Functional identification of genes up- and down-regulated by glucocorticoids in AtT-20 pituitary cells using an enhancer trap

Abstract

The AtT-20/D1 mouse pituitary tumor cell line has been used to study glucocorticoid regulation of POMC. We have used an enhancer trap to determine whether other glucocorticoid-regulated genes exist in AtT-20 cells. An enhancer trap is a recombinant construction containing a selectable marker driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it comes under the influence of an endogenous enhancer. For a selectable marker, we used a fusion gene coding for hygromycin phosphotransferase (Hy) and herpes simplex thymidine kinase. Thus, expression of this gene conferred hygromycin resistance and ganciclovir sensitivity. Suppression resulted in ganciclovir resistance and hygromycin sensitivity. An enhancerless promoter was produced using a truncated, transcriptionally inactive, form of the POMC promoter. AtT-20/D1 cells were transfected with this construct and cultured in medium containing hygromycin to kill any cells not expressing the Hy gene. The survivors were cultured in medium containing ganciclovir and dexamethasone and cloned. Clones in which the transgene was down-regulated by dexamethasone survived and were designated AtT-20/NET (for negative enhancer trap). Northern blot analysis confirmed that the transgene was down-regulated by dexamethasone as expected and that in at least one instance, suppression of the transgene was more complete than suppression of the full-length POMC promoter. Southern blot analysis after restriction enzyme digestion showed that each cell clone contained a single copy of the transgene, and PCR analysis of the promoter region showed that insertion had occurred in two unique sites in at least two cell clones. Another plasmid construct was prepared that contained the selectable gene but lacked any promoter elements. After transfection of AtT-20 cells with this vector, up-regulated enhancers were trapped by selection in hygromycin and dexamethasone followed by ganciclovir alone and designated AtT-20/PET cells (for positive enhancer trap). Up-regulation of the selectable gene in AtT-20/PET cells was confirmed by Northern blot analysis of dexamethasone-treated cells. In summary, glucocorticoid-regulated enhancers have been identified in AtT-20/D1 cells by an enhancer trap strategy that uses sequential selection under conditions that test whether the transgene is active. These results indicate that in addition to the well characterized, down-regulated POMC gene, there are other glucocorticoid-regulated genes in AtT-20/D1 cells that are both up-regulated and down-regulated by glucocorticoids.