DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation
Edited by Anjana Rao, Immune Disease Institute and La Jolla Institute for Allergy and Immunology, Boston, MA, and approved November 18, 2011 (received for review March 9, 2011)
Abstract
The inverse correlation between DNA methylation and lineage-specific gene expression during T helper cell development is well documented. However, the specific functions of the de novo methyltransferases Dnmt3a and Dnmt3b in cytokine gene regulation have not been defined. We demonstrate that the expression of Dnmt3a and Dnmt3b are induced to a greater extent in T helper 2 (Th2) cells than in T helper 1 cells during polarization. Using conditional mutant mice, we determined that Dnmt3a, but not Dnmt3b, regulated expression of T helper cell cytokine genes, with the Il13 gene most prominently affected. Dnmt3a deficiency was accompanied by decreases in DNA methylation and changes in the H3K27 acetylation/methylation status at the Il13 locus. Dnmt3a-dependent regulation of Il13 also occurred in vivo because Dnmt3afl/flCd4cre mice exhibited increased lung inflammation in a murine asthma model, compared with littermate controls. Based on these observations, we conclude that Dnmt3a is required for controlling normal Il13 gene expression and functions as a rate-limiting factor to restrict T helper 2-mediated inflammation.
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Upon encountering antigen in an appropriate cytokine milieu, naïve CD4 cells differentiate into T helper 1 (Th1) or T helper 2 (Th2) cells, which secrete IFN-γ, or IL-4 and IL-13, respectively, and regulate distinct types of inflammation (1). Subset-restricted cytokine gene expression is coordinated by selective activation of transcription factors and epigenetic programming at each locus (2). Stat4 and T-bet are the primary transcription factors for Th1 polarization, whereas Stat6 and Gata3 are required for Th2 differentiation (3).
Studies on T helper cell development demonstrated that epigenetic changes are associated with cytokine gene expression (4–12). Histone modifications that are thought to regulate gene expression primarily occur on the amino termini of core histones, and combinations of modified histone residues constitutes the “histone code” (13, 14). Methylation of mammalian genomic DNA occurs on the cytosine of CpG dinucleotides and correlates with transcriptional inactivation (15, 16). Methylation-mediated gene silencing can be achieved through at least two distinct mechanisms: direct impediment to DNA binding of transcription factors or eliciting a transcriptionally repressive chromatin structure via recruitment of corepressors and chromatin modifiers (17). DNA methylation is essential for normal mammalian development and is required for biological processes such as chromosome X inactivation, gene imprinting, and silencing of transposons and retroviruses (18–21). There are three active mammalian DNA methyltransferases: Dnmt1 (22), Dnmt3a, and Dnmt3b (23). The DNA template-dependent activity of Dnmt1 replicates the parental methylation pattern onto progeny cells, leading to methylation maintenance and heritability. Dnmt3a and Dnmt3b function as de novo methyltransferases (24), establish new methylation by catalyzing the addition of methyl groups to previously unmethylated cytosines in response to developmental stimuli, and are indispensable for mammalian development because targeted deletion of these genes resulted in embryonic lethality (24, 25). The development of naïve CD4 cells toward effector cells was accompanied by lineage-differential DNA methylation at multiple gene loci (7, 11, 26, 27). Promoter methylation at Ifng, Il18r1, and Il4 gene loci is distinct between Th1 and Th2 subsets, raising the possibility that DNA methyltransferases regulate cytokine expression. Although Dnmt1-mediated methylation is critical for normal T-cell homeostasis and proper cytokine gene expression (28), the function of de novo DNA methyltransferases in the regulation of immune gene expression during T helper cell polarization is incompletely understood. In this study, we found that Dnmt3a deficiency led to increased cytokine gene expression and resulted in severe lung inflammation in a murine asthma model.
Results
Dnmt3a Binding at Cytokine Gene Promoters.
To define the function of de novo DNA methyltransferases in T helper cell subsets, we first measured nuclear expression of Dnmt3a, Dnmt3b, and Dnmt1, as well as lineage-determining transcription factors in naïve CD4 T cells, nonpolarized effector cells, and Th1 and Th2 cells. Stat4 and T-bet were present in the nuclei of Th1 cells, whereas Stat6 and Gata3 were detected in the nuclei of Th2 cells, indicative of efficient polarization of these subsets (Fig. 1A). Dnmt1 was equivalently expressed in all subsets. Dnmt3a and Dnmt3b were expressed at low levels in naïve CD4 cells, but expression was increased in nonpolarized cells, in agreement with a previous report that TCR stimulation resulted in up-regulation of de novo methyltransferase expression (29). Notably, Th2 cells expressed more Dnmt3a and Dnmt3b mRNA and protein than nonpolarized or Th1 cells (Fig. 1 A and B).
