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 (5'CCGGAG) 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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antigens, CD / genetics*
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B-Lymphocytes / metabolism
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Binding Sites
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Bone Marrow Cells / metabolism
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CD79 Antigens
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Cell Lineage
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CpG Islands
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DNA Methylation
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DNA-Binding Proteins / deficiency
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / physiology*
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Gene Expression Regulation, Neoplastic
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Humans
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Macromolecular Substances
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Mice
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Mice, Inbred BALB C
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Mice, Inbred C57BL
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Mice, Knockout
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PAX5 Transcription Factor
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Plasma Cells / metabolism
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Plasmacytoma / pathology
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Promoter Regions, Genetic / genetics*
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Proto-Oncogene Protein c-ets-1
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-ets
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Receptors, Antigen, B-Cell / genetics*
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Recombinant Fusion Proteins / physiology
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Specific Pathogen-Free Organisms
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Transcription Factors / deficiency
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Transcription Factors / physiology*
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Transcription, Genetic / genetics*
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Transfection
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Tumor Cells, Cultured
Substances
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Antigens, CD
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CD79 Antigens
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CD79A protein, human
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Cd79a protein, mouse
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DNA-Binding Proteins
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ETS1 protein, human
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Ets1 protein, mouse
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Macromolecular Substances
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PAX5 Transcription Factor
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PAX5 protein, human
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Pax5 protein, mouse
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Proto-Oncogene Protein c-ets-1
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-ets
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Receptors, Antigen, B-Cell
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Recombinant Fusion Proteins
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Transcription Factors