A transcriptomics-based investigation of the landscape and mechanisms of self-antigen splicing in thymic epithelial cells

Thesis

Thymic epithelial cells (TEC) are remarkable for their ability to promiscuously express virtually the entire protein-coding gene repertoire to generate a molecular library of self-antigens for T cell education and selection. This process is essential for the establishment of central T cell tolerance. While the functional relevance of alternative splicing in TEC has been demonstrated for specific genes, the extent and fidelity of peripheral isoform representations of tissue-restricted genes in TEC is only poorly understood. We therefore subjected TEC to deep, stranded, paired-end sequencing and compared their transcriptome to that of peripheral tissues, using matching data from the mouse ENCODE project. In line with previous reports of unusually high splicing entropy in mTEC, I first demonstrated that these cells utilize a broader array of splice junctions than was observed for any of the peripheral tissues examined. Medullary mTEC further transcribed and spliced the highest fraction of known protein-coding gene isoforms. At the same time mTEC do not splice unusually high numbers of novel transcripts identified in a transcriptome assembly of peripheral mouse tissues and mTEC. Using long-read sequencing data from the Oxford Nanopore Technology I confirmed 97 % of highly-expressed novel transcripts expressed in mature mTEC. In this study, I found a limited role for Aire in regulating alternative splicing, but observed a role in modulating transcript usage. Investigating tissue-restricted splicing factor expression in mTEC revealed that a subset of peripheral tissue-restricted splicing factors was present in TEC, which might be regulating the unusual splicing complexity in these cells. However, a set of neuronal splicing factors was not detected in mTEC, which was in line with the absence of a set of neuronal microexons in mTEC.

Authors: Jansen, K

• DOI: 10.5287/ora-y05vazn4r
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford