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Methyl-CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated.

ABSTRACT: 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.

SUBMITTER: Kudo S

PROVIDER: S-EPMC109134 | biostudies-literature | 1998 Sep

REPOSITORIES: biostudies-literature

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Methyl-CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated.

Kudo S S

Molecular and cellular biology 19980901 9

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 ...[more]

PMID: 9710633

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Project description:Mutations in the gene encoding the MECP2 underlies Rett syndrome, a neurodevelopmental disorder in young females. Although reduced pain sensitivity in Rett syndrome patients and in partial MeCP2 deficient mice had been reported, these previous studies focused predominantly on motor impairments. Therefore, it is still unknown how MeCP2 is involved in these sensory defects. In addition, the human disease manifestations where males with mutations in MECP2 gene normally do not survive and females show typical neurological symptoms only after 18 months of age, is profoundly different in MeCP2-deficient mouse where all animals survived, and males but not females displayed Rett syndrome phenotypes at an early age. Thus, the mecp2-deficient zebrafish serves as an additional animal model to aid in deciphering the role and mechanisms of Mecp2 in neurodevelopment. Here, we used two independent methods of silencing expression of Mecp2 in zebrafish to uncover a novel role of Mecp2 in trigeminal ganglion sensory neurons during the embryonic development. mecp2-null mutation and morpholino-mediated silencing of Mecp2 in the zebrafish embryos resulted in defects in peripheral innervation of trigeminal sensory neurons and consequently affecting the sensory function. These defects were demonstrated to be dependent on the expression of Sema5b and Robo2. The expression of both proteins together could better overcome the defects caused by Mecp2 deficiency as compared to the expression of either Sema5b or Robo2 alone. Sema5b and Robo2 were downregulated upon Mecp2 silencing or in mecp2-null embryos, and Chromatin immunoprecipitation (ChIP) assay using antibody against Mecp2 was able to pull down specific regions of both Sema5b and Robo2 promoters, showing interaction between Mecp2 and the promoters of both genes. In addition, cell-specific expression of Mecp2 can overcome the innervation and sensory response defects in Mecp2 morphants indicating that these MeCP2-mediated defects are cell-autonomous. The sensory deficits caused by Mecp2 deficiency mirror the diminished sensory response observed in Rett syndrome patients. This suggests that zebrafish could be an unconventional but useful model for this disorder manifesting defects that are not easily studied in full using rodent models.

Dataset Information

Methyl-CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated.

Publications

Methyl-CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated.

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