Fig. 1.
We next performed chromatin immunoprecipitation (ChIP) to determine the DNA binding of Dnmt3a and Dnmt3b at the promoters of cytokine genes. In contrast to differential binding of lineage transcription factors (Fig. S1A), Dnmt3a was detected at each gene promoter, and Dnmt3b binding was only marginally above background (Fig. 1C) in Th1 and Th2 cells. There was a greater amount of Dnmt3a binding in Th2 cells than in Th1 cells, consistent with greater Dnmt3a expression in Th2 cells. The Il13 promoter region was associated with the most Dnmt3a binding of the promoters examined. Greater binding of Dnmt3a to Il13 was also observed following anti-CD3/anti-CD28 activation in the absence of polarizing conditions, with only marginal effects on Dnmt3b binding, even in the absence of Dnmt3a (Fig. S1 B–D).
Increased Expression of Effector Cytokine Genes in the Absence of Dnmt3a.
To determine the effects of Dnmt3a and Dnmt3b on T helper cell cytokine production, we generated mice that lacked either or both DNA methyltransferases by breeding Dnmt3afl/fl, Dnmt3bfl/fl, or Dnmt3a3bfl/fl mice with transgenic mice (Cd4Cre) expressing Cre recombinase under the control of Cd4 promoter (collectively termed Dnmt3fl/flCd4Cre) facilitating deletion of Dnmt3a, Dnmt3b, or both genes in T cells. The conditional deletion of Dnmt3 was verified by PCR and immunoblot (Fig. S2 A and B). Compared with their respective control mice, Dnmt3fl/flCd4Cre mice displayed normal thymic and splenic cellularity and T-cell populations, and T cell proliferation (Figs. S2C and S3).
To define the repressive effects of Dnmt3a and Dnmt3b on cytokine gene expression, we measured expression of a panel of immune genes, comparing Th1 and Th2 cultures derived from Dnmt3fl/flCd4cre and littermate control mice. Gene expression and secretion of IFNγ, IL-4, and IL-13 were increased in Dnmt3afl/flCd4Cre and Dnmt3a3bfl/flCd4Cre, but not Dnmt3bfl/flCd4Cre, Th1 cells compared with littermate control cells (Fig. 2A and Fig. S4A). The transcription of Il18r1 and Il5 genes were unaffected by Dnmt3a and/or Dnmt3b deletion (Fig. S4A). Dnmt3fl/flCd4Cre cultures did not express cytokine genes of the opposing lineage, suggesting that the Dnmt3a and Dnmt3b deletion was insufficient to derepress cytokine gene expression, and consistent with the expression of lineage-determining transcription factors T-bet and Gata3 being normal in the absence of Dnmt3a (Fig. 2B). However, when Gata3 was ectopically expressed in Th1 cells, it had a greater effect on the induction of IL-13 in the absence of Dnmt3a (Fig. 2C). Together, these results indicated that Dnmt3a, not Dnmt3b, played an inhibitory role on cytokine gene expression, and Il13 was the gene most dramatically affected by the loss of Dnmt3a, consistent with the amount of Dnmt3a binding at the Il13 locus. We also observed increased Il13 expression in Dnmt3a-deficient Th9 cultures, (Fig. S4B).
Fig. 2.
DNA Methylation and Dnmt3a Association at the Il13 Locus.
To determine whether DNA methylation correlated with Dnmt3a–DNA binding at the Il13 locus, we used quantitative PCR (qPCR) to measure DNA methylation-dependent digestion at 11 HpaII sites located within the Il13 locus (Fig. 3A). HpaII site 11, within a CpG island upstream to the Il13 gene body, had very low DNA methylation, consistent with observations that CpG islands are generally hypomethylated (30). HpaII sites 1–10 displayed DNA methylation within the Il13 gene, consistent with a previous report showing gene-body methylation throughout the lymphocyte genome (31). At HpaII sites 1, 2, 5, 7, and 8, Th1 genomic DNA had significantly higher methylation levels than did Th2 DNA (Fig. 3B), indicating that DNA methylation at specific sites of the Il13 locus inversely correlated with the Il13 expression. However, at HpaII sites 1–10, there were greater amounts of Dnmt3a association in Th2 than those in Th1 cells (Fig. 3C).
