In response to the Critique, the scope of this application has been narrowed to answer one central question, """"""""how do glucocorticoids down- regulate genes other than POMC in AtT-20 cells."""""""" While much is known about the mechanism of positive regulation of transcription by glucocorticoids since their receptor is a member of a family of ligand-regulated transcription factors, less is known about negative regulation. The best understood model for negative regulation of transcription by glucocorticoids is pro-opiomelanocortin gene expression in AtT-20 cells which are cultured pituitary cells from an irradiated mouse. Additional genes that are down-regulated by glucocorticoids in AtT-20 cells need to be identified to determine the full range of regulatory mechanisms active in the domain of genes down-regulated by glucocorticoids. I have developed an enhancer-trap strategy to clone additional genes not identified by previous methods which depended on information about the protein expressed by each gene. Using this approach, I have obtained a stable, transformed clone of AtT-20 cells (IDG8) transfected with pA10-NEO in which the NEO mRNA level is down-regulated by Dexamethasone similarly to POMC. The plasmid, pA10- NEO, that contains the selectable marker gene, aminoglycoside 3' phosphotransferase II, which is coupled to an enhancerless SV40 promoter. When transfected, this gene appears to have integrated near active, enhancer which is down-regulated by glucocorticoids. DNA from this cell line (IDG8) will be cloned, mapped, and the specific sequences that control glucocorticoid inhibition of expression identified. The functional glucocorticoid-regulated negative enhancer activity of these sequences will be defined by their insertion into a plasmid containing the chloramphenicol acetyltransferase (CAT) reporter gene and assay of CAT enzyme activity following transfection into wild-type AtT-20 cells. The mechanism of this functional activity will be analyzed to determine if it results from competitive inhibition by the glucocorticoid receptor of binding of another positive transcription factor, from steric hindrance of action of an upstream positive transcription factor, or from protein-protein interaction with another transcription factor. I have in hand a large number of other cell clones form which several such independent clones that are down- regulated by steroid can be identified by decreases in NEO mRNA levels with dexamethasone treatment. These will define a set of genes negatively regulated by glucocorticoids in AtT-20 cells.
| Harrison 3rd, R W; Lippman, S S; VerHoeven, R (1995) Selection of glucocorticoid-resistant mutations from an AtT-20 cell line containing a glucocorticoid-regulated selectable transgene. Biochem Biophys Res Commun 209:18-24 |
