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) and pseudogene (CYP21P) promoters. DNA fragments containing the promoter regions of CYP21 and CYP21P were amplified and cloned into promoterless luciferase reporter plasmids either containing or lacking an enhancer element. Cells of the nonsteroidogenic COS-1 cell line, and the steroidogenic Y-1 cell line were transiently transfected with these recombinant plasmids and a beta-galactosidase cotransfection control plasmid. Cellular lysates were analyzed for luciferase and beta-galactosidase activities. In the nonsteroidogenic system, transfectants with either the CYP21 or CYP21P upstream sequence in enhancer containing plasmids showed a 2.3 fold increase (p < .001) in light production over controls. In the steroidogenic Y-1 cell system, these same CYp21 and CYP21P transfectants showed a 14.3 (+/- 0.8) and 5.2 (+/- 0.6) fold increase in luciferase activity respectively (p < .001) Transfections with recombinant reporter plasmids lacking an enhancer produced light emission which was not significantly different than controls. These observations indicate that 1.) one or more of the 35 nucleotide differences between the CYP21 and CYP21P upstream regions alters a DNA recognition site important for transcriptional activation of this gene in steroidogenic cells, 2.) the steroidogenic milieu has a stimulatory effect on both CYP21 and CYP21P promoter activities, and 3.) based on the minimal promoter activity observed in either cell type transfected with constructs lacking an enhancer element, both of these promoter sequences are enhancer dependent under constitutive conditions in both steroidogenic and nonsteroidogenic cells.
Kyllo
J H
JH
Division of Pediatric Endocrinology University of Iowa College of Medicine, Iowa City 52242, USA.
Collins
M M
MM
Donohoue
P A
PA
D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011544:Pseudogenes / Q000502:physiology*; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D004247:DNA; D008156:Luciferases; D013255:Steroid 21-Hydroxylase