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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.

Hereditary progressive dystonia with marked diurnal fluctuation (HPD; dopa-responsive dystonia, DRD) have been recently found to be caused by a genetic defect in the GTP cyclohydrolase I (GCH1) gene. In this study, we quantified the mRNA level of GCH1 in phytohemagglutinin (PHA)-stimulated mononuclear blood cells from one Japanese family that do not have a mutation in the coding region or splice junctions of the gene. The results showed that the amounts of the GCH1 mRNA were decreased to about 40% of the normal level in both patients and carriers. In addition, we found that the GCH1 mRNA was transcribed from only one allele, indicating that the other allele was in an inactive state. These results suggest that some novel mutations should exist on one of the alleles in some unknown region of the GCH1 gene, and may decrease the GCH1 mRNA causing the HPD/DRD symptoms.

Sepiapterin reductase (SPR) catalyzes the final step of the biosynthetic pathway of tetrahydrobiopterin, which is an essential cofactor for aromatic amino acid hydroxylases and nitric oxide synthases. To aid the analysis of any possible human diseases caused by mutations in SPR, we have cloned and characterized the human SPR gene. The gene is composed of three exons spanning approximately 4 kilobases. The transcriptional starting point was determined around the cytosine nucleotide at position −81 by primer extension and RT-PCR analyses. There was no typical TATA-box within 300 bp from the transcriptional starting point. We found the Sp1-binding consensus sequence in the 5′-flanking region. The human SPR gene was mapped to chromosome band 2p13 by fluorescence in situ hybridization.

Humans produce four different forms of tyrosine hydroxylase (TH) mRNA via alternative splicing of the gene. Here we demonstrate that New- and Old-World monkeys and the gorilla produce only two of the TH isoforms. Comparison among the genomic DNA sequences of various primates revealed that mutations that had accumulated in the genomic DNA created a new exon, resulting in the appearance of two new TH isoforms in man. These findings offer new insight into the sequence of events leading to the evolution of the higher primates into separate species. They also represent what may be the first evidence of a genetic difference between man and primates with respect to a specific brain function.