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1
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Murphy MR, Ramadei A, Doymaz A, Varriano S, Natelson D, Yu A, Aktas S, Mazzeo M, Mazzeo M, Zakusilo G, Kleiman F. Long non-coding RNA generated from CDKN1A gene by alternative polyadenylation regulates p21 expression during DNA damage response. Nucleic Acids Res 2023; 51:11911-11926. [PMID: 37870464 PMCID: PMC10681730 DOI: 10.1093/nar/gkad899] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 09/21/2023] [Accepted: 10/12/2023] [Indexed: 10/24/2023] Open
Abstract
Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during the DNA damage response (DDR). Here, we show that a DDR-activated APA event occurs in the first intron of CDKN1A, inducing an alternate last exon-containing lncRNA. We named this lncRNA SPUD (Selective Polyadenylation Upon DNA Damage). SPUD localizes to polysomes in the cytoplasm and is detectable as multiple isoforms in available high-throughput studies. SPUD has low abundance compared to the CDKN1A full-length isoform under non-stress conditions, and SPUD is induced in cancer and normal cells under a variety of DNA damaging conditions in part through p53. The RNA binding protein HuR binds to and promotes the stability of SPUD precursor RNA. SPUD induction increases p21 protein, but not mRNA levels, affecting p21 functions in cell-cycle, CDK2 expression and cell growth. Like CDKN1A full-length isoform, SPUD can bind two competitive p21 translational regulators, the inhibitor calreticulin and the activator CUGBP1; SPUD alters their association with CDKN1A full-length in a DDR-dependent manner, promoting CDKN1A translation. Together, these results show a new regulatory mechanism by which a lncRNA controls p21 expression post-transcriptionally, highlighting lncRNA relevance in DDR progression and cell-cycle.
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Affiliation(s)
- Michael R Murphy
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
- Biology Program, The Graduate Center, The City University of New York, New York, NY 10016, USA
| | - Anthony Ramadei
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
- Biology Program, The Graduate Center, The City University of New York, New York, NY 10016, USA
| | - Ahmet Doymaz
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
| | - Sophia Varriano
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
- Biology Program, The Graduate Center, The City University of New York, New York, NY 10016, USA
| | - Devorah M Natelson
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
- Biology Program, The Graduate Center, The City University of New York, New York, NY 10016, USA
| | - Amy Yu
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
| | - Sera Aktas
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
| | - Marie Mazzeo
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
| | - Michael Mazzeo
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
| | - George Zakusilo
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
| | - Frida E Kleiman
- Chemistry Department, Hunter College, The City University of New York, New York, NY 10021, USA
- Biology Program, The Graduate Center, The City University of New York, New York, NY 10016, USA
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2
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Walter M, Mayr F, Hanna BMF, Cookson V, Mortusewicz O, Helleday T, Herr P. NUDT22 promotes cancer growth through pyrimidine salvage. Oncogene 2023; 42:1282-1293. [PMID: 36871087 PMCID: PMC10101856 DOI: 10.1038/s41388-023-02643-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2022] [Revised: 02/15/2023] [Accepted: 02/17/2023] [Indexed: 03/06/2023]
Abstract
The NUDIX hydrolase NUDT22 converts UDP-glucose into glucose-1-phosphate and the pyrimidine nucleotide uridine monophosphate but a biological significance for this biochemical reaction has not yet been established. Glucose-1-phosphate is an important metabolite for energy and biomass production through glycolysis and nucleotides required for DNA replication are produced through energetically expensive de novo or energy-efficient salvage pathways. Here, we describe p53-regulated pyrimidine salvage through NUDT22-dependent hydrolysis of UDP-glucose to maintain cancer cell growth and to prevent replication stress. NUDT22 expression is consistently elevated in cancer tissues and high NUDT22 expression correlates with worse survival outcomes in patients indicating an increased dependency of cancer cells to NUDT22. Furthermore, we show that NUDT22 transcription is induced after inhibition of glycolysis, MYC-mediated oncogenic stress, and DNA damage directly through p53. NUDT22-deficient cancer cells suffer from growth retardation, S-phase delay, and slower DNA replication fork speed. Uridine supplementation rescues replication fork progression and alleviates replication stress and DNA damage. Conversely, NUDT22 deficiency sensitizes cells to de novo pyrimidine synthesis inhibition in vitro and reduces cancer growth in vivo. In conclusion, NUDT22 maintains pyrimidine supply in cancer cells and depletion of NUDT22 leads to genome instability. Targeting NUDT22 therefore has high potential for therapeutic applications in cancer therapy.
