Liu W, Wilson J E
Department of Biochemistry, Michigan State University, East Lansing 48824-1319, USA.
Arch Biochem Biophys. 1997 Oct 1;346(1):142-50. doi: 10.1006/abbi.1997.0295.
Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.
已确定编码大鼠己糖激酶I型同工酶的基因有多个转录起始位点（White, J.A., Liu, W., and Wilson, J. E., 《生物化学与生物物理学报》335, 161 - 172, 1996）；这些位点聚集在相对于翻译起始密码子（ATG，A为+1）约-460、-300和-100的位置。用含有-3366至-171位置之间基因组序列的荧光素酶报告基因构建体转染PC12细胞和H9c2细胞。启动子活性显著（85%）降低与-742至-516之间序列的缺失有关。在DNA酶I足迹实验中，PC12细胞核提取物中的蛋白质保护了两个区域，称为P1（-552至-529）和P2（-480至-458）框。P2框的突变或缺失对启动子活性没有影响；该区域的保护作用，包括最上游的转录起始位点簇，归因于RNA聚合酶II或相关因子的结合。相反，P1框中的突变或缺失对PC12细胞核提取物中的启动子活性和蛋白质结合有明显的有害影响。维持位于P1框中心的共有Sp1结合位点对于启动子活性和结合都至关重要。P1框上游的第二个Sp1位点（-570）也被证明具有功能重要性，但在足迹实验中未检测到该区域的保护作用，推测这反映了在所使用条件下该位点的亲和力较低。超迁移实验证明Sp1、Sp3和Sp4参与了与P1框区域形成复合物，并表明这些转录因子参与调节与该区域相关的启动子活性。另一系列报告基因构建体，包括-171至-1之间的序列，允许检测-364下游的额外启动子活性。虽然尚未广泛表征，但已经很明显，影响下游启动子活性的顺式元件与在上游启动子区域表达中确定重要的Sp因子不同。
