Many of the actions of thyroid hormone to promote human development and maintain homeostasis are thought to be exerted by a nuclear protein, the thyroid hormone receptor. Very little is known about the way the thyroid hormone receptor interacts with DNA or operates to control transcription. The Principal Investigator has developed novel techniques for the identification of the exact nucleotides which determine receptor affinity for DNA and can directly assay receptor binding affinities to synthetic oligonucleotides as short as 22 base pairs. Using these techniques, multiple binding regions have been discovered in a hormonally controlled gene, rat growth hormone (rGH). Ten new receptor binding sites have been identified in ten other genes. Site-specific mutations of the rGH sites involving as few as four base pairs affect the ability of the receptor to bind and to promote rGH directed transcription of the chloramphenicol acetyltransferase gene. This proposal is designed to identify the manner by which native thyroid hormone receptors and their proto-oncogene C-erb-alpha counterparts recognize regulatory elements of thyroid hormone controlled genes. Thus, the sites identified to date, and oligonucleotides contain mutations of these sites, will be examined in detail to define the structures that are important for receptor recognition. The critical nucleotide contacts within several of these DNA binding sites of will be identified. DNA phosphate backbone contacts will be determined ethylation protection studies. An examination of other synthetic oligonucleotides will test the effects of altering consensus sequences and the degeneracy of consensus sequences. The possibility of protein-receptor contacts altering the recognition of the receptor binding domain will be explored. Dimerization of the receptor induced DNA flexure by the receptor will be determined. Finally, the function of three receptor binding domains from genes not now known to be responsive to thyroid hormone will be tested. The ability of native receptor protein and human and rat C-erb-a translation products to recognize these sites will be determined. The results of these studies will define the thyroid hormone response element and its action, which are relevant to evaluating thyroid hormone action and the regulation of gene expression in general.
| Robertson, R Paul; Sutherland, David E R; Seaquist, Elizabeth R et al. (2003) Glucagon, catecholamine, and symptom responses to hypoglycemia in living donors of pancreas segments. Diabetes 52:1689-94 |
| King, I N; de Soyza, T; Catanzaro, D F et al. (1993) Thyroid hormone receptor-induced bending of specific DNA sequences is modified by an accessory factor. J Biol Chem 268:495-501 |
| Tansey, W P; Schaufele, F; Heslewood, M et al. (1993) Distance-dependent interactions between basal, cyclic AMP, and thyroid hormone response elements in the rat growth hormone promoter. J Biol Chem 268:14906-11 |
