DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues
Highlights
► AIRE is a key molecule in thymic tolerance to peripheral tissue-specific antigens. ► We studied the CpG methylation and histone modifications in AIRE gene in thymus. ► AIRE promoter has low CpG methylation in thymic epithelial cell subsets. ► AIRE-negative thymomas and peripheral tissues have hypomethylated AIRE promoter. ► AIRE expression in thymic epithelium matches with histone H3 lysine 4 trimethylation.
Introduction
AIRE (AutoImmune REgulator) is a transcriptional activator (Kyewski and Peterson, 2010, Peterson et al., 2008) that is mainly expressed in medullary thymic epithelial cells (mTECs) and secondary lymphoid tissues at low levels (Gardner et al., 2008, Poliani et al., 2010). In mTECs, AIRE promotes the ectopic expression of peripheral tissue-specific antigens (TSAs), such as insulin (Derbinski et al., 2005, Gabler et al., 2007, Kont et al., 2008), which leads to the negative selection of autoreactive T cells (Anderson et al., 2002, Derbinski et al., 2005). The essential role of AIRE in safe-guarding central tolerance to a wide variety of TSAs was first recognized in studies of a rare human autoimmune disease. The autosomal recessive disease called APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, OMIM: 240300) (Consortium, 1997, Nagamine et al., 1997) is caused by mutations in the AIRE gene and is characterized by a loss of immunological self-tolerance towards several endocrine organs (Liston et al., 2003). Similar to humans, Aire-deficient mice develop multiple signs of autoimmunity, such as infiltration of autoreactive T cells into several peripheral tissues and production of autoantibodies with various specificities (Anderson et al., 2002, Ramsey et al., 2002).
The human and mouse AIRE gene promoters have CpG islands, which are highly hypermethylated in several AIRE-negative cell lines (Murumagi et al., 2003). Furthermore, AIRE expression was induced in a methyltransferase-deficient cells, suggesting the regulation via CpG methylation (Klamp et al., 2006). DNA methylation in promoter regions has been proposed to control the expression of tissue-specific genes; for example, the activation of genes such as Maspin (Futscher et al., 2002) and MCJ (Methylation controlled DNAJ) (Strathdee et al., 2004) correlates with CpG island methylation. In addition to CpG methylation, histone modifications represent another level of epigenetic gene regulation. A strong negative correlation between the CpG methylation and the permissive histone H3 lysine 4 (H3K4me3) mark in promoter regions has been shown (Cedar and Bergman, 2009). In a genome-wide study that correlated histone modifications with CpG content, the AIRE promoter was classified as a CpG-rich promoter, displaying permissive H3K4me3 modifications in embryonic stem cells but non-permissive histone H3 lysine 27 methylation (H3K27me3) in mouse embryonic fibroblasts (Mikkelsen et al., 2007). Consistent with this pattern, we earlier reported increased levels of H3K4me3 in the mouse AIRE promoter along with upregulated expression in terminally differentiated mTEC (Org et al., 2009).
In this paper, we analyzed the methylation of AIRE promoter in ex vivo purified human and murine thymic epithelial cells, human thymomas and several non-thymic tissues. When we tested the correlation of methylation and expression levels, we found AIRE promoter to have low levels of methylation in both mTEC and cortical epithelial cells (cTEC) whereas human thymocytes had high level of methylation. We also observed a variable pattern of hypomethylation in the promoter region in thymomas and peripheral tissues, which both lack detectable AIRE protein expression. In contrast, the presence of the permissive and non-permissive histone marks, H3K4me3 and H3K27me3, respectively, did show a correlation with AIRE expression in the respective cell types.
Section snippets
Tissue samples and isolation of thymic cell populations
Normal peripheral tissue samples were obtained from the Tartu University Tissue Bank, Estonia. Thymomas (six WHO type A thymomas, five type AB thymomas and five type B3 thymomas) were supplied by the cryo-archives of the Institute of Pathology, University of Würzburg, Germany. Tumor tissue and the residual non-neoplastic thymus tissue of adult thymoma patients (ranging from 32 to 67 years old) were analyzed. TECs were purified from thymus samples of children undergoing corrective heart surgery
AIRE promoter methylation in human and murine thymic epithelial cells
AIRE expression in thymus is restricted to the terminally differentiated mTECs. We first analyzed the methylation pattern in the CpG islands of the human AIRE gene promoter using bisulfite sequencing of 47 CpG positions (Fig. 1) in 5 normal thymus tissues adjacent to thymomas. The samples showed highly variable methylation patterns from moderate (20–60%) to high levels (80–100%) of methylation in variable positions (Supplementary Fig. 1).
The variable methylation pattern of the AIRE promoter in
Discussion
We investigated the DNA methylation pattern in the AIRE gene promoter in conjunction with AIRE expression in normal and neoplastic thymic tissues and various extrathymic organs. Previous studies showed an induction of AIRE expression upon experimental global hypomethylation and increased AIRE expression levels in DNMT1 and DNMT3b double-deficient cell lines, which suggested that genomic methylation might be a critical mechanism for AIRE gene regulation (Klamp et al., 2006, Murumagi et al., 2003
Role of the funding source
The study was supported by Estonian Science Foundation Grants 8169, the European Regional Fund and Archimedes Foundation and the Estonian Targeted Funding Grant SF0180021s07 (to V.K., K.K., L.T., M.P. and P.P.), the Deutsche Krebshilfe, grant 106430 (P.S. and A.M.) and the Deutsche Forschungsgemeinschaft (SFB 405) and the German Cancer Research Center, Heidelberg, Germany (L-O.T. and B.K.). The funding source(s) had no involvement in study design; in the collection, analysis, and interpretation
Contributions
V.K. performed the experiments and interpretations of the findings. V.K., P.P. and B.K. participated in writing. A.M., L.O.T., S.K., K.W., K.K., M.P. and L.T. participated in performing experiments.
Disclosure statement
The authors declare no conflict of interest.
Acknowledgements
We thank Dr. Ana Rebane and Dr. Martti Laan for critical reading and members of the labs of in B.K., P.P and H.S.S. for helpful discussions and doctors Toomas Aro and Silvia Virro from Tartu University Hospital for kind help in collecting clinical samples. We thank Ms. Laura Tomson for excellent technical assistance.
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Present address: Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.