Target genes regulated by thyroid hormones contain DNA (thyroid hormone response elements or TRE's) sequences to which thyroid hormone receptors (TR's) bind. Binding of TR's to TRE's is facilitated by retinoid X receptors (RXR's and the RXR's also enhance transcriptional effects of thyroid hormone receptors in some but not all target genes. However, the precise mechanisms by which TR's influence genetic transcription are unclear. It is possible that the receptor directly contacts essential transcriptional regulatory proteins. It is also possible that other proteins might be required to mediate the transduction of hormonal signal from the receptors to the transcriptional apparatus. A combination of both mechanisms is also feasible. To address these issues, I have developed systems for the expression and purification from bacteria of human TR's and RXR's. The purified proteins possessed the expected ligand- and DNA-binding characteristics of these receptors. I have also developed a system that produces purified TRbeta labeled with 32P for use as a probe for studying the interactions of TRbeta with other nuclear proteins. Protein interactions were studied by far-western blotting assays. To determine if TRbeta interacts with an essential component of the transcriptional apparatus known to bind to other nuclear receptors (TFIIB), the human TFIIB was expressed in bacteria and tested in far- western blots for TRbeta binding. The recombinant TFIIB bound to purified TRbeta in a region localized to the zinc finger region of TRbeta. The TRbeta also bound to RXRalpha with the expected affinity. Binding of TRbeta to nuclear proteins from various tissues showed tissue-specific patterns particularly with regard to a 75 kDa protein detected in brain. Deletion analysis revealed that several of these tissue nuclear proteins interact with the amino terminal and zinc finger domains of TRbeta. In addition, in GH3 pituitary cells, the levels and/or binding capacities of some of the TRbeta-binding proteins (TRBP's) were negatively regulated by thyroid hormone. These results indicate that TRbeta interacts with several different TRBP's that may participate in thyroid hormone regulation of gene expression. Thus, TRbeta interacts directly with RXR's, an essential transcriptional regulatory protein (TFIIB), and several nuclear proteins yet to be identified. The project proposed here is designed to extend these preliminary findings and to identify the other nuclear proteins with which TR's interact. Using these protein probes, expression libraries will be screened for cDNA's that encode TR-binding proteins. The proteins encoded by the cDNA's will be characterized with respect to their abilities to regulate hormonal responses and with respect to their interactions with TR's, RXR's, TRE's, and possibly other nuclear hormone receptors both in vivo and in vitro. These studies should provide valuable information on mechanisms of thyroid hormone action, signal transduction by nuclear hormone receptors, and transcriptional regulatory mechanisms.
