Activation of the early B-cell-specific mb-1 (Ig-alpha) gene by Pax-5 is dependent on an unmethylated Ets binding site.
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.
Maier
Holly
H
Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, K516B, Denver, CO 80206, USA.
Colbert
Jeff
J
Fitzsimmons
Daniel
D
Clark
Dawn R
DR
Hagman
James
J
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D001854:Bone Marrow Cells / Q000378:metabolism; D051925:CD79 Antigens; D019070:Cell Lineage; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D051757:PAX5 Transcription Factor; D010950:Plasma Cells / Q000378:metabolism; D010954:Plasmacytoma / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D011947:Receptors, Antigen, B-Cell / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013047:Specific Pathogen-Free Organisms; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D051925:CD79 Antigens; C497881:CD79A protein, human; C497882:Cd79a protein, mouse; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; C494042:Ets1 protein, mouse; D046911:Macromolecular Substances; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; C497282:Pax5 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011947:Receptors, Antigen, B-Cell; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors