Cellular and molecular regulation of pGE

Scientists

Chloé Michel, Sheena Pinto, and Kristin Rattay

Project

Figure 3
© dkfz.de

TECs fall into two major subpopulations: cortical (cTECs) and medullary (mTECs). Each population can be further subdivided into phenotypically distinct subsets: immature and mature mTECS. The analysis of pGE in these various subsets by gene arrays and RT-PCR led to the delineation of 4 distinct gene pools. Pool 1 includes genes expressed at comparable levels in cTECs and mTECs; pool 2 includes genes confined to the mTEC lineage irrespective of maturation stage; pool 3 includes genes expressed in mTECs with a mature (MHCIIhi, CD80hi) phenotype. Genes in this latter pool can be further subdivided based on whether their expression is dependent or not on the transcriptional regulator Autoimmune Regulator (Aire) (1). This led us to propose the “terminal differentiation model” (Fig. 3). This model holds that two steps are required to unfold the full extent of pGE, first commitment into the mTEC lineage and second the development of a mature mTEC phenotype. As a consequence, the mTEC subset that is most competent in antigen presentation also shows the highest degree of pGE. This model implies that pGE is superimposed on the developmental program of mTECs. The sequential appearance of mTEC subsets during ontogeny, their inter-conversion in vitro and the co-expression pattern of TRAs in single mTECs support this model (2,3).

The molecular regulation of pGE is presently poorly understood. One molecular determinant - Aire - has been identified to date. The Aire gene, which is invariably mutated in the rare Autoimmune Polyglandular Syndrome-1 (APS-1), has been shown to control the expression of a large set of promiscuously expressed genes in mice. Aire targets and turns on silent gene-loci by binding to hypo-methylated H3K4, which marks promoters in closed chromatin regions. It apparently acts as a docking platform for different, multi-protein complexes known to facilitate transcription by generating local double-strand breaks, relieving stalled RNA polymerase II and promoting mRNA processing. Regulation of pGE is, however more complex, as a multitude of TRAs is still expressed in mTECs in the absence of Aire (1).

Further indirect clues as to the molecular regulation of pGE could be deduced from the in depth analysis of certain gene loci in mice and men (4,5). First, these loci were co-ordinately up regulated in mature mTECs implying that such domains become accessible to transcription upon mTEC differentiation. Second, individual and directly neighbored genes were differentially regulated by Aire (4). These results suggest that the ability of Aire to control transcription may be contingent on epigenetic alterations specific to mTECs. A role for epigenetic control is further implied by the hypo-methylation of CpG sites and histone acetylation of promoters of promiscuously expressed genes (6).

While the entire mTEC population covers a broad, if not almost complete representation of “peripheral” transcripts, each TRA is only expressed by a minor fraction (1–3%) of mTECs at any given time. How this mosaic expression pattern ultimately translates into faithful presentation of thousands of self-antigens in a way that efficient tolerance is ensured remains puzzling. Understanding how this mosaic pattern of pGE is generated and how it is maintained in time and space at the single cell level will be key to eventually comprehend how central tolerance can be so efficient, sensitive and stringent.

In this regard we recently showed that antigen diversity is generated by distinct and fluctuating patterns of gene co-expression in subsets of human mTECs, implying that mTECs transiently display TRAs in certain linkage groups. Such co-expression groups ranged between 100 and 300 TRAs preferentially mapping to particular chromosomes, whereby co-expressed gene loci co-localized within nuclear subdomains, pointing to a level of regulation at the higher genome order (5). These data imply that individual mTECs during their life-time scan a sizeable portion of the genome. The sequential expression and presentation of different sets of genes at the single-cell level would substantially reduce the number of mTECs required to represent the “full” antigen repertoire. Thus, a diverse antigen repertoire might be already displayed in micro-domains of the medulla. It would  suffice for nascent T cells to scan such domains to encounter essentially the full antigen repertoire.

Selected publications

1. Derbinski, J., J. Gabler, B. Brors, S. Tierling, S. Jonnakuty, M. Hergenhahn, L. Peltonen, J. Walter and B. Kyewski. 2005. Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels. J. Exp. Med. 202, 33-45.

2. Gäbler, J., J. Arnold, and B. Kyewski. 2007. Promiscuous gene expression and the developmental dynamics of medullary thymic epithelial cells. Eur. J. Immunol. 37, 3363-3372.

3. Pinto, S., K. Schmidt, S. Egle, H.-J. Stark, P. Boukamp, and B. Kyewski. 2013. An Organotypic Coculture Model Supporting Proliferation and Differentiation of Medullary Thymic Epithelial Cells and Promiscuous Gene Expression. J. Immunol.190: 1085-1093.

4. Derbinski, J., S. Pinto, S. Rösch, K. Hexel, and B. Kyewski. 2008. Promiscuous expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism. Proc. Natl. Acad. Sci. USA 105, 657-662.

5. Pinto, S., C. Michel, H. Schmidt-Glenewinkel, N. Harder, K. Rohr, S. Wild, B. Brors, and B. Kyewski. 2013. Overlapping gene coexpression patterns in human medullary thymic epithelial cells generate self-antigen diversity. Proc. Natl. Acad. Sci. 110: E3497-E3505.

6. Tykocinski, L.O., A. Sinemus, E. Rezavandy, Y. Weiland, D. Baddeley, C. Cremer, S. Sonntag, K. Willecke, J. Derbinski, and B. Kyewski. 2010. Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells. Proc. Natl. Acad. Sci. U S A 107(45):19426-31.

7.  Brennecke, P. A. Reyes, S. Pinto, K. Rattay, M. Nguyen, R. Küchler, W. Huber, B. Kyewski, and L.M. Steinmetz. 2015. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells. Nat. Immunol. 16:933-941.

8. Rattay, K., H.V. Meyer, C. Herrmann, B. Brors, and B. Kyewski. Evolutionary conserved gene co-expression drives generation of self-antigen diversity in medullary thymic epithelial cells.
J. Autoimmun. doi:10.1016/j.jaut.2015.10.001.

 


 

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