Parker M I, Smith A A, Mundell K, Collins M, Boast S, Ramirez F
MRC/UCT Research Unit for the Cell Biology of Atherosclerosis, Department of Medical Biochemistry, University of Cape Town Medical School, Observatory, South Africa.
Nucleic Acids Res. 1992 Nov 11;20(21):5825-30. doi: 10.1093/nar/20.21.5825.
The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.
本研究的目的是确定α2(1)前胶原基因表达是否受到正向或负向反式作用DNA结合蛋白的调节。先前的研究表明，SV40转化的WI-38成纤维细胞（SVWI-38）克隆不产生任何α2(1)前胶原mRNA（Parker等人，(1989)，《生物化学杂志》264，7147 - 7152）。为了阐明导致这种失活的机制，我们检测了转染的野生型COL1A2启动子在SVWI-38细胞中的活性。一组5'启动子缺失片段与氯霉素乙酰转移酶（CAT）基因相连，并转染到SVWI-38和其他表达I型胶原的细胞系中。所得的CAT分析证实了几个上游区域对启动子活性的重要性，并记录了SVWI-38细胞系中外源COL1A2启动子转录活性的降低。用过量的COL1A2启动子DNA片段和恒定数量的COL1A2/CAT构建体进行的竞争实验显示，过量的COL1A2片段与SVWI-38细胞中的CAT活性之间存在线性关系，这表明存在一种可滴定的负效应子。电泳迁移率变动分析显示存在一种特异性DNA-蛋白质复合物，该复合物存在于SVWI-38细胞中，而在对照成纤维细胞中几乎不存在。甲基化干扰分析确定了该因子相对于转录起始位点在核苷酸-80至-72之间的结合区域。因此，所提供的数据有力地证明了存在一种负反式作用因子，它可能在SVWI-38成纤维细胞中COL1A2表达的抑制中起作用。
