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Title: p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. Author: Lipski R, Lippincott DJ, Durden BC, Kaplan AR, Keiser HE, Park JH, Levesque AA. Journal: PLoS One; 2012; 7(8):e42615. PubMed ID: 22905155. Abstract: DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.[Abstract] [Full Text] [Related] [New Search]