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Affiliation(s)
- Melanie Walter
- Department of Oncology and Metabolism, University of Sheffield, Sheffield, S10 2RX, UK
| | - Florian Mayr
- Science for Life Laboratory, Department of Oncology and Pathology, Karolinska Institute, 171 76, Stockholm, Sweden
| | - Bishoy M F Hanna
- Science for Life Laboratory, Department of Oncology and Pathology, Karolinska Institute, 171 76, Stockholm, Sweden
| | - Victoria Cookson
- Department of Oncology and Metabolism, University of Sheffield, Sheffield, S10 2RX, UK
| | - Oliver Mortusewicz
- Science for Life Laboratory, Department of Oncology and Pathology, Karolinska Institute, 171 76, Stockholm, Sweden
| | - Thomas Helleday
- Science for Life Laboratory, Department of Oncology and Pathology, Karolinska Institute, 171 76, Stockholm, Sweden
| | - Patrick Herr
- Department of Oncology and Metabolism, University of Sheffield, Sheffield, S10 2RX, UK.
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3
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Murphy MR, Ramadei A, Doymaz A, Varriano S, Natelson D, Yu A, Aktas S, Mazzeo M, Mazzeo M, Zakusilo G, Kleiman FE. Long Non-Coding RNA Generated from CDKN1A Gene by Alternative Polyadenylation Regulates p21 Expression during DNA Damage Response. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.01.10.523318. [PMID: 36711808 PMCID: PMC9882041 DOI: 10.1101/2023.01.10.523318] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during the DNA damage response (DDR). Here, we show that a DNA damage activated APA event occurs in the first intron of CDKN1A , inducing an alternate last exon (ALE)-containing lncRNA. We named this lncRNA SPUD (Selective Polyadenylation Upon Damage). SPUD localizes to polysomes in the cytoplasm and is detectable as multiple isoforms in available high throughput studies. SPUD has low abundance compared to the CDKN1A full-length isoform and is induced in cancer and normal cells under a variety of DNA damaging conditions in part through p53 transcriptional activation. RNA binding protein (RBP) HuR and the transcriptional repressor CTCF regulate SPUD levels. SPUD induction increases p21 protein, but not CDKN1A full-length levels, affecting p21 functions in cell-cycle, CDK2 expression, and cell viability. Like CDKN1A full-length isoform, SPUD can bind two competitive p21 translational regulators, the inhibitor calreticulin and the activator CUGBP1; SPUD can change their association with CDKN1A full-length in a DDR-dependent manner. Together, these results show a new regulatory mechanism by which a lncRNA controls p21 expression post-transcriptionally, highlighting lncRNA relevance in DDR progression and cellcycle.
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4
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Pan Y, Zong Q, Li G, Wu Z, Du T, Huang Z, Zhang Y, Ma K. Nuclear localization of alpha-synuclein affects the cognitive and motor behavior of mice by inducing DNA damage and abnormal cell cycle of hippocampal neurons. Front Mol Neurosci 2022; 15:1015881. [PMID: 36438187 PMCID: PMC9684191 DOI: 10.3389/fnmol.2022.1015881] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Accepted: 10/24/2022] [Indexed: 01/21/2024] Open
Abstract
Nuclear accumulation of alpha-synuclein (α-syn) in neurons can promote neurotoxicity, which is considered the key factor in the pathogenesis of synucleinopathy. The damage to hippocampus neurons driven by α-syn pathology is also the potential cause of memory impairment in Parkinson's disease (PD) patients. In this study, we examined the role of α-syn nuclear translocation in the cognition and motor ability of mice by overexpressing α-syn in cell nuclei in the hippocampus. The results showed that the overexpression of α-syn in nuclei was able to cause significant pathological accumulation of α-syn in the hippocampus, and quickly lead to memory and motor impairments in mice. It might be that nuclear overexpression of α-syn may cause DNA damage of hippocampal neurons, thereby leading to activation and abnormal blocking of cell cycle, and further inducing apoptosis of hippocampal neurons and inflammatory reaction. Meanwhile, the inflammatory reaction further aggravated DNA damage and formed a vicious circle. Therefore, the excessive nuclear translocation of α-syn in hippocampal neurons may be one of the main reasons for cognitive decline in mice.
