The thymus plays a central role in the induction of self-tolerance, a hallmark of the immune system. During T cell development a highly diverse T cell receptor repertoire is probed against a matching array of self-antigens. While the generation of the T cell receptor repertoire relies on unique genetic recombination mechanisms, the corresponding molecular principles underlying the generation of TCR ligands - self-peptide/MHC complexes - are less well understood; recent data indicate that novel principles of epigenetic control are employed. This proposal aims at understanding a unique feature of thymic epithelial cells (TECs), namely the expression of a multitude of tissue-restricted antigens (TRAs), a phenomenon termed promiscuous gene expression (pGE) in the context of self-tolerance. The integrative approach of this proposal has 4 main objectives (i) a characterization of the molecular control of different pools of pGE beyond the role of the Autoimmune Regulator (Aire) in relation to the developmental biology of TECs (ii) a comprehensive and comparative analysis of expression patterns of TRAs in single cells of different species in relation to epigenetic signatures and higher chromatin order, (iii) translating these findings to the cellular dynamics and topology of the thymic medullary compartment, e.g. individual mTEC clones in situ (iv) the modelling of these different levels of analysis by a system biology approach, e.g. testing our hypothesis that clusters of TRAs in the genome represent an “operational unit” of pGE. We expect from these studies new basic insights into a fascinating and still arcane aspect of the vertebrate immune system, which will also contribute to our understanding of the etiology and pathophysiology of human autoimmune diseases.