Identification of an intronic regulatory element in the human protein C (PROC) gene

Regulatory DNA elements responsible for human protein C (PROC) gene express ion have previously been identified in the upstream promoter region and fir st (untranslated) exon of the gene. Here we show that an additional sequenc e element located more than 500 bp downstream of the core promoter within i ntron 1 further enhances PROC promoter-driven reporter gene expression in h uman hepatoma cells. In common with core promoter constructs used in previo us studies, the activity of this 3'-extended regulatory region is diminishe d by a naturally occurring promoter mutation. However, in contrast tb const ructs lacking intronic sequence, the promoter/intron regulatory region is r epressed rather than activated by the transcription factor HNF-1. Using bot h conventional alignment procedures and complexity analysis to study the hu man and canine PROC sequences, we identified two conserved intronic regions , which were tested for their involvement in gene regulation. High-level ge ne expression from the intron-coupled promoter was dependent upon the integ rity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for ge ne regulation and serve to illustrate that the results of PROC promoter/rep orter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functio nal evaluation of regulatory mutations and polymorphisms.