Molecular Immunology 49 (2011) 518–526 Contents lists available at SciVerse ScienceDirect Molecular Immunology j ourna l ho me pag e: www.elsevier.com/locate/molimm DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues Vivian Kont a , Astrid Murumägi b , Lars-Oliver Tykocinski c , Sarah A. Kinkel d , Kylie E. Webster d,1 , Kai Kisand a , Liina Tserel a , Maire Pihlap a , Philipp Ströbel e , Hamish S. Scott f , Alexander Marx e , Bruno Kyewski c , Pärt Peterson a,∗ a Molecular Pathology Group, Tartu University, 50411 Tartu, Estonia b Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland c Division of Developmental Immunology, Tumor Immunology Program, German Cancer Research Center, Germany d Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research and the Department of Medical Biology, The University of Melbourne, Melbourne, Australia e Institute of Pathology, University Medical Centre Mannheim, University of Heidelberg, Germany f Division of Molecular Pathology, Institute of Medical and Veterinary Medicine, Adelaide, Australia a r t i c l e i n f o Article history: Received 10 January 2011 Received in revised form 11 September 2011 Accepted 29 September 2011 Available online 27 October 2011 Keywords: Thymus Autoimmune regulator Genomic methylation Histone modification Gene regulation a b s t r a c t 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. © 2011 Elsevier Ltd. All rights reserved. 1. 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 sec- ondary lymphoid tissues at low levels (Gardner et al., 2008; Poliani et al., 2010). In mTECs, AIRE promotes the ectopic expres- sion 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 Abbreviations: Q2ChIP, quick and quantitative chromatin immunoprecipitation; H3K4me3, trimethylated histone 3 lysine 4; H3K27me3, trimethylated histone 3 lysine 27; TEC, thymic epithelial cell; mTEC, medullary thymic epithelial cell; cTEC, cortical thymic epithelial cell; TSA, tissue-specific antigen. ∗ Corresponding author. Tel.: +372 7374202. E-mail address: part.peterson@ut.ee (P. Peterson). 1 Present address: Garvan Institute of Medical Research, Darlinghurst, NSW, Australia. 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 polyen- docrinopathy 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 immunologi- cal 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 reg- ulation via CpG methylation (Klamp et al., 2006). DNA methylation in promoter regions has been proposed to control the expres- sion of tissue-specific genes; for example, the activation of genes such as Maspin (Futscher et al., 2002) and MCJ (Methylation 0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2011.09.022