The long range goal of this research is to understand the molecular basis by which the nuclear thyroid hormone receptor (TR) mediates changes in specific gene transcription. TR binds to specific DNA sequences (thyroid hormone response elements or TREs) in both the absence and presence of hormone. Upon binding of hormone, TR undergoes a conformational change that converts it to a transcriptionally active state. The region of TR between the DNA-binding domain and the C-terminal end (residues 170 to 456 of the beta form) is responsible for ligand binding, dimerization and transcriptional activation. Little is currently known regarding these important processes. The focus of this proposal is to identify the critical amino acid residues responsible for these functions and to understand the mechanism of ligand-mediated transcriptional activation by TR. To accomplish these goals, we have established a yeast system in which expression of the reporter gene, beta-galactosidase, is under control of TR. Like many other mammalian transcription factors, TR can function in yeast to activate transcription provided an appropriate DNA recognition site is present. Using the yeast expression system, we have selected for mutations of the C-terminal region of TR that are defective in promoting transcriptional activation. These mutations fall into two general categories: those that fail to bind hormone and those that bind hormone normally, but cannot trans-activate. To evaluate the nature of these functional domains, we will further characterize the mutations leading to defects in hormone-binding and trans-activation. To identify amino acid residues that are in close contact with hormone, we will screen for mutations that alter the ligand-binding specificity of TR (Specific Aim #1). Mutations that bind hormone, but are defective in trans-activation, will be studied to determine whether the defects result from the failure of TR to undergo the ligand-mediated conformational change or lie directly within the trans-activation domain itself (Specific Aim #2). Recently, several laboratories have shown that TR binding and transcriptional activation on certain TREs can be enhanced by heterodimerization with the retinoid X receptor (RXR). We will use the mutations that we have isolated to study the roles of each of the receptor partners in the heterodimer in the activation process (Specific Aim #3). Finally, we will use phenotypic screening in yeast to identify regions of TR that are critical to the process of heterodimerization and to determine whether distinct dimerization domains are involved in interacting with different types of TREs (Specific Aim #4). These studies will allow a better understanding of the process of ligand-mediated transcriptional activation by TR and provide valuable tools for future work to further explore this process.