Fig. 3.
Dnmt3a itself or Dnmt3a-dependent alterations in chromatin can block DNA binding capacities of transcription factors, including GATA3 (16, 32). Although Dnmt3a binding was absent in Dnmt3afl/flCd4Cre+ cells, GATA3 binding was unchanged compared with that in control cells (Fig. 3D and Fig. S5A). We next asked whether Dnmt3 deficiency altered DNA methylation at specific gene loci by assessing methylation levels at the Il13 HpaII sites. Th2 cells demonstrated lower amounts of methylation and restricted effects of Dnmt3a deficiency, with significantly decreased methylation at HpaII sites 3 and 6 (Fig. 3E). We further performed bisulfite sequencing of the Il13 promoter and conserved Gata3 Response Element (CGRE) that overlaps with the CpG island. Although we observed no methylation in the CGRE fragments, consistent with the HpaII-PCR results of site 11, and the lack of change in Gata3 binding in the absence of Dnmt3a (Fig. S5A), there was an increase in frequency of unmethylated cytosines at specific sites in the Il13 promoter. CpG sites at −226 and −270 were completely methylated in wild-type Th2 cells but demonstrated increases in the percentage of unmethylated cytosines in Dnmt3a-deficient Th2 cells, whereas sites at −252 and −277 showed no changes in DNA methylation compared with wild-type cells (Table 1 and Fig. S5B). Decreased DNA methylation at the Il13 promoter was consistent with an increased population of IL-13-hi Th2 cells in the absence of Dnmt3a (Fig. S6A). We then sorted cells into IL-13-hi and -low populations and determined, using HpaII-qPCR, that DNA methylation was significantly decreased in IL-13-hi cells, demonstrating a direct link between Il13 DNA methylation and IL-13 production (Fig. S6 B and C).
Table 1.
| Cytosines unmethylated at mouse Il13 promoter, % | |||||
|---|---|---|---|---|---|
| Cells | CpG site −143 | CpG site −226 | CpG site −252 | CpG site −270 | CpG site −277 |
| Dnmt3afl/flCd4cre− Th2 | 20 | 0 | 13.3 | 0 | 6.7 |
| Dnmt3afl/flCd4cre+ Th2 | 33.3 | 33.3 | 13.3 | 13.3 | 6.7 |
Genomic DNA isolated from Dnmt3afl/flCd4cre− and Dnmt3afl/flCd4cre+ Th2 cells was bisulfite-treated and then subjected to PCR amplification, cloning, and sequencing. The percentage of unmethylated cytosines at each CpG site was calculated based on cytosine methylation status of 15 individual clones. The region interrogated is within the Il13 promoter CNS.
Although modest changes in DNA methylation can lead to changes in gene expression (7, 11, 26, 27), we wanted to determine to what degree that changes in Il13 gene expression in the absence of Dnmt3a were indeed due to the methyltransferase activity of Dnmt3a. Thus, we transduced Dnmt3a-deficient Th2 cultures with control or Dnmt3a-expressing retrovirus, or a retrovirus encoding a catalytically inactive mutant, Dnmt3a C120S (33). Transduction of Dnmt3a into Dnmt3a-deficient cells reduced Il13 expression to wild-type Th2 levels (Fig. 3F). In contrast, Il13 mRNA was not significantly decreased by transduction of the inactive C120S mutant, and amounts were significantly higher than cells transduced with active Dnmt3a (Fig. 3F). Although not significant, there was a trend toward a partial effect of the C120S mutant, suggesting the possibility that Dnmt3a could have functions in addition to DNA methyltransferase activity.
Histone Modification Changes at the Loss of Dnmt3a.
DNA methylation and histone modifications are interdependent (34, 35). To understand whether Dnmt3a deletion had effects on histone modifications, we performed ChIP to evaluate histone modifications at the Il13 HpaII sites, comparing Dnmt3afl/flCd4Cre and control Th1/Th2 cells. Correlating with Il13 expression, more histone H4 acetylation (H4ac) and H3 lysine 27 acetylation (H3K27ac) were present in Th2 than in Th1 cells, and H3 lysine 27 tri-methylation (H3K27me3) was enriched in Th1 cells (Fig. 4A). H4ac in Th2 cells was not dramatically affected by Dnmt3 deficiency. Dnmt3afl/flCd4Cre and Dnmt3a3bfl/flCd4Cre Th2 cultures had more H3K27ac enrichment than control Th2 cells, which paralleled increased Il13 expression in the absence of Dnmt3a. Interestingly, there was increased H3K27me3 in Dnmt3afl/flCd4Cre and Dnmt3a3bfl/flCd4Cre Th1 cells, compared with control Th1 cultures, suggesting that H3K27me3 may function as an alternative mechanism to compensate for reduced repression in the absence of Dnmt3a. Dnmt3a-deficient Th2 cells demonstrated a greater amount of CBP and decreased HDAC2 at the Il13 promoter, compared with controls (Fig. 4B).
