CHARACTERIZATION OF THE IFN-GAMMA-RESPONSIVE ELEMENT IN THE 5'-FLANKING REGION OF THE C1 INHIBITOR GENE

Treatment of a variety of cell lines with IFN-gamma leads to enhanced synthesis and secretion of C1 inhibitor (C1inh). The induction of C1in h synthesis by IFN-gamma is primarily regulated at the transcriptional level, and is controlled by elements in the 5' flanking region and th e first intron of the C1inh gene. Hep3B cells transfected with reporte r constructs containing truncated segments between -738 and -81 of the 5' flanking region and stimulated with IFN-gamma expressed increased levels of chloramphenicol acetyl transferase. These data as well as th e data obtained from studies using constructs with mutated IFN-gamma-a ctivated sequence (GAS) indicate that the most proximal CAS element (G AS 4) that mapped to nucleotides -126 to -118 is responsible for this IFN-gamma responsiveness. Electrophoretic mobility shift assays using GAS 4 yielded a single band that appeared within 5 min after stimulati on with IFN-gamma. In competition experiments, both GAS 4 and consensu s GAS probes, but not a mutated GAS probe, competed for the GAS bindin g protein present in Hep3B and U-937 cell extracts. The identity of th e GAS binding protein was confirmed using anti-STAT-1 alpha Abs in sup ershift assays. The results indicate that STAT-1 alpha binds to GAS 4, which is the primary element in the 5' flanking region responsible fo r IFN-gamma induction of the C1inh gene.