Mechanism for controlled assembly of transcriptional condensates by Aire [ChIP-seq]

Transcriptional condensates play a crucial role in gene expression and regulation, yet their assembly mechanisms remain poorly understood. We here report a multi-layered mechanism for condensate assembly by Autoimmune regulator (Aire), an essential transcriptional regulator (TR) that orchestrates gene expression reprogramming for central T-cell tolerance. Aire condensates assemble on enhancers, stimulating local transcriptional activities and connecting disparate inter-chromosomal loci. This functional condensate formation hinges upon the coordination between three Aire domains: polymerization domain CARD, histone binding domain PHD1 and C-terminal tail (CTT). Specifically, CTT binds coactivators CBP/p300, recruiting Aire to CBP/p300-rich enhancers and promoting CARD-mediated condensate assembly. Conversely, PHD1 binds to the ubiquitous histone mark H3K4me0, keeping Aire dispersed throughout the genome until Aire nucleates on enhancers. Our findings showed that the balance between PHD1-mediated suppression and CTT-mediated stimulation of Aire polymerization is crucial to form transcriptionally active condensates at target sites, providing new insights into controlled polymerization of TRs. Overall design: To investigate the impact of AIRE.CTT–CBP/p300 interaction on AIRE's target site selection, we compared ChIP-seq of WT AIRE to deltaCTT.R3 mutan, which is deficient in CBP/p300 binding. To investigate the exent to which a polymerization defect contributes to AIRE targeting, we compared ChIP-seq of WT AIRE to deltaCARD mutant, which is deficient in polymerization. We also examine what histone marks including H3K27ac, H3K4me1, H3K27me3 are associated with AIRE-enriched sites, and whether p300 enrichment is correlated with AIRE enrichment in the genome. Next, we asked whether p300 inhibitors would affect AIRE targeting.