CONSTITUTIVE HUMAN STEROID 21-HYDROXYLASE PROMOTER GENE AND PSEUDOGENE ACTIVITY IN STEROIDOGENIC AND NONSTEROIDOGENIC CELLS WITH THE LUCIFERASE GENE AS A REPORTER

This study was directed toward initial comparison and characterization of the activities of the human steroid 21-hydroxylase gene (CYP21) an d pseudogene (CYP21P) promoters. DNA fragments containing the promoter regions of CYP21 and CYP21P were amplified and cloned into promoterle ss luciferase reporter plasmids either containing or lacking an enhanc er element. Cells of the nonsteroidogenic COS-1 cell line, and the ste roidogenic Y-1 cell line were transiently transfected with these recom binant plasmids and a beta-galactosidase cotransfection control plasmi d. Cellular lysates were analyzed for luciferase and beta-galactosidas e activities. In the nonsteroidogenic system, transfectants with eithe r the CYP21 or CYP21P upstream sequence in enhancer containing plasmid s showed a 2.3 fold increase (p<.001) in light production over control s. In the steroidogenic Y-1 cell system, these same CYP21 and CYP21P t ransfectants showed a 14.3 (+/-0.8) and 5.2 (+/-0.6) fold increase in luciferase activity respectively (p<.001). Transfections with recombin ant reporter plasmids lacking an enhancer produced light emission whic h was not significantly different than controls. These observations in dicate that 1.) one or more of the 35 nucleotide differences between t he CYP21 and CYP21P upstream regions alters a DNA recognition site imp ortant for transcriptional activation of this gene in steroidogenic ce lls, 2.) the steroidogenic milieu has a stimulatory effect on both CYP 21 and CYP21P promoter activities, and 3.) based on the minimal promot er activity observed in either cell type transfected with constructs l acking an enhancer element, both of these promoter sequences are enhan cer dependent under constitutive conditions in both steroidogenic and nonsteroidogenic cells.