A distal cis-regulatory element, CNS-9, controls NFAT1 and IRF4-mediated IL-10 gene activation in T helper cells

IL-10 is a multifunctional cytokine that plays a critical role in maintaining the balance between immunity and tolerance. Previously, we identified proximal regulatory elements and alterations of chromatin structure in the IL-10 gene loci of Th1 and Th2 cells. We have now characterized a crucial cis-regulatory element, CNS-9, located 9 kb upstream of the transcription start site in IL-10 gene loci. The CNS-9 region is highly conserved in vertebrate genomes, and contains clustered NFAT and IRF binding motifs. In vitro binding of NFAT1 and IRF4 to the CNS-9 region was observed by EMSA. Furthermore, Th2-preferential in vivo binding of NFAT1 and IRF4 to the CNS-9 region was observed by ChIP. Cyclosporine A treatment on wild type Th2 cells or Th2 cells derived from NFAT1 knockout (NFAT1^-/-) mice showed significantly reduced trans-activity of CNS-9. The Th2 subset-specific enhancer activity of CNS-9 was upregulated synergistically by NFAT1 and its partner IRF4. Mutations in the binding sites for NFAT1 and IRF4 abrogated its enhancer activity of CNS-9. Collectively, our results establish crucial roles for enhancer element CNS-9, and NFAT1 and IRF4 that bind to it, for IL-10 expression in differential T helper subsets.