Sugiyama T, Scott D K, Wang J C, Granner D K
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Mol Endocrinol. 1998 Oct;12(10):1487-98. doi: 10.1210/mend.12.10.0187.
The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes three accessory factor-binding sites (AF1, AF2, and AF3), two glucocorticoid receptor-binding sites (GR1 and GR2), and a cAMP response element. All of these elements, and the proteins that bind to them, are required for a complete glucocorticoid response. The PEPCK promoter also contains a retinoic acid response unit (RARU) that consists of two retinoic acid response elements (RARE1 and RARE2) that bind retinoic acid receptor/9-cis-retinoic acid receptor heterodimers. The sequences of RARE1 and RARE2 coincide with those for AF1 and AF3, respectively. Thus, the PEPCK promoter can mediate different hormone responses through hormone response units that utilize common elements, but that bind different sets of proteins. We reasoned that each response might require a unique structural assembly and therefore tested how various arrangements of the PEPCK promoter affect the actions of either glucocorticoids or retinoic acid. The activation of the PEPCK gene in response to glucocorticoids requires a specific set of cis-acting elements that must be precisely positioned within the GRU. The distance between AF2 and GR1 is critical for the glucocorticoid response, and since the AF2 factor, HNF3, interacts with GR in vitro, this protein-protein interaction may be important for the glucocorticoid response. By contrast, the distance and orientation requirements of AF1 and AF3 with respect to GR1 are more flexible. In the case of the RARU, although the relative positions of RARE1 and RARE2 are important for the retinoic acid response, more tolerance for distance and stereospecific alignment between RARE1 and RARE2 is allowed. In addition, we show that the GRU and the RARU can act as a module, within a restricted region, in the context of the PEPCK promoter and in limited contexts of a heterologous promoter. These observations suggest that the structural requirements of the GRU and the RARU are different, and this may have important implications for how multiple hormonal signals are integrated through this promoter.
磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子包含一个糖皮质激素反应单元（GRU），该单元包括三个辅助因子结合位点（AF1、AF2和AF3）、两个糖皮质激素受体结合位点（GR1和GR2）以及一个cAMP反应元件。所有这些元件以及与之结合的蛋白质都是完整的糖皮质激素反应所必需的。PEPCK启动子还包含一个视黄酸反应单元（RARU），它由两个视黄酸反应元件（RARE1和RARE2）组成，这两个元件结合视黄酸受体/9-顺式视黄酸受体异二聚体。RARE1和RARE2的序列分别与AF1和AF3的序列一致。因此，PEPCK启动子可以通过利用共同元件但结合不同蛋白质组的激素反应单元介导不同的激素反应。我们推测每种反应可能需要独特的结构组装，因此测试了PEPCK启动子的各种排列如何影响糖皮质激素或视黄酸的作用。PEPCK基因对糖皮质激素反应的激活需要一组特定的顺式作用元件，这些元件必须精确地定位在GRU内。AF2和GR1之间的距离对糖皮质激素反应至关重要，并且由于AF2因子HNF3在体外与GR相互作用，这种蛋白质-蛋白质相互作用可能对糖皮质激素反应很重要。相比之下，AF1和AF3相对于GR1的距离和方向要求更灵活。就RARU而言，尽管RARE1和RARE2的相对位置对视黄酸反应很重要，但RARE1和RARE2之间对距离和立体特异性排列的耐受性更高。此外，我们表明GRU和RARU可以在PEPCK启动子的背景下以及在异源启动子的有限背景下，在一个受限区域内作为一个模块发挥作用。这些观察结果表明GRU和RARU的结构要求不同，这可能对多种激素信号如何通过该启动子整合具有重要意义。
