In our experiments involving HeLa cells, a cell line which was previously used to study the mechanism of IFN-c action on IDO1 promoter activity , luciferase reporter assays showed that the 1.6-kb fragment upstream of the translation start site is a functional promoter of IDO1, and that both IFN-c and TNF-a induce promoter activity, a greater activation being obtained with IFN-c and a synergy being observed with a combination of the two cytokines. However, no significant difference in promoter activity was seen between *V1 and *V2 alleles under any experimental condition. Based on these results, the absence of any difference in promoter activity between the two alleles, even after synergistic induction, may not have been unexpected, since the VNTR polymorphism does not BYL719 supplier modify any critical BEZ235 customer reviews response element, such as ISRE or GAS. A bioinformatic analysis was undertaken to predict potential cisacting elements in the promoter region of IDO1 in order to investigate whether the VNTR polymorphism could affect interactions between putative binding sites and transcription factors. In addition to the response elements for IFN-c and TNF-a previously described, the in silico analysis predicted that the LEF-1/TCF transcription factor could interact with the promoter region of IDO1 at three different sites, with one putative consensus sequence being located in the 24-bp motif of the VNTR. As TFBS prediction programs cannot assert the existence of a TFBS or infer the functionality of a site , additional experiments were undertaken. Initially, the presence of the three potential binding sites for LEF-1 in the IDO1 promoter was confirmed by specific PCR amplifications using chromatin immunoprecipitation of HeLa cellular extracts. To investigate the functionality of the potential sites, additional gene reporter assays were performed using an overexpression of a functional LEF-1. As a member of the TCF/LEF family of transcription factors, LEF-1 is one of the major nuclear transducers involved in Wnt signaling. However, it has no transcriptional activation potential in isolation and requires binding with b-catenin, the b-catenin activation domain and TCF/LEF DNA-binding domain forming a bipartite transcriptional activator of target genes .