Characterization of a dominant negative mutant form of the HNF-4 orphan receptor.
Characterization of a dominant negative mutant form of the HNF-4 orphan receptor.
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DOI：
10.1093/nar/24.15.2930
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发表时间：
1996-08
影响因子：
14.9
通讯作者：
David G. Taylor;S. Haubenwallner;T. Leff;T. Leff
中科院分区：
文献类型：
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作者：
David G. Taylor;S. Haubenwallner;T. Leff;T. Leff
HNF-4 孤儿受体是转录因子核受体家族的成员，也是碳水化合物和脂质代谢相关基因的主要调节因子。作为表征 HNF-4 在代谢调节中的作用的第一步，我们生成了 HNF-4 的显性失活形式 (DN-HNF-4)，其中包含有缺陷的 DNA 结合结构域。在凝胶迁移率变动测定中，DN-HNF-4 不结合代表 HNF-4 必需结合位点（人 apo CIII 启动子中包含的 C3P）的寡核苷酸探针，但确实阻止了两种重组异构体 HNF-4α1 和 HNF-4α2 以及天然存在的 HNF-4 的结合。 DN-HNF-4 对 PPARgamma-RXRα 异二聚体与 PPAR 反应元件的结合没有影响。在转染的 HepG2 细胞中，DN-HNF-4 显着降低了人 apo CIII 启动子的组成型转录活性，并消除了表达 HNF-4 任一亚型的质粒引起的正转录活性。这些结果表明DN-HNF-4是选择性显性失活突变体，其与野生型HNF-4形成有缺陷的异二聚体，从而阻止HNF-4的DNA结合和随后的转录激活。
The HNF-4 orphan receptor is a member of the nuclear receptor family of transcription factors and a major regulator of genes involved in carbohydrate and lipid metabolism. As an initial step in characterizing the role of HNF-4 in the regulation of metabolism, we have generated a dominant negative form of HNF-4 (DN-HNF-4) that contains a defective DNA-binding domain. In gel mobility shift assays, DN-HNF-4 did not bind an oligonucleotide probe representing an essential HNF-4 binding site, C3P contained in the human apo CIII promoter, but did prevent the binding of two recombinant isoforms, HNF-4alpha1 and HNF-4alpha2, as well as naturally-occurring HNF-4. DN-HNF-4 had no effect on the binding of PPARgamma-RXRalpha heterodimers to a PPAR response element. In transfected HepG2 cells, DN-HNF-4 dramatically reduced constitutive transcriptional activity of the human apo CIII promoter and abolished the positive transcriptional activity caused by plasmids expressing either isoform of HNF-4. These results indicate that DN-HNF-4 is a selective dominant negative mutant which forms defective heterodimers with wild-type HNF-4, thereby preventing DNA binding and subsequent transcriptional activation by HNF-4.