Elsevier

Molecular Immunology

Volume 49, Issue 3, December 2011, Pages 518-526
Molecular Immunology

DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues

https://doi.org/10.1016/j.molimm.2011.09.022Get rights and content

Abstract

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.

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.

References (33)

  • F.-G.A. Consortium

    An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains

    Nat. Genet.

    (1997)
  • J.A. Dahl et al.

    A quick and quantitative chromatin immunoprecipitation assay for small cell samples

    Front. Biosci.

    (2007)
  • J. Derbinski et al.

    Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels

    J. Exp. Med.

    (2005)
  • M. Frommer et al.

    A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands

    Proc. Natl. Acad. Sci. U. S. A.

    (1992)
  • B.W. Futscher et al.

    Role for DNA methylation in the control of cell type specific maspin expression

    Nat. Genet.

    (2002)
  • J. Gabler et al.

    Promiscuous gene expression and the developmental dynamics of medullary thymic epithelial cells

    Eur. J. Immunol.

    (2007)
  • Cited by (31)

    • The Epigenetics of Primary Biliary Cholangitis

      2018, The Epigenetics of Autoimmunity
    • The epigenetics of PBC: The link between genetic susceptibility and environment

      2016, Clinics and Research in Hepatology and Gastroenterology
      Citation Excerpt :

      Global hypomethylation as well as hypo- or hypermethylation of specific loci [79,80] is tipical features of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ulcerative colitis (UC). Moreover, DNA methylation plays essential roles in normal transcriptional control of key factors in T cell differentiation and function, such as the interferon gamma locus [81], and the transcription factors forkhead box P3 (FoxP3) [82] and AIRE [83]. Unfortunately even if numerous global or single gene methylation dysregulation are reported in PBC patients, as discussed in the next paragraphs, any alteration of methylation have been reported to correlate with gene expression in PBC.

    • MicroRNA expression profiling of thymic epithelial tumors

      2014, Lung Cancer
      Citation Excerpt :

      A key challenge for a relevant advance in TET management could derive from a deeper molecular biology characterization of these relatively rare neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. To date, specific genetic and epigenetic aberrations associated to different TET histotypes have been identified, providing insight into the genes involved in the pathogenesis of these tumors [5–9]. Moreover, by microarray-based gene expression analysis, genes related to stage and metastatic potential have been described [10–12] and a nine-gene signature predicting metastatic behavior has been proposed [13].

    • Altered DNA methylation profile in Norwegian patients with Autoimmune Addison's Disease

      2014, Molecular Immunology
      Citation Excerpt :

      The DNA methylation signature is changed in many autoimmune diseases, as exemplified by global hypomethylation as well as hypo- or hypermethylation of specific loci (Lu, 2013; Selmi et al., 2012). Moreover, DNA methylation plays essential roles in normal transcriptional control of key factors in T cell differentiation and function, such as the interferon gamma locus (Sawalha, 2008), and the transcription factors FoxP3 (forkhead box P3 (Lal et al., 2009) and AIRE (Kont et al., 2011). Primary adrenal insufficiency (Addison's disease) is a rare disease with a prevalence of around 100–140 per million inhabitants in European countries (Betterle et al., 2002; Laureti et al., 1999; Lovas and Husebye, 2002; Myhre et al., 2002).

    • Autoimmune regulator (Aire) controls the expression of microRNAs in medullary thymic epithelial cells

      2013, Immunobiology
      Citation Excerpt :

      In addition, Aire may regulate the transcription of genes that do not encode PTAs (Derbinski et al. 2005) and even act as a repressor of other loci in the genome (Mathis and Benoist 2007, 2009). Weakly transcribed genes are targets for the AIRE protein, which interacts with hypomethylated promoter regions in the chromatin through its PHD1 domain (Org et al. 2008; Koh et al. 2008, 2010; Chignola et al. 2009; Kont et al. 2011). According to Giraud et al. (2012), Aire is considered an unusual TF because it does not appear to function as a typical transactivator.

    View all citing articles on Scopus
    1

    Present address: Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

    View full text