Methyl-CpG-Binding Protein MeCP2 Represses Sp1-Activated Transcription of the Human Leukosialin Gene When the Promoter Is Methylated

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.

人类白细胞唾液酸蛋白（Human leukosialin，CD43）的表达具有细胞谱系特异性与分化阶段特异性。由Sp1结合位点（Sp1 binding site）及类似起始子（initiator）的序列组成的白细胞唾液酸蛋白启动子，具备较高的转录潜能。既往研究数据显示，不表达该蛋白的细胞中白细胞唾液酸蛋白基因的表达会通过DNA甲基化被下调。本文报道了DNA甲基化在表达系统中的抑制机制。将白细胞唾液酸蛋白-氯霉素乙酰转移酶（chloramphenicol acetyltransferase，CAT）重组载体经SssI（CpG）甲基化酶进行体外甲基化修饰后，在人HeLa细胞与Jurkat细胞系的稳定转染体系中，其转录活性显著降低。与之相反，在缺乏基因组甲基化系统的黑腹果蝇（Drosophila melanogaster）细胞中，体外甲基化所介导的转录抑制效果并不显著；在这类细胞中，Sp1可同等高效地反式激活未甲基化与甲基化的白细胞唾液酸蛋白启动子。为验证甲基化CpG结合蛋白（methyl-CpG-binding proteins）之一的MeCP2是否参与白细胞唾液酸蛋白基因的转录抑制，本研究克隆了人类MeCP2互补DNA（cDNA，编码486个氨基酸残基）并在果蝇细胞中进行表达。实验发现，当白细胞唾液酸蛋白启动子发生甲基化时，MeCP2可显著抑制Sp1介导的转录激活，且抑制水平与转染的MeCP2表达载体剂量呈正相关。对MeCP2的C端缺失突变体（C-terminal deletion mutants）进行分析后可知，其对Sp1反式激活（Sp1 transactivation）的抑制活性定位于包含氨基酸残基1至193的N端区域（N-terminal region），该区域涵盖了甲基结合结构域（methyl-binding domain）。上述结果提示，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重要机制之一。