Fig. 4.
Dnmt3afl/flCd4Cre Mice Displayed Excessive Lung Inflammation in an Asthma Model.
Th2-biased inflammation is implicated in the development of allergic airway disease in humans and mice (36). To explore whether Dnmt3a deficiency induced Il13 expression in vivo, we sensitized and challenged Dnmt3afl/flCd4cre and control mice to induce allergic airway inflammation. Dnmt3afl/flCd4cre mice had increased airway hyperreactivity compared with littermate control mice upon increasing amounts of methacholine (Fig. 5A and Fig. S7A). The Dnmt3afl/flCd4cre mice exhibited significantly higher numbers of total bronchoalveolar lavage (BAL) cells and eosinophils and greater inflammation and mucus production than control mice (Fig. 5 B–D and Fig. S7B).
Fig. 5.
To determine the effects of Dnmt3a deficiency on cytokine production in vivo, we purified splenocytes from sensitized and challenged Dnmt3afl/flCd4cre and control mice. Cells were either stimulated with ovalbumin (OVA) for 4 h for cytokine gene mRNA analysis or for 3 d for detection of cytokine secretion. OVA-stimulated splenocytes derived from Dnmt3afl/flCD4Cre mice had significantly higher Il13 mRNA than control mice (Fig. 5E), with a concomitant trend toward increased Il4 transcription. Cytokine secretion measured by ELISA also demonstrated that IL-13 expression was significantly increased in Dnmt3afl/flCd4cre cells (Fig. 5F).
Discussion
Transcription of cytokine genes in T helper cell subsets is partly determined by DNA methylation status. In this study, we examined mice that lack de novo DNA methyltransferases in T cells to demonstrate that Dnmt3a, not Dnmt3b, was required for controlling normal expression of Il13 in Th2 cells. Dnmt3a regulated DNA methylation and histone modifications at the Il13 locus. The absence of Dnmt3a resulted in increased IL-13 production in vitro and in vivo and increased allergic inflammation.
Il13 expression was undetectable in Th1 cultures derived from Dnmt3afl/flCd4cre mice despite reduced DNA methylation at the Il13 locus (Fig. S5C), which is consistent with normal expression of the lineage-defining transcription factors, even in the absence of Dnmt3a (Fig. 2B). Although altered DNA methylation was correlated with changes in histone modifications, the Il13 gene in Dnmt3a-deficient Th1 cells did not acquire a Th2 pattern of modification, suggesting that the absence of Dnmt3a alone was not sufficient to generate an active chromatin configuration. Thus, although DNA hypomethylation is an important component of a coordinated regulatory network governing gene expression, effective cytokine gene activation requires the activity of lineage-specific transcription factors. Indeed, when Gata3 was transduced into Dnmt3a-deficient Th1 cells, there was greater induction of IL-13 production than in wild-type cells (Fig. 2C). Our study complements another study showing that in the absence of Dnmt3a, there was greater switching of IL-4–secreting to IFN-γ–secreting cells when culture conditions were altered (29). A model wherein epigenetic repression of cytokine loci represents only one component of subset-specific expression is consistent with our previous work demonstrating that STAT4-dependent induction of Th1 genes only partly depends upon reducing Dnmt3a association with target loci (10, 11). Thus, both permissive epigenetic modifications and appropriate transcription factors are required for lineage-restricted cytokine expression.
