Promiscuous gene expression in the thymic medulla – on regulation at the epigenetic and single cell level

The immune system is delicately balanced by self-antigen driven tolerance and pathogen-driven immunity. Self-tolerance of the T cell repertoire, which is an essential aspect of this balance, is mediated by multiple mechanisms operating both in the thymus (central tolerance) and in peripheral lymphoid and non-lymphoid organs (peripheral tolerance). Central tolerance, thus self-tolerance of the maturing T-cell repertoire in the thymus, is controlled by negative selection and the induction of regulatory T cells (Treg). These processes are mediated via TCR-MHC/peptide contacts between thymocytes and thymic antigen presenting cells (dendritic cells, thymic epithelial cells). The scope of central tolerance is to a large extent dictated by the expression of tissue-restricted antigens (TRA) by medullary thymic epithelial cells (mTEC), a process known as promiscuous gene expression (pGE). pGE encompasses the ectopic expression of TRA from virtually all tissues of the body in the mTEC population. While increasing insight into the tolerance modes linked to pGE has been gained in the last years, the molecular mechanisms involved in the regulation of pGE in mTEC remain largely obscure. The majority of TRA are expressed in the mature CD80high mTEC population (mTEChigh) whereby expression patterns of individual cells are highly heterogeneous. Only 1-15% of mTEChigh express a given antigen and co-expression patterns have been characterized as highly stochastic. A conspicuous feature of promiscuously expressed genes is their co-localization in chromosomal clusters suggesting a regulation of pGE at the epigenetic level. In order to gain insight into the regulation of such clustered gene expression, we exemplarily investigated the epigenetic regulation of pGE at the population and the single cell level of two genomic loci in mTEC: the casein gene locus and the Gad67 locus. For both loci, mechanisms of regulation were directly compared between mTEC subpopulations and the corresponding tissue. We focused on epigenetic regulation mechanisms such as histone tail modifications and certain aspects of nuclear structure, both were analyzed in the casein gene locus. In the Gad67 locus we analyzed allele specific and gene co-expression patterns in a Gad67/eGFP knock in mouse model in addition to epigenetic modifications. We found the expression of Casein beta (Csnb) to correlate both with chromatin decompaction and active histone modifications. Gad67 expression equally correlated with active histone modifications. However, the types of histone modifications differed between Gad67 and Csnb, which is an unusual gene in the casein locus as it is expressed at a particularly high frequency. In the Gad67 locus we additionally performed single cell expression analysis. We found significant discrepancies between protein and mRNA frequencies in the case of TRA. On the basis of these findings we propose a three-step model for the epigenetic opening of the Csnb gene: First, DNA demethylation takes place followed by chromatin decompaction and the introduction of active histone modifications. Furthermore, the discrepancies found between protein and mRNA expression frequency in the case of TRA let us assume that pGE fluctuates in individual cells. This concept potentially increases the diversity of antigen expression in the microenvironment in the thymus and thus may be crucial for the induction of T cell tolerance.