Rabelo R, Reyes C, Schifman A, Silva J E
Department of Medicine, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, Canada.
Endocrinology. 1996 Aug;137(8):3478-87. doi: 10.1210/endo.137.8.8754777.
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.
解偶联蛋白（UCP）对于棕色脂肪组织（BAT）的产热功能至关重要。甲状腺激素通过位于-2300上游且相隔27 bp的两个甲状腺激素反应元件（TRE）刺激大鼠UCP基因。它们是一个反向重复序列（上游TRE或upTRE）和一个正向重复序列（下游TRE或dnTRE）。本研究的目的是探讨这些TRE是否相互作用以及这种相互作用如何有助于解释UCP在体内对T3的反应性。因此，我们旨在确定：T3受体（T3R）的异二聚体伴侣；T3在受体-受体和受体-DNA相互作用中的作用；这种体外相互作用如何与TRE的增强子功能相关；以及两个TRE如何相互作用。研究包括利用T3R和视黄酸X受体（RXR）进行的电泳迁移率变动分析；DNA足迹分析；以及对源自BAT的细胞系HIB-1B细胞的瞬时转染。与许多先前描述的TRE一样，T3R的伴侣是RXR。未结合配体的T3R以同二聚体形式与TRE结合，作为转录抑制因子起作用。T3减少T3R同二聚体的结合，从而解除抑制，并与异二聚体与元件的结合成比例地刺激异二聚体的结合和转录。尽管在这些方面定性相似，但两个TRE之间存在重要的定量差异。与dnTRE相比，upTRE结合更多的T3R同二聚体和更少的T3R-RXR异二聚体，并且T3更容易促进异二聚体与dnTRE而非upTRE的结合。这些体外特征反映在T3通过upTRE解除T3R同二聚体介导的抑制和刺激转录的效率低于通过dnTRE。在受体T3R和RXR的结合以及对T3的反应性方面，两个TRE之间也存在显著的相互作用。单独来看，每个TRE对T3的反应都较为适度，upTRE的敏感性低于传统TRE，dnTRE的敏感性高于传统TRE，而在基因序列的背景下，它们共同介导的反应大于每个TRE单独介导的反应之和，对T3具有中等敏感性。因此，两个不足以解释UCP基因对T3反应性的TRE共同形成了一个适合甲状腺激素调节该基因的复杂单元。这些相互作用代表了TRE塑造基因对甲状腺激素反应性的又一种方式。
