Chen X, Wright K L, Berkowitz E A, Azizkhan J C, Ting J P, Lee D C
Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill 27599-7295.
Oncogene. 1994 Nov;9(11):3179-87.
Transcription from the rat TGF alpha promoter initiates at two predominant sites (-188 and -58) in a G+C-rich region that does not contain TATA or CAAT motifs. Previous studies using transfected reporter constructs implicated the transcription factor Sp1 in active expression from the promoter, particularly from the -58 site (Chen et al., 1992; Shin et al., 1992). In the present report we have examined the functionality of two adjacent clusters of Sp1-like recognition sites that are located in the upstream portion of the promoter from -300 to -273. A double-stranded oligonucleotide, which spanned this region and contained the putative Sp1 elements, demonstrated similar gel-mobility shifts in the presence of both crude HeLa cells nuclear extract and pure Sp1 protein. Mutations that simultaneously altered several of the overlapping Sp1 elements significantly reduced the gel-mobility shift activity of this oligonucleotide probe and, when introduced into the promoter templates, inhibited transcription in vitro from the proximal -188 start site. To confirm the binding of protein to these sites in cells, we carried out an in vivo genomic footprinting analysis of this portion of the TGF alpha promoter in normal and transformed rat liver epithelial cell lines that express the endogenous gene at varying levels. This analysis revealed clear evidence of protein/DNA interaction at Sp1-like sites in the -300 and -273 region in cells actively expressing the gene but not in a normal, parental cell line that expressed very low levels of TGF alpha mRNA. Collectively, these results corroborate the functional importance of Sp1 binding elements in the -300 to -273 region, and together with previous findings, indicate that two clusters of Sp1 binding sites respectively determine levels of transcription from the -188 and -58 start sites. Our additional finding that Sp1 mRNA and protein were present at similar levels in normal and transformed cells that expressed the endogenous TGF alpha gene at markedly different levels, suggests that the activity of the TGF alpha promoter could be regulated via the accessibility of Sp1 protein.
大鼠转化生长因子α（TGFα）启动子的转录起始于富含G+C区域的两个主要位点（-188和-58），该区域不含TATA或CAAT基序。先前使用转染报告基因构建体的研究表明，转录因子Sp1参与启动子的活性表达，特别是来自-58位点的表达（Chen等人，1992年；Shin等人，1992年）。在本报告中，我们研究了位于启动子上游-300至-273区域的两个相邻的Sp1样识别位点簇的功能。一个跨越该区域并包含假定Sp1元件的双链寡核苷酸，在粗制的HeLa细胞核提取物和纯Sp1蛋白存在下表现出相似的凝胶迁移率变化。同时改变几个重叠Sp1元件的突变显著降低了该寡核苷酸探针的凝胶迁移率变化活性，并且当引入启动子模板时，抑制了来自近端-188起始位点的体外转录。为了证实蛋白质在细胞中与这些位点的结合，我们对正常和转化的大鼠肝上皮细胞系中TGFα启动子的这一部分进行了体内基因组足迹分析，这些细胞系以不同水平表达内源性基因。该分析揭示了在积极表达该基因的细胞中，-300和-273区域的Sp1样位点存在蛋白质/DNA相互作用的明确证据，但在表达极低水平TGFα mRNA的正常亲本细胞系中则没有。总体而言，这些结果证实了Sp1结合元件在-300至-273区域的功能重要性，并且与先前的发现一起表明，两个Sp1结合位点簇分别决定了从-188和-58起始位点的转录水平。我们的另外一项发现是，在表达内源性TGFα基因水平明显不同的正常和转化细胞中，Sp1 mRNA和蛋白质的水平相似，这表明TGFα启动子的活性可能通过Sp1蛋白的可及性来调节。
