The goal of this research is to understand hormonal mechanisms that control gene expression and how these molecules influence developmental and physiologic processes. Our previous studies have led to the identification of a superfamily of regulatory proteins that include receptors for steroid hormones, thyroid hormones and the vertebrate morphogen retinoic acid. The molecular interactions of the glucocorticoid receptor and thyroid hormone receptor with their binding sites will be examined using DNA- binding mutants that have altered target gene specificities or altered activational properties. The potential for functional receptor heterodimers will be examined. The DNA-binding domains of the GR and TR will be overproduced in E. coli to solve their three-dimensional structure in solution and in crystals by nuclear magnetic resonance (NMR) and X-ray crystallographic studies, respectively. Activation domains of the thyroid hormone receptors will be defined by deletion, truncation and point mutational analysis. Putative domains will be transferred to hybrid activators and potential interaction of the activator domains with the transcriptional machinery will be characterized through a transcriptional inteference analysis. Proteins interacting with the glucocorticoid and thyroid hormone receptors will be sought by biochemical approaches to identify the encoding genes. Biochemical approaches include chromatography of nuclear extracts to glucocorticoid receptor and thyroid hormone receptor affinity columns and the detection of receptor-DNA complexes with altered electrophoretic mobilities. Recent studies have shown the erbA oncogene is capable of functioning as a thyroid hormone receptor antagonist. The mechanism of this antagonism will be sought be examining the relative binding affinities of the oncogene and the thyroid hormone receptor for their response elements as well as identifying the specific mutations involved in converting the wild-type receptor to its oncogenic form. The thyroid hormone receptor repressor activity will be characterized. In summary, the proposed experiments should provide important information on basic questions such as the molecular nature of protein-DNA interactions in eukaryotic gene regulation, the interactions between activator proteins and the transcription machinery, and molecular interpretation for how steroid and thyroid hormones act to control human physiology as well as pathologic changes associated with reproduction, metabolism and cancer.
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