DNA accessibility is calculated based on a method described previously [56]. Every experiment was repeated at least 3 times
Figure 4
Figure 4
DNA methylation changes at the promoter of signature genes in T cell differentiation. A. Schematic representation of the promoter regions of Ifng, Il4, Il17 and Foxp3 genes, showing the nucleotide distances upstream from the transcription start sites (dark arrows), the locations of the CpG dinucleotides (vertical arrows) and NF-kB binding elements for reference. PCR primers that covered one or multiple CpG dinucleotides were designed for each inducible gene for the MeDIP assay and are shown as horizontal lines underneath the promoters and denoted by capital letters. B. Using a MeDIP approach, DNA methylation levels at the promoter regions of Ifng and Il4 (B), Il17 and Foxp3 (C) were examined by real-time PCR using the primer sets shown in A. The data were normalized to Gapdh, and were then normalized to the unstimulated samples, and are shown as the mean and SEM of three independent experiments
Figure 5
Figure 5
mRNA expression of enzymes and co-factors involved in DNA demethylation in unstimulated CD4+ T cells in two age groups of mice. Total RNA was prepared from unstimulated CD4+ T cells in both age groups of mice. After reverse transcription, quantitative PCR analysis was performed on cDNA with primers designed to detect these enzymes and co-factors. mRNA expression of (A) DNA methyltransferases (Dnmt1, Dnmt3a), (B) deaminases (Apobec3 and Apobec4), glycosylases (Mbd4 and Tdg), Gadd45 family (Gadd45a, Gadd45b and Gadd45g) and (c) ten-eleven translocation family (Tet1, Tet2 and Tet3) was determined by real-time PCR. The gene expression data were normalized to the Ubc housekeeping gene, and are shown as the mean and SEM of three independent experiments
Figure 6
Figure 6
mRNA expression of enzymes and co-factors involved in DNA demethylation in stimulated CD4+ T cells. A - C. mRNA expression of these enzymes and co-factors in CD4+ T cells after 4 hours PMA/I stimulation in both age groups of mice. Expression is presented as fold change relative to unstimulated CD4+ T cells (NS) of the same age in addition to initial Ubc normalization as mentioned earlier. The data are shown as the mean and SEM of three independent experiments
Figure 7
Figure 7
mRNA expression of enzymes and co-factors involved in DNA demethylation in differentiated T cells. A. - C. Comparison of mRNA expression of these enzymes and co-factors in non-differentiated naive T cells and differentiated T cells (Th1, Th2, Th17, nTreg and iTreg). The data were normalized to naive cells in addition to initial Ubc normalization as mentioned earlier, and are shown as the mean and SEM of three independent experiments
Figure 8
Figure 8
Generation of 5-hydroxymethylcytosine at specific gene promoters in CD4+ T cells. A. - E. The MeDIP approach, using a 5-hydroxymethylcytosine (5hmC) antibody (Active Motif) that specifically recognizes 5-hydroxymethylcytosine, was applied to determine the DNA 5hmC levels at individual CpG dinucleotides located at the promoter regions of inducible genes in CD4+ T cells from mice of both age groups during PMA/I stimulation for 0, 0.5, 2 and 4 hours (A. - C.) and T cell differentiation (D. - E.); primers Il2 set B (A) and Il2 set C (B) are located at the Il2 promoter; C . primer Csf2 set A is located at the Csf2 promoter; primer sets A of Ifng and Il4 are located at the Ifng and Il4 promoters, respectively; primers Il17 set A and set B, Foxp3 set A and set B are located at the Il17 and Foxp3 promoters, respectively. The data were normalized to NS, and are shown as the mean and SEM of three independent experiments
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Holliday R. The inheritance of epigenetic defects. Science. 1987;238:163–170. doi: 10.1126/science.3310230. - DOI - PubMed
    1. Morgan HD, Santos F, Green K, Dean W, Reik W. Epigenetic reprogramming in mammals. Hum Mol Genet. 2005;14:R47–R58. doi: 10.1093/hmg/ddi114. - DOI - PubMed
    1. Feng S, Jacobsen SE, Reik W. Epigenetic Reprogramming in Plant and Animal Development. Science. 2010;330:622–627. doi: 10.1126/science.1190614. - DOI - PMC - PubMed
    1. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;293:1089–1093. doi: 10.1126/science.1063443. - DOI - PubMed
    1. Calvanese V, Fernández AF, Urdinguio RG, Suárez-Alvarez B, Mangas C, Pérez-García V, Bueno C, Montes R, Ramos-Mejía V, Martínez-Camblor P. et al.A promoter DNA demethylation landscape of human hematopoietic differentiation. Nucleic Acids Res. 2012;40:116–131. doi: 10.1093/nar/gkr685. - DOI - PMC - PubMed

Publication types

Substances