A hierarchical Rorc(?t) cis-regulatory cascade orchestrates differentiation of ROR?t? innate immune cells [ChIL-seq]
Beyond its well-established role in type 3 immunity, ROR?t+ innate immune cells are also essential for secondary lymphoid organ (SLO) formation and gut homeostasis. However, the transcriptional regulatory mechanisms governing ROR?t 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 ROR?t in these cells. At later stages, CNS9 plays a critical role, particularly in the development of ROR?t? 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. Overall design: ChIL-seq targeting CBFß was performed in MNK3 cells cultured with IL-7, IL-2, and IL-15 in the presence of ATRA. A total of 5,000 cells were used per sample for the analysis.