Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3
To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors. An increase in luciferase activity was observed only when the FOXP3 promoter (-511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A). The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1. Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (-511 to +176) luciferase construct were mutated (Fig. 4 A). In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-beta for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient. 
To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination. No reduction in luciferase activity was observed when the -53 site was mutated and only a slight reduction when either the -287 or -333 site was mutated (Fig. 4 B). However, mutating the -53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter. Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h. This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-beta. Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5). 
