FUNCTIONAL IDENTIFICATION OF GENES UP-REGULATED AND DOWN-REGULATED BYGLUCOCORTICOIDS IN ATT-20 PITUITARY-CELLS USING AN ENHANCER TRAP

The AtT-20/D1 mouse pituitary tumor cell line has been used to study g lucocorticoid regulation of POMC. We have used an enhancer trap to det ermine whether other glucocorticoid-regulated genes exist in AtT-20 ce lls. An enhancer trap is a recombinant construction containing a selec table marker driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it co mes under the influence of an endogenous enhancer. For a selectable ma rker, 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. Suppress ion resulted in ganciclovir resistance and hygromycin sensitivity. An enhancerless promoter was produced using a truncated, transcriptionall y inactive, form of the POMC promoter. AtT-20/D1 cells were transfecte d 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 a nd were designated AtT-20/NET (for negative enhancer trap). Northern b lot analysis confirmed that the transgene was dean-regulated by dexame thasone as expected and that in at least one instance, suppression of the transgene was more complete than suppression of the full-length PO MC 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 occ urred 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 ve ctor, up-regulated enhancers were trapped by selection in hygromycin a nd dexamethasone followed by ganciclovir alone and designated AtT-20/P ET cells (for positive enhancer trap). Up-regulation of the selectable gene in AtT-20/PET cells was confirmed by Northern blot analysis of d examethasone-treated cells. In summary, glucocorticoid-regulated enhan cers have been identified in AtT-20/D1 cells by an enhancer trap strat egy that uses sequential selection under conditions that test whether the transgene is active. These results indicate that in addition to th e well characterized, down-regulated POMC gene, there are other glucoc orticoid-regulated genes in AtT-20/D1 cells that are both up-regulated and down-regulated by glucocorticoids.