The goal of this proposal is to gain a better understanding of the central regulation of the hypothalamicpituitary-thyroid axis (HPT axis). Negative regulation by thyroid hormone is thought to exert the dominant influence on the axis. However, after years of study, the molecular mechanism by which thyroid hormone negatively regulates the HPT axis remains unknown. Prior in vitro studies to address this mechanism have been limited by the use of heterologous cell lines.
In Aim 1, I will employ chromatin immunoprecipitation (ChIP) assays to locate thyroid hormone receptor binding sites (putative negative thyroid regulatory elements, nTREs) in the TSH beta gene of a mouse thyrotroph cell line, T alpha T1.1. I will validate identified putative nTREs using deletion mutagenesis.
In Aim 2, I seek to determine the mechanism by which the thyroid hormone receptor (TR) activates gene expression in the absence of ligand (T3) and represses gene expression in the presence of ligand. This will be accomplished via a candidate approach using ChIP assays as well as gel shift and ABCD assays to determine protein cofactors that interact with the TR at the nTREs identified in Aim 1. The identification and validation of an nTRE in a homologous cell line is a critical step in developing a model to study negative regulation by thyroid hormone. TSH determination is the sole clinically-useful bioassay of thyroid hormone action in humans. Yet TSH measurement is not reliable in several clinical settings, including severe illness, hypothalamic or pituitary disease, and starvation. An improved understanding of TSH regulation may lead to other clinically-useful assays of thyroid hormone action. It may also lead to the development of novel agents for TSH suppression, which has an important role in the treatment of thyroid cancer. ? ?
The proposed experiments will expand our knowledge of how nuclear hormone receptors are negatively regulated, a fundamental biological question with far-reaching potential clinical consequences. ? ? ?
| Machado, Danielle S; Sabet, Amin; Santiago, Leticia A et al. (2009) A thyroid hormone receptor mutation that dissociates thyroid hormone regulation of gene expression in vivo. Proc Natl Acad Sci U S A 106:9441-6 |