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Affiliation(s)
| | | | | | | | | | - Zhangqiong Huang
- Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China
| | - Ying Zhang
- Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China
| | - Kaili Ma
- Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China
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5
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Xie L, Wang Y, Li Q, Ji X, Tu Y, Du S, Lou H, Zeng X, Zhu L, Zhang J, Zhu M. The HIF-1α/p53/miRNA-34a/Klotho axis in retinal pigment epithelial cells promotes subretinal fibrosis and exacerbates choroidal neovascularization. J Cell Mol Med 2021; 25:1700-1711. [PMID: 33438362 PMCID: PMC7875902 DOI: 10.1111/jcmm.16272] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Revised: 12/07/2020] [Accepted: 12/28/2020] [Indexed: 12/19/2022] Open
Abstract
Wet age‐related macular degeneration (wAMD), characterized by choroidal neovascularization (CNV), is a leading cause of irreversible vision loss among elderly people in developed nations. Subretinal fibrosis, mediated by epithelial‐mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells, leads to unsuccessful anti‐vascular endothelial growth factor (VEGF) agent treatments in CNV patients. Under hypoxic conditions, hypoxia‐inducible factor‐1α (HIF‐1α) increases the stability and activation of p53, which activates microRNA‐34a (miRNA‐34a) transcription to promote fibrosis. Additionally, Klotho is a target gene of miRNA‐34a that inhibits fibrosis. This study aimed to explore the role of the HIF‐1α/p53/miRNA‐34a/Klotho axis in subretinal fibrosis and CNV. Hypoxia‐induced HIF‐1α promoted p53 stability, phosphorylation and nuclear translocation in ARPE‐19 cells (a human RPE cell line). HIF‐1α‐dependent p53 activation up‐regulated miRNA‐34a expression in ARPE‐19 cells following hypoxia. Moreover, hypoxia‐induced p53‐dependent miRNA‐34a inhibited the expression of Klotho in ARPE‐19 cells. Additionally, the HIF‐1α/p53/miRNA‐34a/Klotho axis facilitated hypoxia‐induced EMT in ARPE‐19 cells. In vivo, blockade of the HIF‐1α/p53/miRNA‐34a/Klotho axis alleviated the formation of mouse laser‐induced CNV and subretinal fibrosis. In short, the HIF‐1α/p53/miRNA‐34a/Klotho axis in RPE cells promoted subretinal fibrosis, thus aggravating the formation of CNV.
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Affiliation(s)
- Laiqing Xie
- Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Ying Wang
- Department of Ophthalmology, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China.,Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, China
| | - Quan Li
- Center of Stomatology, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Xiaoyan Ji
- Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Yuanyuan Tu
- Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, China
| | - Shu Du
- Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, China
| | - Hui Lou
- Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Xinwei Zeng
- Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Linling Zhu
- Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, China
| | - Ji Zhang
- Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Manhui Zhu
- Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, China
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Mansilla SF, De La Vega MB, Calzetta NL, Siri SO, Gottifredi V. CDK-Independent and PCNA-Dependent Functions of p21 in DNA Replication. Genes (Basel) 2020; 11:genes11060593. [PMID: 32481484 PMCID: PMC7349641 DOI: 10.3390/genes11060593] [Citation(s) in RCA: 73] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Revised: 05/13/2020] [Accepted: 05/15/2020] [Indexed: 12/12/2022] Open
Abstract
p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.
