Molecular Mechanisms of Aire in Transcriptional Regulation

The transcription regulator Aire plays a pivotal role in immune tolerance by enforcing the expression of peripheral tissue antigens (PTAs) in medullary thymic epithelial cells (mTECs), selecting autoreactive T cells for clonal deletion or regulatory T cell differentiation. As such, defects in Aire result in manifestations of autoimmune polyglandular syndrome type 1 (APS-1), a form of primary immune deficiency diseases. The goal of this grant is to elucidate the molecular mechanism by which Aire engages with PTA loci and activates their transcription. Studies in mouse mTECs revealed that Aire preferentially binds to super-enhancers (to be referred as group- 1 targets), but predominantly induces genes located outside these regions (group-2 targets). However, Aire ChIP signals at group-2 loci are weak or undetectable. This raises fundamental questions about the relationship between group-1 and group-2 targets and how group-2 genes are activated by Aire. Our recent study suggest that group-1 and group-2 targets are mechanistically distinct. Group-1 loci are bound by Aire through direct interaction with pre-clustered p300/CBP and exhibit high level of basal transcription, which is modestly increased by Aire. These sites serve as a nucleation platform for Aire to form condensates, connecting multiple active loci into a transcription hub. In contrast, group-2 genes, including many PTA genes, are transcriptionally silent prior to Aire expression, and strongly induced by Aire with ordered stochasticity. Importantly, we now have multiple lines of evidence suggesting that these group-2 genes are not secondary to group-1 gene expression. Instead, Aire-p300/CBP condensates may directly contribute to group-2 gene activation. Based on our preliminary findings, we hypothesize that group-2 genes are also direct targets of Aire but are activated through transient interactions with Aire transcriptional condensates formed at p300/CBP-rich group-1 loci. We here propose two specific aims to test our hypothesis. First, we will determine the spatial relationship between Aire condensates and group-2 loci (Aim 1). Second, we will define the temporal relationship between group-1 and group-2 gene transcription (Aim 2). We expect that the proposed work will address key gaps in our understanding of how Aire regulates PTA gene expression. Furthermore, the proposed research will have a broad impact on the field of transcriptional regulation. Project Number: 1R21AI199018-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Qianxia Zhang | Institution: BOSTON CHILDREN'S HOSPITAL, BOSTON, MA | Award Amount: $267,000 | Activity Code: R21 | Study Section: Molecular and Structural Immunology Study Section[MSI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19901801