In an initial series of studies we generated a series of reporter constructs containing regions of the IL-17 gene upstream of this transcription start site and then assessed the activity of these constructs following their transfection into Jurkat cells and stimulation with PMA and ionomycin. We found that whereas the lineage-specific IL-17 transcription factor, RORgamma-t. is present in a 1.1kb promoter fragment and this factor is necessary for IL-17 transcription, optimal IL-17 transcription requires the presence of a 2kb promoter fragment containing up-stream sequence that does not bind RORgamma-t. In further studies we identified Runx (particularly Runx1) as another IL-17 transcription factor that binds to the IL-17 promoter at an up-stream site. However, whereas this transcription factor was critically necessary for optimal IL-17 transcription it is totally dependent on RORgamma-t binding and activity. In yet other studies we identified at least one distal (up-stream) conserved non-coding sequence (CNS sequence) that contains binding sites for both RORgamma-t and Runx and which is necessary for IL-17 transcription.? ? To substantiate the above findings in CD4+ T cells undergoing Th17 differentiation we conducted extensive studies in which we evaluated the effect of Runx1 (or Runx2) down-regulation of IL-17 expression. These studies involved the transfection of cells with Runx-specific siRNA or the transduction of cells with a retrovirus expressing either a short-hairpin siRNA or a dominant-negative Runx construct. The results of these studies were congruent in that they showed that down-regulation of Runx led to greatly decrease IL-17 production. However, as presaged by the reporter studies discussed above, down-regulation of Runx1 in cells cultured under conditions leading to the absence of RORgammat or only small amounts of RORgammat, had no or little effect on IL-17 expression. In related studies we showed using ChIP assays that both RORgamma-t and Runx1 bind to the IL-17 promoter and the CNS enhancer of T cells undergoing Th17 differentiation.? ? Recently it has been shown that Foxp3, the lineage-specific factor of regulatory T cells interacts with RORgamma-t and thereby down-regulates the latters capacity to induce IL-17 expression. To explore the regulatory activity of Foxp3 in relation to the role of Runx in IL-17 transcription we performed co-transfection studies of CD4+ T cells of Runx and Foxp3 mutants. We found that whereas wild type Foxp3 suppressed RORgamma-t-induced IL-17 expression, a Foxp3 mutant lacking the ability to bind to Runx1 had no effect. These data revealed the critical fact that Foxp3 inhibition of Th17 depends both on its ability to bind to Runx and to RORgamma-t. In a final series of studies related to such binding we performed co-immunoprecipitation studies in which we showed first that Runx1 physically interacts with RORgammat and second that Foxp3 intereacts with RORgt. These data, coupled with previous data that Foxp3 physically interacts with Runx1 lead to the conclusion that a complex, three-way interaction between Runx1, Foxp3 and RORgamma-t determines the differentiation of CD4 cells into Th17 cells, or alternatively into regulatory T cells. In addition, they strongly imply that regulation of Runx1 is another way that IL-17 transcription is regulated.
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