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7
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Sheng C, Mendler IH, Rieke S, Snyder P, Jentsch M, Friedrich D, Drossel B, Loewer A. PCNA-Mediated Degradation of p21 Coordinates the DNA Damage Response and Cell Cycle Regulation in Individual Cells. Cell Rep 2019; 27:48-58.e7. [DOI: 10.1016/j.celrep.2019.03.031] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2018] [Revised: 01/03/2019] [Accepted: 03/08/2019] [Indexed: 12/29/2022] Open
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8
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di Gennaro A, Damiano V, Brisotto G, Armellin M, Perin T, Zucchetto A, Guardascione M, Spaink HP, Doglioni C, Snaar-Jagalska BE, Santarosa M, Maestro R. A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness. Cell Death Differ 2018; 25:2165-2180. [PMID: 29666469 PMCID: PMC6262018 DOI: 10.1038/s41418-018-0103-x] [Citation(s) in RCA: 77] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2017] [Revised: 02/20/2018] [Accepted: 03/09/2018] [Indexed: 12/16/2022] Open
Abstract
Inactivation of p53 contributes significantly to the dismal prognosis of breast tumors, most notably triple-negative breast cancers (TNBCs). How the relief from p53 tumor suppressive functions results in tumor cell aggressive behavior is only partially elucidated. In an attempt to shed light on the implication of microRNAs in this context, we discovered a new signaling axis involving p53, miR-30a and ZEB2. By an in silico approach we identified miR-30a as a putative p53 target and observed that in breast tumors reduced miR-30a expression correlated with p53 inactivation, lymph node positivity and poor prognosis. We demonstrate that p53 binds the MIR30A promoter and induces the transcription of both miRNA strands 5p and 3p. Both miR-30a-5p and -3p showed the capacity of targeting ZEB2, a transcription factor involved in epithelial–mesenchymal transition (EMT), tumor cell migration and drug resistance. Intriguingly, we found that p53 does restrain ZEB2 expression via miR-30a. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and distal spreading and impinges upon miR-200c expression. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds support to the notion that miRNAs represent key elements of the complex network whereby p53 inactivation affects TNBC clinical behavior.
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Affiliation(s)
- Alessandra di Gennaro
- Oncogenetics and Functional Oncogenomics Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Valentina Damiano
- Oncogenetics and Functional Oncogenomics Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Giulia Brisotto
- Oncogenetics and Functional Oncogenomics Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Michela Armellin
- Oncogenetics and Functional Oncogenomics Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Tiziana Perin
- Pathology Unit, CRO Aviano National Cancer Institute, Aviano (PN), via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Antonella Zucchetto
- Unit of Cancer Epidemiology, CRO Aviano National Cancer Institute, Aviano (PN) via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Michela Guardascione
- Medical Oncology Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy
| | - Herman P Spaink
- Molecular Cell Biology Department, Institute of Biology, Leiden University, Leiden, 2333CC, The Netherlands
| | - Claudio Doglioni
- Ateneo Vita-Salute, Department of Pathology, IRCCS Scientific Institute San Raffaele, Milan, 20132, Italy
| | - B Ewa Snaar-Jagalska
- Molecular Cell Biology Department, Institute of Biology, Leiden University, Leiden, 2333CC, The Netherlands
| | - Manuela Santarosa
- Oncogenetics and Functional Oncogenomics Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy.
| | - Roberta Maestro
- Oncogenetics and Functional Oncogenomics Unit, CRO Aviano National Cancer Institute, via F. Gallini 2, Aviano, 33081, PN, Italy.
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9
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Klusmann I, Rodewald S, Müller L, Friedrich M, Wienken M, Li Y, Schulz-Heddergott R, Dobbelstein M. p53 Activity Results in DNA Replication Fork Processivity. Cell Rep 2017; 17:1845-1857. [PMID: 27829155 DOI: 10.1016/j.celrep.2016.10.036] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Revised: 09/03/2016] [Accepted: 10/12/2016] [Indexed: 12/18/2022] Open
Abstract
p53 induces cell death upon DNA damage, but this may not confer all of its tumor suppressor activity. We report that p53 activation enhances the processivity of DNA replication, as monitored by multi-label fiber assays, whereas removal of p53 reduces fork progression. This is observed in tumor-derived U2OS cells but also in murine embryonic fibroblasts with heterozygous or homozygous p53 deletion and in freshly isolated thymocytes from mice with differential p53 status. Mdm2, a p53-inducible gene product, similarly supports DNA replication even in p53-deficient cells, suggesting that sustained Mdm2-expression is at least one of the mechanisms allowing p53 to prevent replicative stress. Thus, p53 helps to protect the genome during S phase, by preventing the occurrence of stalled or collapsed replication forks. These results expand p53's tumor-suppressive functions, adding to the ex-post model (elimination of damaged cells) an ex-ante activity; i.e., the prevention of DNA damage during replication.
