Project description:The aim of this study is to analyze the transcriptional effects of Aire deficiency in the thymus, using the Affymetrix MoGene platform to analyze variation in exon usage MECs were isolated from 4-6 wk-old WT or Aire KO ((B6xNOD)F1 background) mice. Three WT and three Aire-KO mice taken individually were used.
Project description:Background: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells (mTECs) that have a unique capacity to express, and subsequently present a large fraction of body antigens. While the scope of promiscuously expressed genes by mTECs is well established, relatively little is known about the expression of variants that are generated by co- and post-transcriptional processes. Results: Our study reveals that in comparison to other cell types, mTECs display significantly higher levels of alternative splicing, as well as A-to-I and C-to-U RNA editing, which thereby further expand the diversity of their self-antigen repertoire. Interestingly, Aire, the key mediator of mTECs promiscuous gene expression, plays a limited role in the regulation of these transcriptional processes. Conclusions: Our results highlight RNA processing as another layer by which the immune system assures a comprehensive self-representation in the thymus which is required for the establishment of self-tolerance and prevention of autoimmunity. Identification of the number of genes expressed in Aire-KO MEChi
Project description:The aim of this study is to analyze the transcriptional effects of Aire deficiency in the thymus, using the Affymetrix MoGene platform to analyze variation in exon usage
Project description:The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire?s primary targets while distinguishing them from the secondary targets.
Project description:Promiscuous gene expression (pGE) of numerous self-antigens in thymic epithelial cells (TEC) enables the elimination of self-reactive T cells. The autoimmune regulator (Aire) is the only known molecular determinant driving pGE in the thymus but the existence of Aire-independent mechanisms has been inferred. Here, we analyzed the poly(A)+ transcriptome of TEC populations by RNA-sequencing (RNA-seq) in order to reveal differential features of Aire-induced vs. –independent pGE. We report an unanticipated effect of Aire deletion on the proliferation and differentiation of cortical TEC. Moreover, the RNA-seq data reveal the breath of Aire-induced and –independent pGE in medullary TEC (mTEC) subsets and the extent of thymic peripheral tissue representation. The results suggest that Aire-induced promiscuously expressed transcripts affect several functions with far reaching biological consequences in mTEC. High-throughput characterization of TEC transcriptomes will enable progress in understanding TEC biology and the establishment of self-tolerance. The mRNA profiles of cTEC, mTEClo and mTEChi from 6-8 week-old wild type (WT) and Aire-/- (KO) mice were generated by RNA-sequencing using Illumina HiSeq2000.
Project description:Promiscuous gene expression (pGE) of numerous self-antigens in thymic epithelial cells (TEC) enables the elimination of self-reactive T cells. The autoimmune regulator (Aire) is the only known molecular determinant driving pGE in the thymus but the existence of Aire-independent mechanisms has been inferred. Here, we analyzed the poly(A)+ transcriptome of TEC populations by RNA-sequencing (RNA-seq) in order to reveal differential features of Aire-induced vs. –independent pGE. We report an unanticipated effect of Aire deletion on the proliferation and differentiation of cortical TEC. Moreover, the RNA-seq data reveal the breath of Aire-induced and –independent pGE in medullary TEC (mTEC) subsets and the extent of thymic peripheral tissue representation. The results suggest that Aire-induced promiscuously expressed transcripts affect several functions with far reaching biological consequences in mTEC. High-throughput characterization of TEC transcriptomes will enable progress in understanding TEC biology and the establishment of self-tolerance.
Project description:Aire in medullary thymic epithelial cells plays an essential role in the negative selection through expression of broad arrays of tissue-restricted antigens. We asked whether Aire could also activate the expression of tissue-restricted antigens in cortical thymic epithelial cells. We established a semi-knockin strain of NOD-background mice expressing Aire under control of the promoter of β5t, a thymoproteasome expressed exclusively in the cortex. We extracted RNA from cortical thymic epithelial cells ectopically expressing Aire and hybridization was performed on Affymetrix. microarrays.