Chu H M, Fischer W H, Osborne T F, Comb M J
Laboratory of Molecular Neurobiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.
Nucleic Acids Res. 1991 May 25;19(10):2721-8. doi: 10.1093/nar/19.10.2721.
A short region of the human proenkephalin promoter has been shown previously to mediate transcriptional regulation in response to activation of the cAMP, TPA, and Ca+ + dependent intracellular signalling pathways. Two adjacent DNA elements, CRE-1 and CRE-2, are essential for this regulation although neither element alone is sufficient for inducible expression. The CRE-2 element consists of overlapping binding sites for the transcription factors AP-1 and AP-4. The CRE-1 element has been shown to interact with a DNA binding factor called ENKTF-1. Here we characterize proteins from bovine brain which bind the CRE-1 element of the human proenkephalin gene. Interactions between proteins binding the CRE-1 and CRE-2 elements are characterized in vitro using affinity purified DNA binding proteins. We demonstrate that CRE-1 binding proteins from bovine brain consist of three different polypeptides each belonging to the NF-I family of transcription factors. Point mutation analysis of the contacts of these proteins with the CRE-1 element indicate that NF-I proteins contact the inducible enhancer at the sequence CTGGCxxxxxxCCT which overlaps the CRE-1 element (underlined) defined by in vivo point mutation analysis. Cotransfection of one of the three NF-I proteins purified from bovine brain, NF-I/Red1, together with a proenkephalin/bacterial chloramphenicol acetyl transferase (CAT) fusion gene repressed protein kinase A or forskolin stimulated CAT expression.
先前已表明，人前脑啡肽原启动子的一个短区域可介导对cAMP、佛波酯（TPA）和钙离子依赖性细胞内信号通路激活的转录调控。两个相邻的DNA元件，即CRE-1和CRE-2，对于这种调控至关重要，尽管单独任何一个元件都不足以实现诱导型表达。CRE-2元件由转录因子AP-1和AP-4的重叠结合位点组成。已表明CRE-1元件可与一种名为ENKTF-1的DNA结合因子相互作用。在此，我们对来自牛脑的与人前脑啡肽原基因CRE-1元件结合的蛋白质进行了表征。使用亲和纯化的DNA结合蛋白在体外对结合CRE-1和CRE-2元件的蛋白质之间的相互作用进行了表征。我们证明，来自牛脑的CRE-1结合蛋白由三种不同的多肽组成，每种多肽都属于转录因子NF-I家族。对这些蛋白质与CRE-1元件接触的点突变分析表明，NF-I蛋白在与体内点突变分析确定的CRE-1元件（下划线）重叠的序列CTGGCxxxxxxCCT处与诱导型增强子接触。从牛脑纯化的三种NF-I蛋白之一NF-I/Red1与前脑啡肽原/细菌氯霉素乙酰转移酶（CAT）融合基因共转染，可抑制蛋白激酶A或福斯可林刺激的CAT表达。
