Delineation of thyroid hormone-responsive sequences within a critical enhancer in the rat uncoupling protein gene.
Delineation of thyroid hormone-responsive sequences within a critical enhancer in the rat uncoupling protein gene.
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DOI：
10.1210/endo.136.3.7867554
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发表时间：
1995-03
期刊：
影响因子：
4.8
通讯作者：
R. Rabelo;A. Schifman;Amalia Rubio;Xiaoyang Sheng;J. E. Silva
中科院分区：
文献类型：
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作者：
R. Rabelo;A. Schifman;Amalia Rubio;Xiaoyang Sheng;J. E. Silva
解偶联蛋白(UCP)对于棕色脂肪组织(BAT)的产热、冷适应和能量平衡是必不可少的。交感神经系统，通过去甲肾上腺素和cAMP，以及甲状腺激素似乎是UCP表达的主要调节因素。T3增强去甲肾上腺素的作用，对这种蛋白质对寒冷的适应反应是必不可少的。本研究的目的是研究T3是否如体内结果所提示的那样直接刺激大鼠UCP基因的转录，如果是这样的话，鉴定和表征所涉及的序列。我们在JEG-3和BAT来源的HIB-1B细胞中进行了瞬时转染分析，检测了114-3623之间的基因序列。这一3.7千碱基的UCP插入使报告基因对cAMP(4倍)、T3(4倍)或两者结合(12倍)做出反应。我们鉴定了一个位于-2317和-2399之间的82个碱基对的限制性片段，我们称之为甲状腺激素反应序列(THRs)，它赋予T3对UCP最小启动子(4-12倍)和胸苷激酶启动子(3-6倍)的反应性。T3受体在体外与THR结合，在电泳迁移率改变分析中延缓其迁移。THRs的足迹显示两个潜在的甲状腺激素反应元件(TRE)相隔27个碱基：upTRE，-2391/-2376，5‘ACCCCTACTGAGGCAA和dnTRE，-2348/-2334，5’AGGGCAGCAAGGTCA。这些假定的TRE的突变导致了T3受体结合和转录激活的丢失。对突变体的分析还表明，两个tre对UCP基因的t3反应的贡献比例相似，dntre对于t3增强cAMP效应是必要的。这两个TRE都位于先前确定的212个碱基的增强子元件内，两侧是被认为对BAT表达和去甲肾上腺素反应至关重要的序列。虽然它们不调节甲状腺激素的反应，但THR两侧的序列增加了基础报告基因的表达，并增强了对T3的反应。综上所述，我们的结果表明，T3可以刺激UCP基因的转录，并放大cAMP直接作用于该基因的作用。在一个对基因控制至关重要的位置上存在两个功能TRE，这支持了甲状腺激素对其表达的重要性，并暗示了在基因水平上相互作用的可能性，这可能解释了体内UCP调控的复杂性。
Uncoupling protein (UCP) is essential to brown adipose tissue (BAT) thermogenesis and, hence, to cold adaptation and energy balance. The sympathetic nervous system, via norepinephrine and cAMP, and thyroid hormone seem to be the major regulators of UCP expression. T3 potentiates the effect of norepinephrine and is essential for the adaptive response of this protein to cold. The goal of the present studies was to investigate whether T3 directly stimulates the transcription of the rat UCP gene, as suggested by in vivo results, and if so, to identify and characterize the sequences involved. We examined the gene sequence between 114 and -3623 by transient transfection analysis in JEG-3 and HIB-1B cells, a BAT-derived cell line. This 3.7-kilobase UCP insert makes the reporter gene responsive to cAMP (4-fold), T3 (4-fold), or both combined (12-fold). We identified an 82-basepair (bp) restriction fragment between -2317 and -2399, which we called thyroid hormone response sequence (THRS), that conferred T3 responsiveness to the UCP minimal promoter (4- to 12-fold) as well as to the thymidine kinase promoter (3- to 6-fold). T3 receptor bound to THRS in vitro, retarding its migration in electrophoretic mobility shift assays. Footprinting of THRS revealed two potential thyroid hormone response elements (TRE) separated by 27 bp: upTRE, -2391/-2376, 5'ACCCCTACTGAGGCAA; and dnTRE, -2348/-2334, 5'AGGGCAGCAAGGTCA. The mutation of these putative TREs caused loss of both T3 receptor binding and transactivation by T3. The analysis of the mutants also demonstrated that both TREs contribute in similar proportion to the T3 responsiveness of the UCP gene and that dnTRE is necessary for the potentiation of the cAMP effect by T3. Both TREs are located within a previously identified 212-bp enhancer element, flanked by sequences considered essential for BAT expression and norepinephrine responsiveness. Although they do not mediate thyroid hormone responsiveness, the sequences flanking THRS increase basal reporter expression and enhance the responses to T3. In conclusion, our results indicate that T3 can stimulate the transcription of the UCP gene and amplify the effect of cAMP acting directly on the gene. The presence of two functional TREs in a location critical to the control of the gene supports the importance of thyroid hormone for its expression and suggests the potential for interactions at the gene level that may explain the complexity of UCP regulation in vivo.