This project's objective is to use a genetic approach to identify components of the glucocorticoid response mechanism. The strategy will be to introduce a gene construct into AtT-20 cells that results in cells that are growth-arrested in the presence of the neomycin analog, G418, and glucocorticoids. Preliminary studies have used AtT-20/D1.IDG8 cells which contain the neomycin resistance gene (neor) under negative glucocorticoid regulation. These cells grow in the presence of G418 or dexamethasone alone but are growth-arrested in the presence of dexamethasone + G418. The cells were treated with a chemical mutagen and glucocorticoid-resistant clones identified as large colonies on soft agar containing dexamethasone + G418. Fourteen clones were studied. In all clones tested, negative regulation of neor mRNA by dexamethasone was abolished. In ten of fourteen clones, regulation of an endogenous gene, pro-opiomelanocortin (POMC) was retained indicating that the mutation was local to the neor promoter, a cis-mutation. In the other four clones, regulation of POMC and of a reporter gene, prolactin-CAT, was lost indicating that the mutation was of a common, global factor, a trans-mutation. One of four lacked receptor mRNA indicating that it was a receptor-minus, trans-mutation. The other three contained receptor mRNA of normal size and abundance indicating that they might be normal-receptor, trans-mutations. They will be analyzed further by co-introduction of a receptor expression vector and a reporter gene. If the deficient regulation is not corrected by expression of normal receptor, their normal-receptor, trans-mutation status will be confirmed. These studies indicate that genetic selection of informative mutations can be accomplished by this approach. Phase one of the project will be establishment of a cell line stably-transfected with the neomycin resistance gene under control of the POMC promoter (the """"""""POMNEO"""""""" gene). In phase two, mutant clones will be produced, isolated and sorted into receptor defects, other trans defects and cis defects. Phase three will characterize the cis- and trans-mutants. The POMNEO promoter of cis- mutants will be sequenced to identify altered nucleotide sequences. Since a trans-mutation could result in a change of a DNA binding protein other than the receptor, or alteration in a protein-protein interaction necessary for receptor function, trans-mutant cells will be tested to determine if the pattern of protein binding to the POMNEO promotor has been altered. Successful completion of this project will: identify cell processes important to receptor function; and, identify and characterize cis elements of the glucocorticoid response mechanism that recognize trans-factors other than the receptor.
