Activation of the Early B-Cell-Specific mb-1 (Ig-α) Gene by Pax-5 Is Dependent on an Unmethylated Ets Binding Site

Abstract

Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG methylation does not directly affect binding of the promoter by Pax-5. Instead, methylation of an adjacent CpG interferes with assembly of ternary complexes comprising Pax-5 and Ets proteins. In electrophoretic mobility shift assays, recruitment of Ets-1 is blocked by methylation of the Ets site (5CCGGAG) on the antisense strand. In transfection assays, selective methylation of a single CpG within the Pax-5-dependent Ets site greatly reduces mb-1 promoter activity. Prior demethylation of the endogenous mb-1 promoter is required for its activation by Pax-5 in transduced cells. Although B-lineage cells have only unmethylated mb-1 genes and do not modulate methylation of the mb-1 promoter during development, other tissues feature high percentages of methylated alleles. Together, these studies demonstrate a novel DNA methylation-dependent mechanism for regulating transcriptional activity through the inhibition of DNA-dependent protein-protein interactions.The development of specialized cells from multipotent progenitors is the result of a complicated array of events involving the initiation of new gene expression and silencing of unnecessary or inappropriate genes. Transcriptional regulation of genes occurs at several levels. Tissue-specific and temporally regulated transcription factors activate and/or repress genes that define lineage-and differentiation stage-specific characteristics. However, accessibility of genes to these factors is imparted by chromatin architecture, which is itself controlled through two known mechanisms: cytosine methylation within CpG dinucleotides, and chromatin remodeling initiated by histone acetyltransferases and histone deacetylases.DNA methylation has long been recognized as a contributor to epigenetic regulation of transcription (reviewed in reference 2). In higher eukaryotes, transcriptionally active genes and their promoters are generally hypomethylated, while silenced genes are hypermethylated. Two models have been proposed to account for the effects of cytosine methylation on transcription. The first suggests that methylation at CpG dinucleotides blocks binding of transcription factors to their sites through steric hindrance (7). However, this model is limited to a select set of protein-DNA interactions, as many regulatory target sites of DNA-binding proteins do not contain CpG dinucleotides and are not affected by DNA methylation. In addition, some DNA-binding proteins, such as Sp1, are able to bind DNA regardless of its methylation status (23). The second model suggests that specialized methyl-binding proteins bind methylated DNA and recruit chromatin remodelin...

Transcription of the Caveolin-1 Gene Is Differentially Regulated in Lung Type I Epithelial and Endothelial Cell Lines

“…6b). The higher molecular weight bands seen in ETS-1 and PEA3 likely represent phosphorylated forms of these transcription factors; similar doublets for ETS-1 associated with ETS-1 phosphorylation have been described previously (32,33).…”

“…In contrast, a single 72-kDa immunoreactive band is observed in the MFLM-4 cell line. Based on published data, the higher molecular weight band detected in E10 cells likely represents a phosphorylated product (32,33). c, 50 g/lane of nuclear protein immunoprecipitated for ERM was analyzed by immunoblot assay using a polyclonal anti-ERM antibody.…”

Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5

“…We demonstrated previously that enforced expression of Pax5 is sufficient for activation of endogenous mb-1 gene transcription in the M.10 subclone of 558L M plasmacytoma cells (21). On average, each of these cells possesses 1 hypomethylated mb-1 promoter that is accessible to Pax5 in the absence of EBF.…”

“…To generate cells expressing EBF:ER, we expressed the fusion protein stably in the 558L M subclone M.2 (21). Unlike M.10 cells, M.2 cells are refractory to activation by Pax5 because their mb-1 promoters are uniformly hypermethylated in relatively inaccessible chromatin (21,22).…”

Genomic analyses reveal global functional alterations that promote tumor growth and novel tumor suppressor genes in natural killer-cell malignancies

“…Also, while methylation of a TF-binding site may affect TF binding, there is still some controversy regarding the universality of this effect. [47][48][49][50] The significance of these TF-binding sites requires further investigation. PRDM1 expression was very low in all three cell lines with methylation and treatment of cell lines with DNA methyltransferase inhibitor increases the expression of PRDM1.…”