A novel, intergenic enhancer contributes to Il2 gene expression via chromosomal looping.

Abstract

Abstract IL-2 is a key immunoregulatory cytokine with pleotropic effects on many immune cell types. Its expression is highly regulated in T cells through multiple signaling pathways, chromatin remodeling, DNA methylation, and cooperative transcription factor binding. Recently, we identified an evolutionarily conserved intergenic enhancer 83 kb upstream of the Il2 gene that augments transcription from the Il2 promoter and upstream regulatory region (URR) by >50-fold in recombinant reporter assays. Upon TCR/CD28 co-stimulation of primary CD4+ T cells, a long-range chromosomal loop is established between the −83 kb CNS and the endogenous Il2 promoter, and both regions become epigenetically marked by histone acetylation and methylation. To determine the relative contribution of the URR vs. this new distal element to inducible transcription of the Il2 gene, we deleted the URR or the −83 kb CNS in mice using CRISPR/CAS genome editing. Deletion of the URR (~500 bp beyond the TATA element) completely abolished the Il2 transcription by CD4+ T cells, while a ~500 bp deletion of the −83 kb CNS resulted in a 2- to 4-fold decrease in IL-2 production. Young mice deficient for the −83 kb enhancer exhibited no major defects in lymphoid development, however, mutant CD4+ T cells showed reduced proliferative capacity in vitro. Furthermore, TGFB-induced iTreg differentiation from mutant conventional CD4+ precursors was strongly impaired, but could be fully restored by addition of exogenous IL-2. These results show that induction of Il2 depends upon cooperation between the promoter-URR and at least one intergenic enhancer, and suggest mechanisms by which graded expression of Il2 might be achieved through ‘modular’ recruitment of distinct enhancer elements.