Mechanism for controlled assembly of transcriptional condensates by Aire [CRISPR]

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. To understand the molecular mechanisms by which AIRE C-terminal tail (AIRE.CTT) contributes to the TAD-like activity, we investigated genetic interaction partners of AIRE.CTT. A genome-wide CRISPR screen was performed using a modified reporter system, wherein the screening reporter mKate2 was induced upon expression of Gal4DBD-CTT fused with the expression reporter GFP via a self-cleavable peptide 2A.