The long-term objective of this study is to define the molecules and the mechanisms involved in steroid hormone regulation of gene expression. In this context, the glucocorticoid hormone-regulated accumulation of rat Alpha2 urinary globulin (RUG) mRNA, in a cultured mammalian cell line, is examined in three ways. 1) Mouse L cell transformants bearing stably integrated and hormonally inducible copies of recombinant RUG DNA sequences will be prepared. Biochemical characterization of RUG transcripts will test whether the hormone-induced increase in RUG mRNA levels involves mechanisms that affect transcriptional events, posttranscriptional events or both. 2) A series of recombinant vectors containing one or more segment of RUG DNA and flanking sequences will be constructed. The biological assays of the constructs will identify those segments of RUG DNA and/or flanking sequences that are involved in mediation of hormonal response. The ability of such regulatory segments to act as enhancer elements and to cooperate in induction of RUG mRNA will be monitored. 3) Nitrocellulose filter binding and nuclease footprinting assays will be used to examine, in vitro, the interaction of purified rat liver glucocorticoid receptors with recombinant RUG DNA and flanking sequences. If selective receptor:DNA interaction is detected, biological assays will evaluate their significance. In addition, a hypothesis that selective recognition of RUG DNA by the receptor may require the participation of a discrete receptor-associated factor will be examined. The importance of steroid hormones as developmental and physiological regulators stems from their ability to control the activity of specific gene networks in a cell-specific manner. In case of direct responses, glucocorticoid receptors, upon association with their cognate steroid ligands, bind to specific DNA sequences, activate receptor-dependent transcriptional enhancer elements and, in turn, stimulate initiation from nearby promotors. The indirect responses have not been biochemically characterized, and these will be examined in this research proposal. Elucidation of the molecular mechanism of steroid hormone regulation of gene expression should contribute to our understanding of the action of steroids as immunosuppressants, cancer chemotherapeutic agents, and anti-inflammatory drugs, as well as increase our understanding of the role of steroids in the pathogenesis of a number of human diseases and developmental defects.
| Hess, P; Payvar, F (1992) Hormone withdrawal triggers a premature and sustained gene activation from delayed secondary glucocorticoid response elements. J Biol Chem 267:3490-7 |
| Chan, G C; Hess, P; Meenakshi, T et al. (1991) Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u-globulin DNA. J Biol Chem 266:22634-44 |
| Hess, P; Meenakshi, T; Chan, G C et al. (1990) Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment that mediates a delayed secondary response to glucocorticoids in vivo. Proc Natl Acad Sci U S A 87:2564-8 |
