Espinás M L, Roux J, Ghysdael J, Pictet R, Grange T
Institut Jacques Monod du CNRS, Université Paris, France.
Mol Cell Biol. 1994 Jun;14(6):4116-25. doi: 10.1128/mcb.14.6.4116-4125.1994.
We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.
我们先前已经表明，大鼠酪氨酸转氨酶（TAT）基因的两个远端糖皮质激素反应元件（GRU）包含多个转录因子家族的结合位点，包括糖皮质激素受体（GR）。我们在此报告在其中一个GRU中鉴定出转录因子Ets家族成员的两个新结合位点。其中一个结合位点与主要的GR结合位点（GRBS）重叠，而另一个位于其附近。后一个结合位点的失活导致糖皮质激素反应降低两倍，而与GRBS重叠的位点失活则没有可检测到的影响。体内足迹分析表明，在表达TAT的细胞系中，即使该活性位点位于存在糖皮质激素依赖性核小体结构改变的位置，它也以糖皮质激素非依赖性方式被占据。在不表达TAT但表达Ets相关DNA结合活性的细胞系中，同一个位点未被占据，这表明在核小体之上的层次水平存在染色质结构的抑制作用。即使在表达TAT的细胞中，与GRBS重叠的无活性Ets结合位点也未被占据。然而，当从GRU中分离并复制时，这个相同的重叠位点可以赋予Ets依赖性对基础水平和糖皮质激素诱导水平的刺激。COS细胞中的Ets-1表达模拟了肝癌细胞中存在的Ets相关活性的活性。这些Ets结合位点可能参与TAT基因的糖皮质激素反应与其他非甾体细胞外刺激触发的信号转导途径的整合。
