Radioisotope Research Center, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan;
Division of Theoretical Nursing and Genetics, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan.
J Immunol. 2019 Jun 1;202(11):3198-3210. doi: 10.4049/jimmunol.1800672. Epub 2019 Apr 26.
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 gene expression are still unclear. In this study, we have identified a crucial -regulatory element located 32 kb upstream of the mouse 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 γt (RORγt). Mutation of these motifs significantly abrogated the reporter activity, suggesting a role for both factors in the control of enhancer-mediated expression. Runx1 and RORγt 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 RORγt 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 RORγt. Conversely, short hairpin RNA-mediated knockdown of core-binding factor β, 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 RORγt and then partners with RORγt to direct expression through their targeting of the enhancer.
白细胞介素-22（IL-22）是一种细胞因子，在调节屏障表面组织稳态方面发挥着关键作用，由活化的 CD4+Th 细胞产生。目前，调节基因表达的分子机制尚不清楚。在本研究中，我们鉴定了一个位于小鼠启动子上游 32kb 的关键调控元件，称为保守非编码序列（CNS）-32。我们证明 CNS-32 在报告基因实验中作为增强子发挥作用，并且包含 Runt 相关转录因子（Runx）1 和维甲酸相关孤儿受体γt（RORγt）的结合基序。这些基序的突变显著削弱了报告基因的活性，提示这两个因子在控制增强子介导的表达中起作用。在初始 T 细胞分化为产生白细胞介素-22 的 Th22 细胞时，CNS-32 上可见 Runx1 和 RORγt 的结合以及组蛋白 H4 的乙酰化升高。Runx1 和 RORγt 的结合以及 CNS-32 处组蛋白 H4 的乙酰化升高，在初始 T 细胞分化为产生白细胞介素-22 的 Th22 细胞时很明显。Runx1 的过表达通过诱导 RORγt 和白细胞介素-23 受体促进 IL-22 的产生，这些都是诱导 Th22 细胞产生所必需的。虽然 Runx1 单独在 Th22 细胞中增强 IL-22 的产生，但在存在 RORγt 的情况下会进一步增强。相反，核心结合因子β（Runx1 活性所必需的辅助因子）的短发夹 RNA 介导的敲低可有效限制 IL-22 的产生。综上所述，我们的研究结果表明，IL-22 的产生受一个调控回路的控制，其中 Runx1 诱导 RORγt，然后与 RORγt 合作，通过靶向 增强子来指导的表达。
