MECHANISM OF STEROID SPECIFIC GENE ACTIVATION

The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors activate the same DNA element in vitro. Despite their functional diversity, glucocorticoid, androgen, progesterone, and mineralocorticoid receptors (GR, AR, PR, MR) are structurally uniform and recognize the same consensus hormone response element (HRB). To elucidate how specific transcription is imposed, we are studying a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens and not by glucocorticoids. Potent androgen induction requires both a consensus HRE and nonreceptor-binding elements present within a 120 base pair DNA fragment. In contrast to the strong response invoked by AR, GR can bind the HRE within the enhancer but cannot transactivate from that site. Thus specific transcriptional response to androgens, and lack of response to glucocorticoids, derives from selective protein-protein interactions that are determined by the context of the receptor binding site rather than by its distinct sequence. Our goal is to identify the accessory factors that impose this hormonal specificity. Strategies for isolation of these proteins and for functional proof of their significance benefit from the dual nature of specificity, which must as stringently prevent the action of some receptors as enhance the effectiveness of others. Specifically, we will: I) identify domains of AR and GR that confer differential response, by testing the ability of mutant receptors to activate specific and nonspecific enhancers in transfection; II) generate probes of protein-protein interaction by expressing these receptor domains in bacteria, and generate DNA probes by greater definition of nonreceptor protein-DNA interaction; III) isolate accessory factor cDNA clones from expression libraries by screening with both protein and DNA probes; W) prove the functional significance of these factors by their ability to alter differential responsiveness of the specific enhancer in distinct host cells, and demonstrate physical interaction of these factors in ternary complexes with the enhancer and receptor. These experiments will be complemented by assays in hormonally- treated and transgenic mice to validate the physiological significance of the nonreceptor factors in regulation. Steroid-specific transcription is implicit in normal development, homeostasis, and reproduction. Understanding the mechanism by which specific hormone response is elicited from nonspecific receptor binding sites may allow novel insights for combatting hormone-dependent tumors, such as those of the breast and prostate. For prostate cancer particularly, identification of cell-specific factors required for androgen action may have crucial value for future prognosis and therapies.