Interactions between the cytomegalovirus promoter and the estrogen response element: implications for design of estrogen-responsive reporter plasmids.
We aimed to produce an estrogen-responsive reporter plasmid that would permit monitoring of estrogen receptor function in the uterus in vivo. The plasmid pBL-tk-CAT(+)ERE was induced by estrogen in bovine endometrial stromal cells. When the CAT gene was replaced by the secreted alkaline phosphatase SeAP, the resulting construct pBL-tk-SeAP(+)ERE remained estrogen responsive. However when the tk promoter was replaced by the cytomegalovirus (cmv) promoter, the resulting plasmid (pBL-cmv-SeAP(+)ERE) was not estrogen responsive. Inhibition of ERE function was not due to an effect in trans or due to lack of estrogen receptor. It was not due to an interaction between the cmv promoter and the SeAP gene. cmv promoter function was dependent on NF-kappaB, and mutagenesis in the NF-kappaB sites reduced basal reporter expression without imparting responsiveness to estrogen. A mutation in the TATA box also failed to impart estrogen responsiveness. Modeling of DNA accessibility indicated the ERE was inserted at a site accessible to transcription factors. We conclude that the cmv promoter inhibits ERE function in cis when the two sequences are located in the same construct, and that this effect does not involve an interaction between cmv and reporter gene, NF-kappaB sites or the TATA box, or DNA inaccessibility.
Derecka
K
K
Division of Animal Physiology, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK. Kamila.Derecka@nottingham.ac.uk
Wang
C K
CK
Flint
A P F
AP
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics*; D004967:Estrogens; D005818:Genetic Engineering; D005822:Genetic Vectors; D010957:Plasmids; D011401:Promoter Regions, Genetic; D020218:Response Elements
D004967:Estrogens