DNA demethylation regulates gene expression in IgE‐activated mouse mast cells
Authors: Aida Paivandy, Mirjana Grujic, Nima Rafati, Gunnar Pejler
Journal: Allergy (2020)

Mast cells (MCs) have a major detrimental impact on various allergic conditions. MCs express the high affinity receptor for IgE (FcεRI) and are sensitized by the binding of antigen-specific IgE to such receptors. Upon encounter of specific antigen, FcεRI cross-linking occurs, which leads to degranulation and to activated transcription of genes coding for pro-inflammatory cytokines and chemokines.1, 2 Epigenetics refers to the effects on gene transcription that are unrelated to changes in DNA sequence. Major epigenetic mechanisms include DNA methylation, histone modification, and effects of noncoding RNAs.3-6 Generally, DNA methylation of gene promoters leads to gene repression whereas DNA demethylation typically leads to activated gene transcription. Considering the major effects of IgE receptor cross-linking on gene transcription in MCs, it appears plausible that epigenetic mechanisms may have an impact on such processes, but this has received relatively little attention.7, 8 To provide additional insight into this issue, we here investigated whether IgE-mediated MC activation affects global DNA methylation in MCs and whether modulation of DNA methylation/demethylation affects gene transcription. We used fully mature mouse peritoneal cell-derived MCs with a phenotype similar to that of human mast cells9 (Figure S1A). MCs were sensitized with DNP-specific IgE, followed by IgE receptor cross-linking by the exposure to DNP-conjugated human serum albumin. This led to substantial MC degranulation (Figure 1A and Figure S1). To assess the effect of IgE receptor cross-linking on global DNA methylation, we used an ELISA based on a monoclonal antibody to 5-methylcytosine. As seen in Figure 1B, IgE receptor cross-linking caused a substantial drop in global DNA methylation, which was most prominent 2 hours after IgE receptor cross-linking. Next, we assessed the effects of DNA methyltransferase (5-azacitidine; inhibitor of DNMT enzymes) and demethylase (Octyl-ɑ-hydroxyglutarate [2-HG]; inhibitor of Ten-eleven translocation [TET] enzymes) inhibitors on gene expression in MCs. 2-HG was nontoxic for MCs at concentrations up to 150 µmol/L, and 5-azacitidine was nontoxic up to 10 µmol/L (Figure 1C-D). Next, we tested the effects of 5-azacitidine and 2-HG (at nontoxic concentrations) on the expression of Il6, Nr4a3, Il3, and Tnf. IgE receptor cross-linking induced an upregulated expression of all these genes. As shown in Figure 1E, the expression of Il6 was profoundly attenuated by the DNA demethylase inhibitor and, conversely, inhibition of DNA methyltransferase activity potentiated the Il6 expression. Similarly, the expression of Il3 was blunted by DNA demethylase inhibition and potentiated by inhibition of DNA methyltransferase activity (Figure 1F). Neither Tnf nor Nr4a3 expression was significantly repressed by DNA demethylase inhibition (Figure 1G-H). The expression of classical MC markers (Mcpt4, Mcpt6) was not affected by interference with DNA demethylase/ methyltransferase activity (Figure S2). To obtain deeper insight into how DNA demethylase inhibition affects gene transcription pathways in MCs, we performed an unbiased transcriptomic analysis, using the AmpliSeq platform. As shown in Figure S3, IgE-mediated MC activation caused upregulated expression of numerous genes. These included several pro-inflammatory genes, transcription factors, growth factors, and receptors. After interference with DNA demethylation, the expression of a substantial number of IgE-induced genes was blunted (altogether 126 genes; genes with ≥ fourfold repression are highlighted in Figure 2). These included Mrgprx1, S1pr2 (coding for sphingosine-1-phosphate receptor 2), Ccl3, Myc, and Il6. DNA demethylase inhibition also caused an increased expression of several genes in the IgE-activated MCs, many of which having anti-inflammatory properties (eg, Gdf9, Hspa1a (HSP72), Wnt4, Egr1, Tnfaip2, and Shb). DNA demethylase inhibition also affected the expression of numerous genes at baseline conditions. Among the genes that were induced by DNA demethylase inhibition under baseline conditions, many were also highly induced under conditions of IgE-mediated MC activation. These included Hspa1a, Hspa1b, Mrgprb8, Cnnm4, Mospd4, Egr1, Klf4, and Ell2 (Figure S4). In addition, several genes were downregulated by DNA demethylase inhibition under baseline conditions. Of these, several were also highly downregulated (≥fourfold) by DNA demethylase inhibition in IgE-activated MCs (eg, S1pr2, Lhfpl2, and Nef2). To confirm that findings from the transcriptome analysis were replicated with an independent method, we used qPCR analysis where we focused on selected genes: Mrgprx1, Myc, and Ccl3. We also assessed the expression of Mrgprx2, a close relative to Mrgprx1. As seen in Figure S5, the qPCR analysis confirmed a profound upregulation of Mrgprx1 in response to IgE-mediated MC activation and also confirmed that DNA demethylase inhibition suppressed the stimulatory effect of IgE receptor cross-linking on Mrgprx1 expression. A small stimulatory effect of IgE receptor cross-linking on Mrgprx2 expression was also seen, and this effect was blocked by DNA demethylase inhibition. The qPCR analysis also confirmed the induction of Ccl3 and Myc by IgE receptor cross-linking, as well as the inhibitory effects of DNA demethylase blockade on the induction of these genes (Figure S5). Altogether, our findings reveal that gene transcription events occurring downstream of IgE receptor cross-linking in MCs can be regulated by epigenetic mechanisms. Notably, interference with DNA demethylation blunted only a fraction of those genes that were induced by IgE receptor cross-linking. When examining the nature of the genes that were downregulated by DNA demethylase blockade, we found that many of these have pro-inflammatory properties (eg, Myc, S1pr, Il6, and Il3). DNA demethylase inhibition also caused upregulated expression of numerous genes in the IgE-activated MCs. Intriguingly, many of these latter genes can have anti-inflammatory functions, for example, Hspa1a (HSP72), Wnt4, Egr1, Shb, Crtam, and Tnfaip2. Hence, our findings suggest that interference with DNA demethylation in IgE-activated mouse MCs may dampen selected pro-inflammatory pathways and, at the same time, potentiate various anti-inflammatory events. We may therefore propose that interference with epigenetic mechanisms can represent a novel strategy for intervening with pathologies mediated by IgE-activated MCs. However, future studies will be needed to extrapolate the findings to the effects on primary human mast cells. Moreover, such strategies should be adopted with caution, given the possibilities of detrimental side effects associated with global inhibition of DNA demethylation/methylation. AmpliSeq analysis was performed at the National Genomics Infrastructure Uppsala (Uppsala Genome Center) of the Science for Life Laboratory, Sweden. The authors declare no conflict of interest in relation to this work. This work was supported by grants from The Swedish Heart and Lung Foundation, The Swedish Research Council, The Swedish Cancer Foundation, and The Knut and Alice Wallenberg Foundation. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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