Fibroblast growth factor 15 (Fgf15) is expressed in the medial region of diencephalon and midbrain by the seven-somite stage. In the previous studies, we showed that Sonic hedgehog signaling through Gli protein is required for Fgf15 expression in this region. The Fgf15 expression domain overlapped with that of Gli2 and the Gli-binding site (GliBs) is located in the 3.6-kb 5'-flanking enhancer/promoter region of the Fgf15 gene. In this study, we identified the two additional Gli-binding sites in row, called Gli-responsive elements (GliREs). Chromatin immunoprecipitation assay indicated that Gli2 directly binds to GliREs. The results from luciferase assays indicated that the Gli2 activator form binds to the GliBS and that the Gli2 repressor form binds to the GliREs. These findings suggest that the repressor form of Gli2 preferentially binds to the GliREs to control Fgf15 expression. (c) 2008 Elsevier Inc. All rights reserved.