p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.
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.
Department of Biology, University of Hartford, West Hartford, Connecticut, United States of America.
Robert
R
Lipski
Daniel J
DJ
Lippincott
Brittany C
BC
Durden
Anne R
AR
Kaplan
Hilary E
HE
Keiser
Jung-Ho
JH
Park
Aime A
AA
Levesque
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D019926:Cyclin B / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D019281:Dimerization; D015972:Gene Expression Regulation, Neoplastic; D016158:Genes, p53; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D000077146:Irinotecan; D020218:Response Elements; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
D000972:Antineoplastic Agents, Phytogenic; D003432:Cross-Linking Reagents; D019926:Cyclin B; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D000077146:Irinotecan; D005976:Glutaral; D002166:Camptothecin