Identification of an enhancer region for immune activation in the human GTP cyclohydrolase I gene

Abstract

GTP cyclohydrolase I (GCH) catalyzes the first and rate limiting step reaction for the de novo synthesis of 5,6,7,8-tetrahydrobiopterin (BH4). The expression of GCH is dramatically elevated by immune activation, while the mechanism remains to be elucidated. In this study, we investigated the transcription mechanism of the GCH gene using lipopolysaccharide (LPS) to stimulate mouse macrophage RAW264.7 cells. With luciferase assay, we found a highly conserved enhancer region spanning approximately 300 bp in intron 1 of GCH gene as a response element to LPS stimulation. The same enhancer region was also responsible for the induction of the GCH gene by IFN-γ and TNF-α in HUVECs. With electrophoresis mobility shift assay (EMSA) and site directed mutation analysis, we identified two key fragments containing C/EBP and Ets binding motifs within the enhancer. Furthermore, C/EBP-β was involved in LPS activated GCH transcription through direct binding to the enhancer shown by supershift, chromatin immunoprecipitation, and RNA interference experiments. In conclusion, our findings uncovered a novel mechanism of GCH transcriptional regulation by immune activation.

Keywords: BH4; C/EBP; CCAAT enhancer binding protein; Enhancer; GCH; GTP cyclohydrolase I; HUVEC; IFN; LPS; Lipopolysaccharide; NOS; TNF; Tetrahydrobiopterin; human umbilical vein endothelial cell; interferon; lipopolysaccharide; nitric oxide synthase; tetrahydrobiopterin; tumor necrosis factor.