Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA
Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA
复制标题
DOI：
10.1210/me.13.11.1896
复制
发表时间：
1999-11-01
影响因子：
--
通讯作者：
Burnstein, KL
中科院分区：
文献类型：
--
作者：
Grad, JM;Dai, JL;Burnstein, KL
雄激素受体(AR)基因受AR(自动调节)转录调控，但在AR启动子或5‘-侧翼区域未发现此过程所需的雄激素反应元件(ARs)。我们先前的研究表明，AR基因包含参与AR基因自身调节的ARs，并且在异源启动子的驱动下，表达人AR基因的PC3细胞(人前列腺癌PC3细胞株)可复制自身(上调)调控基因。AR基因的350bp片段包含必需的ARE(ARE-1和ARE-2)，当连接到报告基因的上游时，以细胞特异性的方式赋予雄激素诱导性。在这里，我们报告，虽然含有ARE-1和ARE-2沉默突变的AR cDNA产生转录活性的AR，但由这个突变的cDNA编码的AR mRNA在雄激素处理的PC3细胞中不上调。因此，在该模型中，ARE-1和ARE-2在雄激素介导的AR mRNA上调中是必不可少的。由于ARE-1和ARE-P位于AR基因中不同的外显子(外显子D和E)上，我们在其自然背景下对这些ARE进行了评估，这是一个6.5kb的AR基因组片段。雄激素对6.5kb的AR基因组片段和AR基因的350bp区域的调控水平相当，这表明外显子D和E的序列可能参与了雄激素介导的天然AR基因的上调。此外，雄激素调节U2OS细胞中的两个反应区，U2OS细胞是一种人类成骨细胞系，表现出雄激素介导的天然AR mRNA的上调。DNase I用重组AR(DNA和配体结合域)标记350-bp区域的足迹表明存在额外的ARs。凝胶漂移分析和突变研究表明，最大的雄激素调节和AR结合取决于四个ARE(ARE-1、ARE-1A、IVSARE和ARE-P)的完整性。虽然在其他雄激素调节的增强子中存在多个非共识ARE是常见的，但AR基因的雄激素响应区是独特的，因为它包含外显子ARE。用核提取液进行DNA结合研究，以确定非AR转录因子是否对350-bp区域的雄激素调节起作用。这些研究结合突变分析和对Myc和Max显性阴性表达载体的报告基因分析，表明Myc和Max与Myc共识位点的相互作用是雄激素调节350-bp片段所必需的。这些结果代表了AR和Myc蛋白家族之间的一种新的相互作用，并支持AR和Myc家族通过AR和Myc家族与含有多个外显子调控序列的独特内部雄激素反应区域相互作用来控制AR mRNA的雄激素生成模型。
The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-P are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-P). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.