Dnmt3a enzymatic activity was assessed using Il13 since expression was markedly affected by Dnmt3a deficiency and the Il13 gene locus was associated with an upstream CpG island and multiple HpaII (CCGG) sites within the gene body. Our results indicated an inverse relationship between the Il13 expression and DNA methylation during Th1 and Th2 development, compatible with previous findings on differential methylation at Th2 cytokine gene loci (7). However, the presence of Dnmt3a at the Il13 locus was not the only determinant of DNA methylation, because Th2 cells had greater amounts of Dnmt3a bound to the locus than Th1 cells. These results suggest that other factors present within a Dnmt3 complex impact enzymatic activity and are required for DNA methylation. However, Dnmt3a methyltransferase activity is required because complementation of Dnmt3a-deficient Th2 cells with a catalytically inactive Dnmt3a did not significantly repress Il13 expression (Fig. 3F), and diminished levels of DNA methylation are present in the Il13 gene in cells producing the highest amounts of IL-13 (Fig. S6). Thus, de novo methyltransferases are responsible, at least in part, for establishing this pattern of DNA methylation. Presumably, de novo methyltransferase-independent methylation is attributable to activity of Dnmt1, the maintenance methyltransferase expressed in equivalent amounts in both T helper cell subsets. Therefore, our study provides evidence to support the possibility that de novo DNA methylation acts by fine tuning gene locus-specific methylation in response to developmental instruction.
Histone modifications and DNA methylation are believed to be interdependent with respects to modulating chromatin structure and controlling gene expression. Increased Il13 expression in the absence of Dnmt3a was associated with increased H3K27ac enrichment, which correlated with increased CBP and decreased HDAC2 occupancy at the gene locus. These data also suggested that Dnmt3a, or DNA methylation, might enhance recruitment of HDACs and block the binding of HATs to suppress gene expression at specific loci. Together, our observations provide further evidence for a link between DNA methylation and histone modifications.
DNA methyltransferases can be recruited to target gene loci through two mechanisms: direct DNA binding via PWWP domain of DNA methyltransferase (37) or recruitment by interaction with transcriptional repressors (38). Our data also suggest that increased expression of DNA methyltransferases impacts the association with target genes. We observed that greater expression of Dnmt3a in Th2 cells, compared with Th1 cells, is associated with greater binding to the Il13 locus. Similarly, induced expression of Dnmt3a following antigen receptor stimulation results in greater association with cytokine loci. The antigen stimulation-induced expression and DNA binding capacity of DNA methyltransferases has biological relevance in that naïve CD4 cells, upon antigen stimulation, might take advantage of methylation-mediated gene-silencing effects to limit immune responses. Similarly, Dnmt3a-expressing T helper cells may use the same strategy to prevent immune overreaction. This notion was supported by our observation that Dnmt3afl/flCd4cre mice exhibited increased lung inflammation in a murine asthma model, compared with control mice, indicating that Dnmt3a is a rate-limiting factor restricting inflammatory reactions. Whether de novo methyltransferase-mediated negative regulation of gene expression functions as a general mechanism to manipulate immune responses needs further investigation.
Materials and Methods
Dnmt3 conditional mutant mice were established by breeding Dnmt3afl/fl, Dnmt3bfl/fl, or Dnmt3a3bfl/fl mice with Cre transgenic mice (Cd4cre). Dnmt3afl/fl mice were on a C57BL/6 genetic background. Dnmt3bfl/fl and Dnmt3a3bfl/fl mice were on a mixed C57BL/6 × 129 genetic background. Mice were housed in specific pathogen-free conditions in the Laboratory Animal Resource Center at Indiana University School of Medicine. All experiments were performed in accordance with procedures approved by the Indiana University Animal Care and Use Committee. Primers used for Il13 locus analysis are indicated in Fig. S8.
Further details can be found in SI Materials and Methods.
Acknowledgments
We thank Dr. En Li (Novartis Pharmaceutical Inc.) for generously providing Dnmt3afl/fl and Dnmt3bfl/fl mice. This work was supported by Public Health Service Awards R01 AI45515 and AI057459 (to M.H.K.) and R01 AI085046 (to B.Z.). Q.Y. was supported by National Institutes of Health Grants T32CA111198 and T32HL091816.
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Published online: December 21, 2011
Published in issue: January 10, 2012
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Acknowledgments
We thank Dr. En Li (Novartis Pharmaceutical Inc.) for generously providing Dnmt3afl/fl and Dnmt3bfl/fl mice. This work was supported by Public Health Service Awards R01 AI45515 and AI057459 (to M.H.K.) and R01 AI085046 (to B.Z.). Q.Y. was supported by National Institutes of Health Grants T32CA111198 and T32HL091816.
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This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation, Proc. Natl. Acad. Sci. U.S.A.
109 (2) 541-546,
https://doi.org/10.1073/pnas.1103803109
(2012).
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