Unique distance- and DNA-turn-dependent interactions in the human protein C gene promoter confer submaximal transcriptional activity

Recent studies on the regulation of protein C gene transcription revealed t he presence of three transcription-factor binding sites in the close proxim ity to the transcription start site. The proximal 40 bp upstream of the tra nscription-initiation site contain two, partly overlapping, binding sites f or the liver-enriched hepatocyte nuclear factor (HNF)-3 and one binding sit e to which HNF-1 and HNF-6 bind in a mutually exclusive manner. In order to examine the functionality of the tight alignment of transcription-factor b inding sites around the transcription-initiation site, we performed inserti onal mutagenesis experiments. Sequences were inserted at position -21, sepa rating both HNF-3 binding sites from the HNF-1-HNF-6 binding site, and posi tion -5, separating the HNF-3-HNF-1-HNF-6 complex from the transcription st art site. All insertions were made in the context of the protein C gene -38 6/+107 promoter region and tested for activity by transient transfection ex periments. Insertions at position -21 resulted in a combined distance- and DNA-turn-dependent increase in protein C gene expression. Insertions of var iable length at position -5 decreased protein C gene expression in a DNA-tu rn-dependent manner. However, this turn-dependent decrease was accompanied by a distance-dependent increase in promoter activity. This is the first re port in which changing the spacing between adjacent transcription-factor bi nding sites results in enhanced transcription, indicating the submaximal al ignment of promoter elements in the wild-type protein C gene promoter regio n.