Kudo S
Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan.
Mol Cell Biol. 1998 Sep;18(9):5492-9. doi: 10.1128/MCB.18.9.5492.
Human leukosialin (CD43) is expressed in a cell lineage-specific as well as a differentiation stage-specific fashion. The leukosialin promoter, made up of an Sp1 binding site and a sequence similar to that of an initiator, possesses high transcriptional potential. Previous data have demonstrated that the leukosialin gene is down-regulated in nonproducing cells by DNA methylation. In this paper the repressive mechanism of DNA methylation in expression systems is reported. In vitro DNA methylation with SssI (CpG) methylase of leukosialin-chloramphenicol acetyltransferase (CAT) constructs drastically reduced transcriptional activities in stable transfection systems with the human HeLa and Jurkat cell lines. On the other hand, the transcriptional repression by in vitro methylation was less pronounced in Drosophila melanogaster cells, which lack genomic methylation. In these cells, Sp1 could transactivate equally well both the unmethylated and methylated leukosialin promoter. In order to test whether one of the methyl-CpG-binding proteins, MeCP2, is responsible for transcriptional repression of the leukosialin gene, I isolated the human MeCP2 cDNA (encoding 486 amino acid residues) and expressed it in Drosophila cells. I found that MeCP2 substantially inhibited Sp1-activated transcription when the leukosialin promoter was methylated. The level of repression was directly proportional to the amount of MeCP2 expression vector transfected. Analysis of C-terminal deletion mutants of MeCP2 showed that repressive activity of Sp1 transactivation is localized to the N-terminal region consisting of amino acid residues 1 to 193, which encompass the methyl-binding domain. These results suggest that interference with Sp1 transactivation by MeCP2 is an important factor in the down-regulation of leukosialin gene expression by DNA methylation.
人白细胞唾液酸蛋白（CD43）以细胞谱系特异性以及分化阶段特异性的方式表达。白细胞唾液酸蛋白启动子由一个Sp1结合位点和一个与起始子序列相似的序列组成，具有很高的转录潜能。先前的数据表明，在不产生该蛋白的细胞中，白细胞唾液酸蛋白基因通过DNA甲基化而下调。本文报道了DNA甲基化在表达系统中的抑制机制。用白细胞唾液酸蛋白-氯霉素乙酰转移酶（CAT）构建体的SssI（CpG）甲基转移酶进行体外DNA甲基化，在人HeLa和Jurkat细胞系的稳定转染系统中显著降低了转录活性。另一方面，在缺乏基因组甲基化的果蝇细胞中，体外甲基化引起的转录抑制作用不太明显。在这些细胞中，Sp1对未甲基化和甲基化的白细胞唾液酸蛋白启动子的反式激活作用相同。为了检测甲基化CpG结合蛋白之一MeCP2是否负责白细胞唾液酸蛋白基因的转录抑制，我分离了人MeCP2 cDNA（编码486个氨基酸残基）并在果蝇细胞中表达。我发现当白细胞唾液酸蛋白启动子甲基化时，MeCP2能显著抑制Sp1激活的转录。抑制水平与转染的MeCP2表达载体的量成正比。对MeCP2的C末端缺失突变体的分析表明，Sp1反式激活的抑制活性定位于由氨基酸残基1至193组成的N末端区域，该区域包含甲基结合结构域。这些结果表明，MeCP2对Sp1反式激活的干扰是DNA甲基化下调白细胞唾液酸蛋白基因表达的一个重要因素。
