AUXILIARY PROTEINS IN THE RESPONSE TO THYROID HORMONE

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Thyroid hormones are essential for normal development and maintenance of normal metabolism. They affect metabolic rate, intermediary metabolism, and growth and development of the lungs, skeletal system, and central nervous system. The effects of thyroid hormone are largely mediated by nuclear thyroid hormone-receptors (TR), which bind to specific segments of DNA (thyroid hormone response elements; TRE) and regulate gene transcription by an unknown mechanism. A nuclear protein, called TR auxiliary protein (TRAP), has recently been shown to enhance the ability of TR to bind to DNA. TRAP is a sequence-specific DNA-binding protein which appears to form heterodimers with TR. The long-term goal of this project is to understand the role of TRAP in the function of TR. The first specific aim is to characterize the DNA sequence to which TRAP binds. The interactions of TRAP with DNA will be studied by footprint analysis, and the relative positions of the TRAP DNA-binding site and the TR DNA-binding site will be examined in several TREs. The second specific aim is to isolate and characterize a TRAP cDNA clone. The clone will be isolated by screening 1gt11 expression libraries with the TRAP DNA-binding sequence; or by purification of TRAP, microsequencing the protein, and screening libraries with oligonucleotides based on the protein sequence. The sequence of the cDNA will be examined for similarity to families of transcription factors. The third specific aim is to study the role of TRAP in the response to T3. Transfection studies will examine the ability of different TREs to regulate expression of a reporter gene, and the effect of point mutations within the TRA DNA-binding site. Also, the effect of TRAP on the response to T3 will be investigated by transfection of the TRAP cDNA, or expression of TRAP antisense RNA. The fourth specific aim is to study the expression of TRAP mRNA in different tissues and the possibility of a family of TRAP mRNAs. The fifth specific aim is to examine the physiological regulation of expression of TRAP mRNA. The data from all these studies will help define the structure of the TRAP-TR-DNA complex, and the role of TRAP in the T3 response.