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.
Kudo
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Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan. kudos@pref.iph.hokkaido.jp
D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002868:Chromosomal Proteins, Non-Histone; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D051917:Leukosialin; D051783:Methyl-CpG-Binding Protein 2; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D015703:Antigens, CD; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D051917:Leukosialin; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C465014:SPN protein, human; D012795:Sialoglycoproteins; D016329:Sp1 Transcription Factor; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D015500:Chloramphenicol O-Acetyltransferase