RNF4 Is a Coactivator for Nuclear Factor Y on GTP Cyclohydrolase I Proximal Promoter

GTP cyclohydrolase I (GCH) is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH4) that is essential for the synthesis of nitric oxide and catecholamines including dopamine and serotonin. Therefore, the regulation of GCH expression is important in determining the catecholamine levels in the brain under pathophysiological conditions. During the study of human disease dopa-responsive dystonia, we found that coactivator RNF4 is involved in the GCH gene expression. Through serial deletion and mutagenesis studies of the GCH promoter, we defined the RNF4-responsive element on GCH proximal promoter as a CCAAT box. RNF4 did not possess specific DNA binding activity toward this CCAAT box, which suggests that RNF4 may be a coactivator of the CCAAT box-binding protein nuclear factor Y (NF-Y). Cotransfection of a dominant-negative mutant of NF-Y resulted in a significant reduction in RNF4-mediated CCAAT box activation. In addition, overexpression of RNF4 could not activate the CCAAT box in Drosophila melanogaster SL2 cells, which are devoid of endogenous NF-Y, whereas overexpression of RNF4 and NF-Y could. Furthermore, immunoprecipitation experiments revealed the physical association between RNF4 and the NF-Y complex. These data indicate that RNF4 imposes functional importance on GCH promoter.