Achatz G, Hölzl B, Speckmayer R, Hauser C, Sandhofer F, Paulweber B
First Department of Internal Medicine, General Hospital of Salzburg, Austria.
Mol Cell Biol. 1997 Sep;17(9):4914-32. doi: 10.1128/MCB.17.9.4914.
The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.
孤儿受体ARP-1/COUP-TFII是核受体鸡卵清蛋白上游启动子转录因子（COUP-TF）亚家族的成员，它能强烈抑制众多基因的转录活性，包括几个载脂蛋白编码基因。最近已证明，COUP-TFs降低转录活性的机制涉及主动抑制和反式抑制。为了确定ARP-1/COUP-TFII抑制活性所需的结构域，对该蛋白进行了详细的缺失分析。将ARP-1/COUP-TFII羧基末端的不同片段与GAL4 DNA结合结构域融合的嵌合蛋白用于表征其主动抑制结构域。发现赋予异源DNA结合结构域主动抑制活性的最小片段包含210至414位氨基酸残基。该结构域涵盖了ARP-1/COUP-TFII中与核受体超家族其他成员最近发表的晶体结构中的螺旋3至12相对应的区域。它包括由螺旋12形成的AF-2 AD核心结构域，但不包括铰链区，而铰链区对于甲状腺激素和视黄酸受体与共抑制因子的相互作用至关重要。将猿猴病毒40大T抗原的核定位信号（Flu标签）连接到ARP-1/COUP-TFII的氨基末端，消除了其与DNA结合的能力，而不影响其抑制活性。通过使用一系列Flu标签突变体，确定了该蛋白反式抑制活性所需的结构域。它们包括DNA结合结构域和跨越193至399位氨基酸残基的片段。ARP-1/COUP-TFII可抑制由肝脏富集的转录激活因子如肝细胞核因子3（HNF-3）、C/EBP或HNF-4诱导的转录活性，而与其同源结合位点的存在无关，而基础转录或由ATF或Sp1诱导的转录活性不受该蛋白的干扰。总之，我们的结果表明，ARP-1/COUP-TFII主动抑制和反式抑制所需的结构域不一致。此外，它们强烈表明反式抑制是ARP-1/COUP-TFII抑制活性的主要机制。这种机制很可能涉及该蛋白与一种或几种转录共激活蛋白的相互作用，这些蛋白被各种肝脏富集的转录激活因子所利用，而不是被诸如Sp1或ATF等普遍存在的因子所利用。
