Functional dissection of the Il2ra superenhancer reveals developmentally regulated and cell type specific elements

Abstract

The expression of the IL-2 receptor a chain is tightly regulated during early T cell development and in mature lymphoid populations by either cytokine stimulation or T cell receptor interactions. IL-2Ra expression can be either constitutive, as in some double negative thymocytes and T regulatory cells, or inducible, as in CD4⁺ and CD8⁺ mature T cells. Previously, our lab identified a super-enhancer in the mouse Il2ra locus that extensively bound STAT5 and controlled expression of this gene in mature T cells. To further dissect developmental and cell type specific expression of this super-enhancer, Crispr-Cas9 mediated deletion was used to generate mutant mice lacking either individual or groups of STAT5 binding sites in either the upstream region or the first intron. Deletion of the upstream region led to the absence of CD25 expression in DN2 and DN3 thymocytes as well as a significant reduction of CD25 expression on regulatory T cells, but minimal effect on inducible CD25 expression in mature T cells. Analysis of individual upstream elements in reporter assays revealed a specific element that controlled high activity in DN3 cells and a Treg cell line, with markedly diminished activity in the presence of Notch inhibitors. In contrast, deletion of an intronic region markedly diminished both IL-2 and TCR-induced expression of CD25 in vitro, while having little if any effect on CD25 expression in DN thymocytes or Treg cells. These data reveal that the Il2rasuper-enhancer is composed of multiple elements that preferentially mediate either constitutive or inducible Il2ragene expression. Further analysis of these elements will define mechanisms for regulation and potentially manipulation of CD25 expression in autoimmune and other disease states.