Abstract
Transcription from the B-myb (MybL2 gene) promoter is strictly cell cycle-regulated by repression mediated through an E2F site during G(0)/early G(1). We report here the characterization of a corepressor site (downstream repression site (DRS)) required for this activity that is closely linked to the E2F site. Systematic mutagenesis of the DRS enabled a consensus to be derived, and it is notable that this sequence is compatible with cell cycle gene homology region sequences associated with cell cycle-dependent elements in the cyclin A, cdc2, and CDC25C promoters. The B-myb promoter is inappropriately active during G(0) in mouse embryo fibroblasts lacking the p107 and p130 pocket proteins, and we show that the ability of transfected p107 and p130 to re-impose repression on the promoter is dependent on the DRS. In contrast, transfected Rb was unable to repress the B-myb promoter. Consistent with the notion that Rb.E2F complexes are unable to bind the B-myb promoter E2F site in vivo, footprinting showed that this site is unoccupied in cells lacking p107 and p130. Chromatin immunoprecipitation assays showed a requirement for the DRS in recruiting p107 and p130 complexes to the B-myb promoter, indicating that in vivo the DRS governs the occupancy of the adjacent E2F site by transcriptional repressors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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Animals
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Cell Cycle / physiology
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cells, Cultured
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DNA-Binding Proteins / genetics*
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DNA-Binding Proteins / metabolism
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E2F Transcription Factors
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Embryo, Mammalian / physiology
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Fibroblasts / physiology
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Genes, Reporter
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Humans
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Mice
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Mice, Knockout
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Mutation
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Oncogene Proteins / genetics
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Oncogene Proteins / metabolism
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Promoter Regions, Genetic*
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Proteins*
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Response Elements
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Retinoblastoma Protein / genetics
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Retinoblastoma Protein / metabolism
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Retinoblastoma-Like Protein p107
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Retinoblastoma-Like Protein p130
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Trans-Activators / genetics*
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Trans-Activators / metabolism
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcription, Genetic*
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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E2F Transcription Factors
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MYBL2 protein, human
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Mybl2 protein, mouse
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Nuclear Proteins
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Oncogene Proteins
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Phosphoproteins
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Proteins
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RBL1 protein, human
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RBL2 protein, human
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Rbl1 protein, mouse
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Rbl2 protein, mouse
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Repressor Proteins
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Retinoblastoma Protein
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Retinoblastoma-Like Protein p107
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Retinoblastoma-Like Protein p130
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Trans-Activators
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Transcription Factors