Interactions among receptors, thyroid hormone response elements, and ligands in the regulation of the rat uncoupling protein gene expression by thyroid hormone.
Uncoupling protein (UCP) is essential to the thermogenic function of brown adipose tissue (BAT). Thyroid hormone stimulates the rat UCP gene through two thyroid hormone response elements (TRE) located upstream of -2,300 and separated by 27 bp. They are an everted repeat (upstream TRE or upTRE) and a direct repeat (down-stream TRE or dnTRE). The goal of the present studies was to investigate whether these TREs interact and how such an interaction could contribute to explain the UCP responsiveness to T3 in vivo. We therefore aimed to define: the heterodimeric partner of the T3 receptor (T3R); the role of T3 in the receptor-receptor and receptors-DNA interactions; how such in vitro interactions relate to the enhancer function of TREs; and how the two TREs interact. Studies included electrophoretic mobility shift assays, utilizing T3R and retinoid X receptors (RXR); DNA footprinting; and transient transfections of HIB-1B cells, a BAT-derived cell line. As in many previously described TREs, the partner of the T3R is RXR. The unliganded T3Rs bind to the TREs as homodimers, which act as repressors of transcription. T3 reduces the binding of T3R homodimers, hence relieving the repression, and stimulates the binding of heterodimers and transcription in proportion to the heterodimer binding to the elements. Although qualitatively similar in these regards, there were important quantitative differences between both TREs. The upTRE binds more T3R homodimers and less T3R-RXR heterodimers than the dnTRE, and T3 more readily facilitates heterodimer binding to the dn- than to the upTRE. These in vitro characteristics are reflected in a lower efficiency of T3 to relieve T3R homodimer-mediated repression and to stimulate transcription through up-than through dnTRE. There were also significant interactions between the two TREs both in the binding of the receptors, T3R and RXR, and in the responsiveness to T3. By itself, each TRE responded modestly to T3, upTRE with lower sensitivity and dnTRE with higher sensitivity than traditional TREs, whereas together, in the context of the gene sequence, they mediated a response greater than the sum of those mediated by each TRE separately, with an intermediate sensitivity to T3. Thus, two TREs that are inadequate to explain the responsiveness of the UCP gene to T3, together form a complex unit appropriate for the regulation of the gene by thyroid hormone. These interactions represent yet another way TREs can shape up the responsiveness of genes to thyroid hormone.