Laherty C D, Gierman T M, Dixit V M
Department of Pathology, University of Michigan Medical School, Ann Arbor, 48109.
J Biol Chem. 1989 Jul 5;264(19):11222-7.
Thrombospondin (TSP) is an extracellular matrix glycoprotein whose synthesis and secretion by mesenchymal cells is regulated at the level of gene transcription by platelet-derived growth factor. To examine the transcriptional regulation of the TSP gene at the molecular level, a genomic clone containing the human TSP promoter and flanking sequence was isolated and characterized. A 3.8-kilobase pair (kb) DNA fragment containing the first three exons, the first two introns, and 2.2 kb of 5'-flanking region was sequenced, and the site of transcription initiation was determined by both primer extension and S1 nuclease mapping. Consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, including a TATA box consensus sequence, TTTAAAA, located 24 base pairs upstream from the transcription start site. A chimeric gene was constructed containing the first intron, the first exon, and 2.0 kb of 5'-flanking sequence of the TSP gene fused to the promoterless gene for chloramphenicol acetyltransferase. When transfected into COS-1 or NIH3T3 cells this gene construct was transcribed, indicating the presence of a functional promoter in the TSP sequence. Transient transfection studies using deletion mutants of this TSP-chloramphenicol acetyltransferase construct were performed to locate cis-acting regulatory sequences. The deletion of flanking sequence 5' to position -234 had little or no effect on transcriptional activity, whereas deletion of 5'-flanking sequence extending further in the 3' direction resulted in the gradual loss of transcriptional activity. The removal of the first intron resulted in a 4-fold decrease in transcript levels, indicating the presence of a cis-acting positive element(s) in the first intron of the human TSP gene. This element(s) was further localized to the region between position +576 and position +727.
血小板反应蛋白（TSP）是一种细胞外基质糖蛋白，间充质细胞对其合成和分泌在基因转录水平上受血小板衍生生长因子调控。为了在分子水平上研究TSP基因的转录调控，分离并鉴定了一个包含人TSP启动子及侧翼序列的基因组克隆。对一个3.8千碱基对（kb）的DNA片段进行了测序，该片段包含前三个外显子、前两个内含子以及2.2 kb的5'侧翼区域，并通过引物延伸和S1核酸酶图谱分析确定了转录起始位点。在5'侧翼序列中发现了几个潜在调控元件的共有序列，包括一个TATA盒共有序列TTTAAAA，位于转录起始位点上游24个碱基对处。构建了一个嵌合基因，它包含TSP基因的第一个内含子、第一个外显子以及2.0 kb的5'侧翼序列，并与无启动子的氯霉素乙酰转移酶基因融合。当转染到COS-1或NIH3T3细胞中时，这个基因构建体能够转录，表明TSP序列中存在一个功能性启动子。使用该TSP - 氯霉素乙酰转移酶构建体的缺失突变体进行瞬时转染研究，以定位顺式作用调控序列。删除-234位点上游的侧翼序列对转录活性几乎没有影响，而删除向3'方向进一步延伸的5'侧翼序列则导致转录活性逐渐丧失。去除第一个内含子导致转录水平下降4倍，表明人TSP基因的第一个内含子中存在一个顺式作用阳性元件。该元件进一步定位于+576位点和+727位点之间的区域。
