Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance. [Display omitted] •Enhancers activated at Foxp3-CNS0 and -CNS3 are indispensable for thymic Treg generation•CNS0 and CNS3 interact with other enhancers and Foxp3 promoter during Treg development•Deletion of both CNS0 and CNS3 causes lethal autoimmunity•CNS0 is responsible for IL-2-dependent induction and maintenance of Foxp3 expression. The Foxp3+ Treg generation process in the thymus has not been fully understood, especially in the perspective of genomic enhancer coordination. Kawakami et al. reveal that two independently activated enhancers, Foxp3-CNS0 and Foxp3-CNS3, cooperatively induce and maintain Foxp3 expression for the establishment of self-tolerance.