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Affiliation(s)
- Ina Klusmann
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Sabrina Rodewald
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Leonie Müller
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Mascha Friedrich
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Magdalena Wienken
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Yizhu Li
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Ramona Schulz-Heddergott
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany
| | - Matthias Dobbelstein
- Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany.
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Chillemi G, Kehrloesser S, Bernassola F, Desideri A, Dötsch V, Levine AJ, Melino G. Structural Evolution and Dynamics of the p53 Proteins. Cold Spring Harb Perspect Med 2017; 7:cshperspect.a028308. [PMID: 27091942 DOI: 10.1101/cshperspect.a028308] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The family of the p53 tumor suppressive transcription factors includes p73 and p63 in addition to p53 itself. Given the high degree of amino-acid-sequence homology and structural organization shared by the p53 family members, they display some common features (i.e., induction of cell death, cell-cycle arrest, senescence, and metabolic regulation in response to cellular stress) as well as several distinct properties. Here, we describe the structural evolution of the family members with recent advances on the molecular dynamic studies of p53 itself. A crucial role of the carboxy-terminal domain in regulating the properties of the DNA-binding domain (DBD) supports an induced-fit mechanism, in which the binding of p53 on individual promoters is preferentially regulated by the KOFF over KON.
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Affiliation(s)
- Giovanni Chillemi
- CINECA, SCAI-SuperComputing Applications and Innovation Department, Rome 00185, Italy
| | - Sebastian Kehrloesser
- Institute of Biophysical Chemistry, Goethe University, 60438 Frankfurt am Main, Germany
| | - Francesca Bernassola
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata," 00133 Rome, Italy
| | | | - Volker Dötsch
- Institute of Biophysical Chemistry, Goethe University, 60438 Frankfurt am Main, Germany
| | - Arnold J Levine
- Institute for Advanced Study, Princeton, New Jersey 08540.,Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey 08903
| | - Gerry Melino
- Medical Research Council, Toxicology Unit, Leicester University, Leicester LE1 9HN, United Kingdom
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11
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Xiao H, Tong R, Yang B, Lv Z, Du C, Peng C, Ding C, Cheng S, Zhou L, Xie H, Wu J, Zheng S. TAZ regulates cell proliferation and sensitivity to vitamin D3 in intrahepatic cholangiocarcinoma. Cancer Lett 2016; 381:370-379. [PMID: 27554639 DOI: 10.1016/j.canlet.2016.08.013] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2016] [Revised: 08/12/2016] [Accepted: 08/15/2016] [Indexed: 02/05/2023]
Abstract
The transcriptional coactivator with PDZ binding motif (TAZ) is reported as one of the nuclear effectors of Hippo-related pathways. TAZ is found overexpressed in many primary tumors and could regulate many biological processes. However, little is known about the role of TAZ in Intrahepatic Cholangiocarcinoma (ICC). In this study, we found that TAZ is expressed more in ICC tissues than in peritumoral tissue, and a robust expression of TAZ is correlated with a lower overall survival rate of ICC patients after hepatectomy. TAZ knockdown results in an increase in cell apoptosis, a promotion of cell-cycle arrest and a decrease in tumor size and weight in vivo through an increased expression of p53. Vitamin D3 can also inhibit cell proliferation by promoting p53 expression in ICC cells. A reduction in TAZ can also enhance the sensitivity of tumor cells to vitamin D by regulating the p53/CYP24A1 pathway. In conclusion, TAZ is associated with the proliferation and drug-resistance of ICC cells, and could be a novel therapeutic target for the treatment of ICC.
