Mechanism RORγt gene expression control clarified by Kyushu University

A research group led by Professor Shinichiro Sawa of the Medical Institute of Bioregulation and JSPS Research Fellow Takuma Fukui of the Graduate School of Medical Sciences at Kyushu University, has clarified that during the differentiation process into type 3 innate lymphoid cells (ILC3), which are responsible for innate immunity, the RORγt gene is activated in a stepwise manner by different cis-regulatory regions at each stage of differentiation. This demonstrates the existence of a hierarchical gene regulatory mechanism that underlies innate lymphoid cell differentiation.

Type 3 immune responses is a collective term for immune responses that are involved not only in defense against bacterial and fungal infections, primarily through neutrophils, but also in the pathogenesis of autoimmune diseases. Appropriately controlling immune responses in opposing directions, enhancing infection defense through immune activation while preventing autoimmune disease through immune suppression, is an extremely important challenge in clinical medicine.

Innate immune lymphocytes called ILC3, as well as adaptive immune lymphocytes such as Th17 cells, function as the command center of type 3 immune responses. The transcription factor RORγt is known as a master regulator that plays an essential role in the differentiation of these lymphocytes. However, the regulatory mechanism by which the RORγt gene is expressed had not previously been clarified in detail.

The research group used ATAC-seq to analyze, in a time-course and cross-cell-type manner, how chromatin accessibility in the RORγt gene region changes during the differentiation process of each immune cell type in both the innate and adaptive immune systems. They further used bioinformatics analysis to identify the upstream transcription factor network that directly regulates RORγt gene expression.

The results indicated that the cis-regulatory regions used to control RORγt gene expression may differ between innate immune cells, including ILC3, their closely related fetal lymphoid tissue inducer (LTi) cells, and RORγt-positive antigen-presenting cells (APCs), and adaptive immune cells such as Th17 cells and regulatory T cells. The group further generated various mouse lines carrying mutations in candidate cis-regulatory regions and examined their function.

The results revealed that binding of the transcription factor complex RUNX/CBFβ to a cis-regulatory region called CNS11 in the RORγt gene locus in undifferentiated cells is essential for the differentiation of all RORγt-positive immune cells other than thymocytes and plays an important role in the rapid establishment of type 3 immune responses.

On the other hand, binding of transcription factors including RUNX/CBFβ to CNS9 was found to be essential not only for constitutive RORγt expression in ILC3 in which the direction of differentiation has been established, but also for the differentiation of RORγt-positive APCs.

It was confirmed in mice carrying mutations in CNS9 that differentiation of RORγt-positive regulatory T cells (Tregs) in the intestinal tract was impaired and that Oxazolone-induced colitis, a model of ulcerative colitis in which type 2 immune responses predominate, was exacerbated.

These results demonstrate that RORγt, the master regulator of type 3 immune responses, is induced through a hierarchical gene regulatory mechanism in innate immune cells and also suggest the possibility that it indirectly suppresses type 2 immune responses through the induction of regulatory T cell differentiation.

The present study demonstrates a new gene regulatory principle, a hierarchical cis-regulatory cascade of the RORγt gene and clarifies the possibility that it also indirectly controls type 2 immune responses.

This discovery brings new understanding to the molecular basis of innate lymphoid cell differentiation and may lead to new approaches targeting the control of mucosal immunity for the prevention of infectious diseases and for the treatment of inflammatory bowel disease and allergic diseases.

Sawa commented: "I was able to confront an essential question about ILC3, which I was involved in discovering 16 years ago, namely the mechanism controlling the expression of the master regulator RORγt, and I spent an enjoyable time doing this research. Catching a glimpse of the intricately controlled scheme by which RORγt expression is precisely regulated through different mechanisms in different cell types, I was once again moved by the profound depth of the immune system and developmental mechanisms."

Journal Information
Publication: Immunity
Title: A hierarchical Rorc(γt) cis-regulatory cascade orchestrates differentiation of RORγt⁺ innate immune cells
DOI: 10.1016/j.immuni.2026.02.002