A hierarchical Rorc(γt) cis-regulatory cascade orchestrates differentiation of RORγt⁺ innate immune cells [ChIL-seq]

Beyond its well-established role in type 3 immunity, RORγt+ innate immune cells are also essential for secondary lymphoid organ (SLO) formation and gut homeostasis. However, the transcriptional regulatory mechanisms governing RORγt expression in these cells, including ILC3s, LTi cells, and antigen-presenting cells (APCs), remain largely unknown. Here, we identify two key cis-regulatory elements within conserved non-coding sequences (CNS) 9 and 11 in the Rorc locus, which are sequentially utilized during their differentiation. Initially, Runx-binding sites in CNS11 establish chromatin accessibility as early as the HSC stage. Notably, disruption of this chromatin priming prevents subsequent transcriptional activation, thereby abolishing the initial induction of RORγt in these cells. At later stages, CNS9 plays a critical role, particularly in the development of RORγt⁺ APCs, contributing to colonic pTreg induction. This hierarchical transcriptional regulation is essential for SLO formation, postnatal type 3 immunity, and restraining excessive intestinal type 2 immune responses through pTreg induction.