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Affiliation(s)
- Heng Xiao
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China
| | - Rongliang Tong
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China
| | - Beng Yang
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China
| | - Zhen Lv
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China
| | - Chengli Du
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China
| | - Chuanhui Peng
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China
| | - Chaofeng Ding
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China
| | - Shaobing Cheng
- The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China
| | - Lin Zhou
- Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China
| | - Haiyang Xie
- Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China
| | - Jian Wu
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China.
| | - Shusen Zheng
- Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou, China.
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12
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Beckerman R, Yoh K, Mattia-Sansobrino M, Zupnick A, Laptenko O, Karni-Schmidt O, Ahn J, Byeon IJ, Keezer S, Prives C. Lysines in the tetramerization domain of p53 selectively modulate G1 arrest. Cell Cycle 2016; 15:1425-38. [PMID: 27210019 DOI: 10.1080/15384101.2016.1170270] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest.
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Affiliation(s)
| | - Kathryn Yoh
- a Department of Biological Sciences , Columbia University , New York , NY , USA
| | | | | | - Oleg Laptenko
- a Department of Biological Sciences , Columbia University , New York , NY , USA
| | - Orit Karni-Schmidt
- a Department of Biological Sciences , Columbia University , New York , NY , USA
| | - Jinwoo Ahn
- b Department of Structural Biology , University of Pittsburgh , Pittsburgh , PA , USA
| | - In-Ja Byeon
- b Department of Structural Biology , University of Pittsburgh , Pittsburgh , PA , USA
| | - Susan Keezer
- c Cell Signaling Technology, Inc. , Danvers , MA , USA
| | - Carol Prives
- a Department of Biological Sciences , Columbia University , New York , NY , USA
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13
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Chen Y, Pan K, Wang P, Cao Z, Wang W, Wang S, Hu N, Xue J, Li H, Jiang W, Li G, Zhang X. HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis. J Biol Chem 2016; 291:12688-12705. [PMID: 27129219 PMCID: PMC4933444 DOI: 10.1074/jbc.m116.714147] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2016] [Indexed: 01/09/2023] Open
Abstract
The activity of the CDK inhibitor p21 is associated with diverse biological activities, including cell proliferation, senescence, and tumorigenesis. However, the mechanisms governing transcription of p21 need to be extensively studied. In this study, we demonstrate that the high-mobility group box-containing protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/β-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/β-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/β-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state.
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Affiliation(s)
- Yifan Chen
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Kewu Pan
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Pingzhang Wang
- the Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
| | - Zhengyi Cao
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Weibin Wang
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Shuya Wang
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Ningguang Hu
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Junhui Xue
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Hui Li
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Wei Jiang
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Gang Li
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and
| | - Xiaowei Zhang
- From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Peking University Health Science Center, Beijing 100191 and.
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14
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Rosso M, Polotskaia A, Bargonetti J. Homozygous mdm2 SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death. Oncotarget 2015; 6:34573-91. [PMID: 26416444 PMCID: PMC4741474 DOI: 10.18632/oncotarget.5312] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Accepted: 09/07/2015] [Indexed: 11/25/2022] Open
Abstract
A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma. Surprisingly, following etoposide treatment transcription initiation occurred at the compromised target genes in MANCA and A875 cells similar to the T/T ML-1 cell line. In all cell lines tested there was equally robust recruitment of total and initiated RNA polymerase II (Pol II). We found that knockdown of MDM2 in G/G cells moderately increased expression of subsets of p53 target genes without increasing p53 stability. Importantly, etoposide and actinomycin D treatments increased histone H3K36 trimethylation in T/T, but not G/G cells, suggesting a G/G correlated inhibition of transcription elongation. We therefore tested a chemotherapeutic agent (8-amino-adenosine) that induces p53-independent cell death for higher clinically relevant cytotoxicity. We demonstrated that T/T and G/G mdm2 SNP309 cells were equally sensitive to 8-amino-adenosine induced cell death. In conclusion for cancer cells overexpressing MDM2, targeting MDM2 may be less effective than inducing p53-independent cell death.
