Estrogen receptor alpha (ERα) is a ligand‐dependent transcription regulator, containing two transactivation functional domains, AF‐1 and AF‐2. The selective estrogen receptor modulators (SERMs), including 4‐hydroxytamoxifen (4OHT), activate AF‐1 preferentially rather than AF‐2. However, it is unclear whether this specific function is related to the tissue‐selective functionality of SERMs. Moreover, there is no information determining AF‐1–dependent estrogenic‐genes existing in tissues. We sought to identify AF‐1–dependent estrogenic‐genes using the AF‐2 mutated knock‐in (KI) mouse model, AF2ERKI. AF2ER is an AF‐2 disrupted estradiol (E2)‐insensitive mutant ERα, but AF‐1–dependent transcription can be activated by the estrogen‐antagonists, fulvestrant (ICI) and 4OHT. Gene profiling and ChIP‐Seq analysis identified Klk1b21 as an ICI‐inducible gene in AF2ERKI uterus. The regulatory activity was analyzed further using a cell‐based reporter assay. The 5′‐flanking 0.4k bp region of Klk1b21 gene responded as an ERα AF‐1–dependent estrogen‐responsive promoter. The 150 bp minimum ERα binding element (EBE) consists of three direct repeats. These three half‐site sequences were essential for the ERα‐dependent transactivation and were differentially recognized by E2 and 4OHT for the gene activation. This response was impaired when the minimum EBE was fused with a thymidine‐kinase promoter but could be restored by fusion with the 100 bp minimum transcription initiation element (TIE) of Klk1b21, suggesting that the cooperative function of EBE and TIE is essential for mediating AF‐1–dependent transactivation. These findings provide the first in vivo evidence that endogenous ERα AF‐1 dominant estrogenic‐genes exist in estrogen‐responsive organs. Such findings will aid in understanding the mechanism of ERα‐dependent tissue‐selective activity of SERMs.

中文翻译：

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雌激素受体 α 反式激活功能 1 调节基因的基因组调控元件

雌激素受体α (ERα) 是一种配体依赖性转录调节因子，包含两个反式激活功能域，AF-1 和 AF-2。选择性雌激素受体调节剂 (SERM)，包括 4-羟基三苯氧胺 (4OHT)，优先激活 AF-1 而不是 AF-2。然而，尚不清楚这种特定功能是否与 SERM 的组织选择性功能有关。此外，没有确定组织中存在 AF-1 依赖性雌激素基因的信息。我们试图使用 AF-2 突变的敲入 (KI) 小鼠模型 AF2ERKI 来鉴定 AF-1 依赖性雌激素基因。AF2ER 是一种 AF-2 破坏的雌二醇 (E2) 不敏感突变体 ERα，但 AF-1 依赖性转录可以被雌激素拮抗剂氟维司群 (ICI) 和 4OHT 激活。基因分析和 ChIP-Seq 分析将 Klk1b21 鉴定为 AF2ERKI 子宫中的 ICI 诱导基因。使用基于细胞的报告基因分析进一步分析了调节活性。Klk1b21 基因的 5' 侧翼 0.4k bp 区域作为 ERα AF-1 依赖性雌激素反应启动子起反应。150 bp 最小 ERα 结合元件 (EBE) 由三个直接重复组成。这三个半位点序列对于 ERα 依赖性反式激活至关重要，并且被 E2 和 4OHT 差异识别以进行基因激活。当最小 EBE 与胸苷激酶启动子融合时，这种反应会受到损害，但可以通过与 Klk1b21 的 100 bp 最小转录起始元件 (TIE) 融合来恢复，这表明 EBE 和 TIE 的协同功能对于介导 AF 至关重要‐1 依赖性反式激活。这些发现提供了第一个体内证据，证明内源性 ERα AF-1 主要雌激素基因存在于雌激素反应器官中。这些发现将有助于理解 SERM 的 ERα 依赖性组织选择性活性的机制。