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 wh...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3414442?pdf=render |
| _version_ | 1818927957450686464 |
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| author | Robert Lipski Daniel J Lippincott Brittany C Durden Anne R Kaplan Hilary E Keiser Jung-Ho Park Aime A Levesque |
| author_facet | Robert Lipski Daniel J Lippincott Brittany C Durden Anne R Kaplan Hilary E Keiser Jung-Ho Park Aime A Levesque |
| author_sort | Robert Lipski |
| collection | DOAJ |
| description | 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. |
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| format | Article |
| id | doaj.art-9bb815b69d5f4ab8b85027c93c2a69c0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T03:21:16Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-9bb815b69d5f4ab8b85027c93c2a69c02022-12-21T19:55:13ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0178e4261510.1371/journal.pone.0042615p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.Robert LipskiDaniel J LippincottBrittany C DurdenAnne R KaplanHilary E KeiserJung-Ho ParkAime A LevesqueDNA 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.http://europepmc.org/articles/PMC3414442?pdf=render |
| spellingShingle | Robert Lipski Daniel J Lippincott Brittany C Durden Anne R Kaplan Hilary E Keiser Jung-Ho Park Aime A Levesque p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. PLoS ONE |
| title | p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. |
| title_full | p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. |
| title_fullStr | p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. |
| title_full_unstemmed | p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. |
| title_short | p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription. |
| title_sort | p53 dimers associate with a head to tail response element to repress cyclin b transcription |
| url | http://europepmc.org/articles/PMC3414442?pdf=render |
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