TRANSCRIPTIONAL REGULATION OF THE GENE CODING FOR HUMAN PROTEIN-C

The promoter for the gene coding for human protein C has been characte rized as to nucleotide sequences that regulate the synthesis of mRNA. The major transcription start site was found 65 nucleotides upstream f rom the first intron/exon boundary along with two minor sites, Functio nal characterization of 1528 base pairs at the 8'-end of the gene was then carried out by chloramphenicol acetyltransferase reporter assays, protection from DNase I digestion, and electrophoretic mobility shift assays employing HepG2 and HeLa cells. One of the upstream regions (n ucleotides -25 to +9) contained binding sites for at least two differe nt transcription factors, including a hepatic nuclear factor 1-binding site (-10 to +9) and two overlapping and oppositely oriented hepatic nuclear factor 3-binding sites (-25 to -11). A second major region (PC E1) (+12 to +30) appeared to be a unique, liver-specific regulatory se quence, An Sp1-binding site in exon I (+58 to +65) was also recognized by cotransfection experiments with an Spl expression plasmid. Specifi c mutations in these promoter elements reduced transcriptional activit y and abolished the binding of hepatic nuclear proteins. Finally, a st rong silencer element (PCS1) (between -162 and -82) and two possible l iver-specific enhancer regions (PCE2 and PCE3), which interact coordin ately with the promoter elements, were also found (between -1462 and - 162).