Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor
Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor
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DOI：
10.1074/jbc.275.14.10638
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发表时间：
2000-04-07
影响因子：
4.8
通讯作者：
Edwards, PA
中科院分区：
文献类型：
--
作者：
Laffitte, BA;Kast, HR;Edwards, PA
法尼醇X激活受体（FXR; NR 1H 4）是核激素受体超家族的成员，与9-顺式视黄酸受体（RXR）形成异源二聚体。为了确定FXR/RXR异二聚体的最佳DNA结合序列，我们利用了选择和扩增的结合序列印记技术。该技术在体外鉴定了许多与FXR /RXR相互作用的相关序列。共有序列含有序列AGGTCA的反向重复，具有1个碱基对间隔（IR-1）。该序列被证明是体外FXR/RXR的高亲和力结合位点，并通过FXR/RXR赋予异源启动子配体依赖性转录激活。电泳迁移率变动分析和瞬时转染分析被用来调查的核心半位点序列，间隔核苷酸，侧翼序列，和方向和间隔的核心半位点的DNA结合和配体依赖性转录激活FXR/RXR的重要性。这些研究表明，FXR/RXR异二聚体以最高亲和力结合共有IR-1序列，尽管FXR/RXR可以结合并通过多种元件激活，包括核心半位点序列、间隔核苷酸和侧翼核苷酸发生变化的IR-1元件。此外，FXR/RXR可以结合并通过直接重复反式激活。鉴定了三个基因，其近端启动子中含有IR-1序列。这些元件显示出在体外结合FXR/RXR，并且响应于胆汁酸或合成类维生素A而将FXR/RXR依赖性转录激活赋予异源启动子。这些基因之一磷脂转移蛋白的内源mRNA水平显示出由FXR和FXR配体诱导。IR-1和相关元件作为FXR/RXR的高亲和力结合位点和功能反应元件的鉴定以及FXR/RXR靶基因的鉴定应有助于鉴定由FXR/RXR调节的其他基因。
The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis-retinoic acid receptor (RXR). In order to determine the optimal DNA binding sequence for the FXR/RXR heterodimer, we have utilized the selected and amplified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR /RXR in vitro. The consensus sequence contained an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1). This sequence was shown to be a high affinity binding site for FXR/RXR in vitro and to confer Ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter. Electrophoretic mobility shift assays and transient transfection assays were used to investigate the importance of the core half-site sequences, spacing nucleotide, flanking sequences, and orientation and spacing of the core half-sites on DNA binding and ligand-dependent transcriptional activation by FXR/RXR. These studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Three genes were identified that contain IR-1 sequences in their proximal promoters. These elements were shown to bind FXR/RXR in vitro and to confer FXR/RXR-dependent transcriptional activation to a heterologous promoter in response to a bile acid or synthetic retinoid, The endogenous mRNA levels of one of these genes, phospholipid transfer protein, were shown to be induced by FXR and FXR ligands. The identification of the IR-1 and related elements as high affinity binding sites and functional response elements for FXR/RXR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR.