RBFOX splicing factors contribute to a broad but selective recapitulation of peripheral tissue splicing patterns in the thymus.

Thymic epithelial cells (TEC) control the selection of a T cell repertoire reactive to pathogens but tolerant of self. This process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire, but the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, remain largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific microexons in mTEC. We did not find unusual numbers of novel transcripts in TEC, and we show that Aire, the facilitator of promiscuous gene expression, promotes the generation of long "classical" transcripts (with 5' and 3' UTRs) but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of nonpromiscuously expressed SF genes, among which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss-of-function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the RBFOX family, to generate a broad but selective representation of the peripheral splice isoform repertoire.

Comprehensively Profiling the Chromatin Architecture of Tissue Restricted Antigen Expression in Thymic Epithelial Cells Over Development.

Thymic epithelial cells (TEC) effect crucial roles in thymopoiesis including the control of negative thymocyte selection. This process depends on their capacity to express promiscuously genes encoding tissue-restricted antigens. This competence is accomplished in medullary TEC (mTEC) in part by the presence of the transcriptional facilitator AutoImmune REgulator, AIRE. AIRE-regulated gene transcription is marked by repressive chromatin modifications, including H3K27me3. When during TEC development these chromatin marks are established, however, remains unclear. Here we use a comprehensive ChIP-seq dataset of multiple chromatin modifications in different TEC subtypes to demonstrate that the chromatin landscape is established early in TEC differentiation. Much of the chromatin architecture found in mature mTEC was found to be present already over earlier stages of mTEC lineage differentiation as well as in non-TEC tissues. This was reflected by the fact that a machine learning approach accurately classified genes as AIRE-induced or AIRE-independent both in immature and mature mTEC. Moreover, analysis of TEC specific enhancer elements identified candidate transcription factors likely to be important in mTEC development and function. Our findings indicate that the mature mTEC chromatin landscape is laid down early in mTEC differentiation, and that AIRE is not required for large-scale re-patterning of chromatin in mTEC.

Dynamic spatio-temporal contribution of single ß5t+ cortical epithelial precursors to the thymus medulla.

Intrathymic T-cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit ß5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that ß5t(+) TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage-tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual ß5t(+) cortical progenitors located at the cortico-medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio-temporal dynamics that control the growth of the thymic medulla.

Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.