The promoter of the C1 inhibitor gene contains a polypurine center dot polypyrimidine segment that enhances transcriptional activity

The CI inhibitor (C1INH) promoter is unusual in two respects: 1) It contain s no TATA sequence, but instead contains a TdT-like initiator element (Inr) at nucleotides -3 to +5; 2) it contains a polypurine polypyrimidine tract between nucleotides -17 and -45, Disruption of the Inr by the introduction of point mutations reduced promoter activity by 40%. A TATA element inserte d at nucleotide -30 in the wild-type promoter and in promoter constructs co ntaining the mutated Inr led to a 2-fold increase in basal promoter activit y. Previous studies suggested that the potential hinged DNA-forming polypur ine polypyrimidine tract might be important in the regulation of C1INH prom oter activity. The present studies indicate that this region is capable of such intramolecular triple helix formation. Disruption of the polypurine po lypyrimidine sequence by substitution of 5 of the 23 cytosine residues with adenine prevented triple helix formation. Site-directed mutagenesis experi ments demonstrate that the regulation of promoter activity is independent o f hinged DNA-forming capacity but requires an intact AC box (ACCCTNNNNNACCC T) or the overlapping PuF binding site (GGGTGGG), The C1INH gene also conta ins a number of potential regulatory elements, including an Sp-l and an hep atocyte nuclear factor-1 binding site and a CAAT box. The role of these ele ments in regulation of the C1INH promoter was examined. Elimination of the hepatocyte nuclear factor-1 site at nucleotides -94 to -81 by truncation re duced the activity of the promoter by similar to 50%. Similarly, site-direc ted mutations that disrupt this site reduce promoter activity by 70%.