Runx1 and RORγt Cooperate to Upregulate IL-22 Expression in Th Cells through Its Distal Enhancer

IL-22 is a cytokine that plays a pivotal role in regulating tissue homeostasis at barrier surfaces and is produced by activated CD4(+) Th cells. Currently, the molecular mechanisms regulating Il22 gene expression are still unclear. In this study, we have identified a crucial cis-regulatory element located 32 kb upstream of the mouse Il22 promoter, termed conserved noncoding sequence (CNS)-32. We demonstrated that CNS-32 acts as an enhancer in reporter assays and contains binding motifs for Runt-related transcription factor (Runx)1 and retinoic acid-related orphan receptor gamma t (ROR gamma t). Mutation of these motifs significantly abrogated the reporter activity, suggesting a role for both factors in the control of enhancer-mediated Il22 expression. Runx1 and ROR gamma t occupancy and elevated histone H4 acetylation at CNS-32 were evident, as naive T cells differentiated into IL-22-producing Th22 cells. Overexpression of Runx1 promoted IL-22 production by inducing ROR gamma t and IL-23 receptor, all critical to Th22 cell induction. Although Runx1 alone enhanced IL-22 production in Th22 cells, it was further enhanced in the presence of ROR gamma t. Conversely, short hairpin RNA-mediated knockdown of core-binding factor beta, a cofactor essential for Runx1 activity, was effective in limiting IL-22 production. Collectively, our results suggest that IL-22 production is controlled by a regulatory circuit in which Runx1 induces ROR gamma t and then partners with ROR gamma t to direct Il22 expression through their targeting of the Il22 enhancer.