IL-2 is a crucial cytokine with roles in both immunity and tolerance, and IL-21 mediates inflammation by promoting B cell and CTL differentiation. These cytokines are genetically linked, but are produced by CD4+ T cells in a lineage-specific manner. To better understand the transcriptional regulation of the il2 and il21 genes, we analyzed eleven conserved non-coding sequences (CNS) in the 100 kb of intergenic space between the two loci in the mouse genome. One of these CNS, located 80 kb upstream of the il2 gene, can enhance il2 promoter-driven transcription in transgenic promoter-reporter assays, while three distinct CNS act to enhance il21 promoter-driven transcription. We also define a functional insulator/boundary element located between these two differentially expressed genes. The -80 kb il2 enhancer exhibits an epigenetically silenced pattern of histone modification (H3k27me3) in non-IL-2-producing cells, while IL-2-producing cells show increased DNA accessibility, HAT/co-activator occupancy, and a poised histone modification pattern (H3k4m3) in this region. As expected of a bona fide distal enhancer, the -80 kb CNS was shown by 3-dimensional chromosome conformation (3C) analysis to loop back and interact with the il2 promoter in a manner associated with il2 transcription. These studies define a long-range, cis-regulatory architecture that helps to explain the complex and differential regulation of these cytokine loci during immunity vs. tolerance.

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