GLUCOCORTICOID RECEPTOR STRUCTURE SUBUNIT ARCHITECTURE

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Glucocorticoid-regulated genes contain one or more specific elements that bind glucocorticoid receptor (GR) and, when associated with other genes make them glucocorticoid responsive. This particular element has teen named the glucocorticoid response element or GRE. This proposal asserts that are genes in a issue or cell that are regulated by GR interaction with a GRE can be identified and characterized using a novel approach which does not require foreknowledge of the regulated gene's protein products. The basis for this approach is the discovery that a 17,000 MW tryptic fragment of the receptor (17-K GR) binds GRE-containing DNA with higher affinity and specificity than does the intact GR. A spharose-protein A-monoclonal antibody matrix will be used to immobilize the 17-K GR thus resulting in an affinity column with specificity for genomic domains containing a GRE. This column will be used to extract GRE-containing fragments of genomic DNA (GRE- DNA). The proposed experiments will utilize AtT-20/D1 cells stably transfected with an LTR hybrid to yield an up-regulated gene to complement the down-regulated gene, POMC. The GRE-DNA fragments will be cloned and functional genes selected by determining which clones are complementary to RNA whose bundance is changed by glucocorticoid treatment. The corresponding structural genes will also be isolated and used to determine which gene is first affected by glucocorticoids. Further characterization of the genomic and structural DNAs will be done starting with the gene whose transcription is modified earliest by glucocorticoid treatment. The gene will be subcloned and sequenced to determine the elements common to other regulated genes. Functional characterization, not all of which can be accomplished in this granting period will include transfection of functional genes, transfection of anti- sense genes, and expression of the genes and characterization of their proteins (if any). The successful completion of these experiments will, for the first time, allow the unambiguous identification of all genes regulated by glucocorticoids. Logical extensions of this experimental paradigm to other tissues, such as, for example, lymphocytes will lead to a fundamental understanding of the mechanism of glucocorticoid-induced cell death.