Kudo S, Fukuda M
La Jolla Cancer Research Foundation, CA 92037.
Eur J Biochem. 1994 Jul 15;223(2):319-27. doi: 10.1111/j.1432-1033.1994.tb18997.x.
Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell-type specific and differentiation-stage-specific manners. Previously we found that the sequence from -53 to -40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483-8489]. A transient-expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin-producing and non-producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel-mobility-shift assays. The nuclear extracts from both leukosialin-producing Jurkat cells and non-producing Hela cells showed a footprint on the 5' flanking region from -58 to -34. Gel-mobility-shift assays revealed that DNA-protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Sp1-binding consensus sequence. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA-protein complex. In addition, the footprint produced by the purified Sp1 transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Sp1 to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophila Schneider cells demonstrated that cotransfection with Sp1 expression plasmid increased the transcriptional activity. These results establish that Sp1 can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non-producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue-specific expression is probably achieved by suppression of the basal transcriptional activity in non-producing cells.
人白细胞涎酸蛋白（CD43）以细胞类型特异性和分化阶段特异性的方式在造血细胞表面表达。此前我们发现，从-53至-40的序列对启动子功能至关重要[工藤，S. & 福田，M.（1991年）《生物化学杂志》266，8483 - 8489]。使用氯霉素乙酰转移酶报告基因进行的瞬时表达分析表明，该启动子可在产生白细胞涎酸蛋白的细胞和不产生白细胞涎酸蛋白的细胞中均赋予高基础转录活性。通过DNA酶I足迹法和凝胶迁移率变动分析确定了与启动子序列相互作用的转录因子。来自产生白细胞涎酸蛋白的Jurkat细胞和不产生白细胞涎酸蛋白的Hela细胞的核提取物在从-58至-34的5'侧翼区域均显示出一个足迹。凝胶迁移率变动分析表明，两种核提取物均形成了DNA - 蛋白质复合物，并且这些复合物的形成被含有Sp1结合共有序列的寡核苷酸所抑制。在此分析中，抗Sp1抗体与核提取物预先孵育会导致DNA - 蛋白质复合物条带出现超迁移。此外，纯化的Sp1转录因子产生的足迹与Jurkat细胞和Hela细胞核提取物产生的足迹相同。突变分析表明，Sp1与突变启动子序列的结合亲和力与这些启动子序列的转录活性平行。在果蝇Schneider细胞中的瞬时表达分析表明，与Sp1表达质粒共转染可增加转录活性。这些结果表明，Sp1可与启动子结合并正向调节白细胞涎酸蛋白基因的表达。即使在不产生白细胞涎酸蛋白的Hela细胞中稳定表达CAT构建体也显示出高转录活性。因此，白细胞涎酸蛋白的表达似乎受独特机制调控，即抑制高基础转录活性而非激活基础转录水平。组织特异性表达可能是通过抑制不产生白细胞涎酸蛋白的细胞中的基础转录活性来实现。
