Aire-dependent transcripts escape Raver2-induced splice-event inclusion in the thymic epithelium.

Area of Science:

• Immunology
• Molecular Biology
• Epigenetics

Background:

• Medullary thymic epithelial cells (mTECs) are crucial for central tolerance by presenting self-antigens.
• Aire facilitates the expression of numerous self-antigens in mTECs.
• The role of alternative splicing events (ASEs) in Aire-induced transcripts within mTECs is largely uncharacterized.

Purpose of the Study:

• To investigate the extent and regulation of alternative splicing events (ASEs) in Aire-sensitive and Aire-neutral genes in mTECs.
• To identify factors and epigenetic mechanisms that control ASEs in mTECs, particularly in response to Aire.
• To understand the implications of ASEs in Aire-induced transcripts for T cell tolerance.

Main Methods:

• Comparative analysis of alternative splicing events in Aire-sensitive and Aire-neutral genes in mTECs.
• Identification and characterization of splicing-related factors, such as Raver2, in mTECs.
• Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36 trimethylation (H3K36me3) profiling.
• Analysis of gene expression and splicing patterns in relation to epigenetic marks and splicing factors.

Main Results:

• Aire-sensitive genes display significantly fewer ASEs in mTECs compared to Aire-neutral genes.
• Raver2, a splicing factor overexpressed in mTECs and dependent on H3K36me3, promotes ASEs in Aire-neutral genes but not Aire-sensitive genes.
• H3K36me3 is depleted at Aire-sensitive genes, suggesting an epigenetic mechanism that precedes Aire expression and limits ASEs in these transcripts.
• Approximately 25% of observed ASEs in Aire-sensitive genes are also found in peripheral tissues.

Conclusions:

• Aire-induced transcripts in mTECs are characterized by a low frequency of alternative splicing events.
• Epigenetic modifications, specifically H3K36me3 depletion, at Aire-sensitive genes limit their alternative splicing, potentially mediated by the splicing factor Raver2.
• The reduced diversity of Aire-induced self-antigen isoforms due to limited ASEs may lead to incomplete negative selection of autoreactive T cells.
• Complementary peripheral tolerance mechanisms are essential to prevent the activation of autoreactive T cells.