Vaulont S, Puzenat N, Kahn A, Raymondjean M
Unité de Recherches en Génétique et Pathologie Moléculaires, INSERM U. 129-CHU Cochin, Paris, France.
Mol Cell Biol. 1989 Oct;9(10):4409-15. doi: 10.1128/mcb.9.10.4409-4415.1989.
A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.
一个相对于大鼠L型丙酮酸激酶（L-PK）基因帽位点跨越核苷酸-183至-4的DNA片段含有至少四个假定转录因子的结合位点：肝细胞核因子1（HNF1）、肝因子A1（LF-A1）、核因子1（NF1）和主要晚期转录因子（MLTF）。该片段用于在细胞提取物中指导报告序列（无G盒）的转录。这个L-PK启动子在肝细胞核提取物中具有活性，但在非肝组织的提取物中没有活性。使用仅包含HNF1结合位点且缺失了部分序列的L-PK启动子，其活性降低了50%。相反，缺失HNF1结合位点会使启动子失活超过90%。这些结果通过合成寡核苷酸的滴定实验得到了证实。滴定HNF1导致转录活性降低85%，而滴定LF-A1仅导致转录活性降低40%。在这个系统中，NF1和MLTF的影响似乎很小。因此L-PK基因的近端5'侧翼序列在体外似乎作为一个有效的肝脏特异性启动子发挥作用，它需要肝因子HNF1结合并且也受到另一个肝脏特异性因子LF-A1结合的刺激。
