Promiscuous Gene Expression in Thymic Medullary Epithelial Cells : Scope, Phylogenetic Conservation and Regulation at the Single Cell Level

Abstract

In the thymus a specific subset of thymic stromal cells - medullary thymic epithelial cells (mTECs) - express a highly diverse set of tissue-restricted antigens (TRAs) representing essentially all tissues of the body, which is known as promiscuous gene expression (pGE). This allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing T-cells thus, rendering them tolerant to most self-antigens. The scope of central tolerance is to a large extent dictated by this pool of promiscuously expressed genes. Lack of a single TRA can result in spontaneous organ-specific autoimmunity. Therefore, it is important to define the scope of pGE and parameters/mechanisms that regulate this gene pool. Promiscuously expressed genes display two prominent features: they are highly clustered in the genome and show a preference for TRAs. To link these features we focused on studying genes which are up-regulated in mature mTECs. The analysis was performed in mouse, rat and human in order to assess evolutionary conservation of pGE. Our analysis proceeded from the bioinformatic definition of TRA clusters, gene clustering and homology mapping via gene expression analysis using whole genome arrays to the in depth analysis of selected TRA clusters by RT-PCR at the population level. The mTEC compartment represents a mosaic of clonally derived mTEC clusters undergoing continuous renewal, whereby the sets of genes expressed in single mTECs ultimately add up to a complete representation of the promiscuous gene pool at the population level. Hence, we wanted to elucidate what dictates pGE at the single cell level, i.e. whether it was random or subject to rules of co-expression. We observed that TRAs per se are clustered in the genome in all three species irrespective of structural relatedness or antigenic properties. Most of the clusters are localized in syntenic regions. In the thymus, the promiscuously expressed genes are enriched in TRAs that are partitioned into clusters, again conserved between species. These clusters harbor both TRAs and non-TRAs that are interspersed among each other. TRAs are preferentially regulated over non- TRAs during mTEC differentiation. Moreover, genes within a particular gene cluster are subject to partial co-regulation. Based on these data, we propose these clusters to be the “operational genomic unit” of pGE in the thymus. Single cell studies of a mTEC subpopulation expressing a particular antigen revealed a deterministic component in the regulation of pGE. Co-expression groups in single cells not only defined intra-chromosomal but also inter-chromosomal (e.g. chromosome 1 and 19) gene coregulation. Strikingly, these co-expression patterns correlated with in situ co-localization of the respective chromosomal domains upon mTEC maturation as analyzed by fluorescence in situ hybridization. Taken together, our data show that pGE is highly conserved between species, maps to gene clusters and is governed by certain co-expression rules at the single cell level.