Aguanno A, Afar R, Albert V R
Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USA.
J Biol Chem. 1996 Feb 23;271(8):4528-38. doi: 10.1074/jbc.271.8.4528.
The rat aromatic l-amino acid decarboxylase (AADC) gene contains alternative promoters which direct expression of neuronal and nonneuronal mRNAs that differ only in their 5'-untranslated regions (UTRs). We have analyzed the expression of the nonneuronal promoter of the rat AADC gene in the kidney epithelial cell line LLC-PK1 and in cells which do not express the nonneuronal form of AADC by transient transfection. These studies revealed that the first 1.1 kilobases of the nonneuronal promoter, including the nonneuronal-specific 5'-UTR (Exon 1), contains sufficient information to direct tissue-specific expression. Serial deletions of this promoter localized the cis-active element to a region between -52 and -28 base pairs upstream of the nonneuronal transcription start site. An A/T-rich sequence, within this region which we have termed KL-1, was found to bind a kidney and liver-specific factor by DNase footprint analysis and was capable of directing tissue-specific expression from a heterologous promoter. Moreover, when the KL-1 sequence was mutated in the context of the entire promoter sequence, all transcriptional activity was abolished. DNA sequence comparison revealed that the KL-1 fragment is highly homologous to the binding site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not express the nonneuronal form of AADC resulted in activation of transfected AADC nonneuronal promoter constructs. These results strongly suggest that the transcription factor which regulates the tissue-specific expression of the nonneuronal form of AADC mRNA is HNF-1.
大鼠芳香族L-氨基酸脱羧酶（AADC）基因含有多个可变启动子，这些启动子指导神经元和非神经元mRNA的表达，它们仅在5'-非翻译区（UTR）有所不同。我们通过瞬时转染分析了大鼠AADC基因非神经元启动子在肾上皮细胞系LLC-PK1以及不表达非神经元形式AADC的细胞中的表达情况。这些研究表明，非神经元启动子的前1.1千碱基，包括非神经元特异性的5'-UTR（外显子1），包含指导组织特异性表达的足够信息。该启动子的系列缺失将顺式作用元件定位到非神经元转录起始位点上游-52至-28碱基对之间的区域。通过DNA酶足迹分析发现，我们称为KL-1的该区域内富含A/T的序列可结合肾脏和肝脏特异性因子，并且能够指导来自异源启动子的组织特异性表达。此外，当KL-1序列在整个启动子序列的背景下发生突变时，所有转录活性均被消除。DNA序列比较显示，KL-1片段与肝细胞核因子-1（HNF-1）的结合位点高度同源。利用抗HNF-1抗体进行的迁移率变动研究表明HNF-1与KL-1片段结合，并且将HNF-1 cDNA共转染到不表达非神经元形式AADC的细胞中会导致转染的AADC非神经元启动子构建体被激活。这些结果强烈表明，调节AADC mRNA非神经元形式组织特异性表达的转录因子是HNF-1。