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Affiliation(s)
- Melissa Rosso
- The Department of Biological Sciences Hunter College at The Belfer Research Building and The Graduate Center Biology PhD Program, CUNY, New York, NY 10021, USA
| | - Alla Polotskaia
- The Department of Biological Sciences Hunter College at The Belfer Research Building and The Graduate Center Biology PhD Program, CUNY, New York, NY 10021, USA
| | - Jill Bargonetti
- The Department of Biological Sciences Hunter College at The Belfer Research Building and The Graduate Center Biology PhD Program, CUNY, New York, NY 10021, USA
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15
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Molina B, Marchetti F, Gómez L, Ramos S, Torres L, Ortiz R, Altamirano-Lozano M, Carnevale A, Frias S. Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2015; 56:457-467. [PMID: 25663157 DOI: 10.1002/em.21938] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 01/05/2015] [Indexed: 06/04/2023]
Abstract
Fanconi's anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin-C (MMC) on FA patient-derived lymphoblastoid cell lines from groups FA-A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC-induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well-known role in replication fork stalling during S-phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457-467, 2015. © 2015 Wiley Periodicals, Inc.
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Affiliation(s)
- Bertha Molina
- Laboratorio de Citogenética, Instituto Nacional de Pediatría, México
| | | | - Laura Gómez
- Laboratorio de Citogenética, Instituto Nacional de Pediatría, México
| | - Sandra Ramos
- Laboratorio de Citogenética, Instituto Nacional de Pediatría, México
| | - Leda Torres
- Laboratorio de Citogenética, Instituto Nacional de Pediatría, México
| | - Rocio Ortiz
- Laboratorio de Citometría de Flujo, Universidad Autónoma Metropolitana, Iztapalapa, Mexico
| | | | - Alessandra Carnevale
- Subdirección de Genómica Poblacional, Instituto Nacional de Medicina Genómica, México
| | - Sara Frias
- Laboratorio de Citogenética, Instituto Nacional de Pediatría, México
- Departamento de Medicina, Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, México
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16
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FURUKAWA K. Challenge to the suppression of tumor growth by the β4-galactosyltransferase genes. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2015; 91:1-16. [PMID: 25743061 PMCID: PMC4405391 DOI: 10.2183/pjab.91.1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Accepted: 11/14/2014] [Indexed: 06/04/2023]
Abstract
It has been well established that structural changes in glycans attached to proteins and lipids are associated with malignant transformation of cells. We focused on galactose residues among the sugars since they are involved in the galectin-mediated biology, and many carbohydrate antigens are frequently expressed on this sugar. We found changes in the expression of the β4-galactosyltransferase (β4GalT) 2 and 5 genes in cancer cells: decreased expression of the β4GalT2 gene and increased expression of the β4GalT5 gene. The growth of mouse melanoma cells showing enhanced expression of the β4GalT2 gene or reduced expression of the β4GalT5 gene is inhibited remarkably in syngeneic mice. Tumor growth inhibition is probably caused by the induction of apoptosis, inhibition of angiogenesis, and/or reduced MAPK signals. Direct transduction of human β4GalT2 cDNA together with the adenovirus vector into human hepatocellular carcinoma cells grown in SCID mice results in marked growth retardation of the tumors. β4GalT gene-transfer appears to be a potential tool for cancer therapy.
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Affiliation(s)
- Kiyoshi FURUKAWA
- Laboratory of Glycobiology, Graduate School of Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan
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17
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Pan LZ, Ahn DG, Sharif T, Clements D, Gujar SA, Lee PWK. The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target. Cell Cycle 2014; 13:1041-8. [PMID: 24552824 DOI: 10.4161/cc.28128] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
NAD(+) metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD(+) metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD(+) biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD(+) synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD(+) levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD(+) synthesis is congruent with p53's emerging role as a key regulator of metabolism and related cell fate.
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Affiliation(s)
- Lu-Zhe Pan
- Department of Microbiology and Immunology; Dalhousie University; Halifax, Nova Scotia, Canada
| | - Dae-Gyun Ahn
- Department of Microbiology and Immunology; Dalhousie University; Halifax, Nova Scotia, Canada
| | - Tanveer Sharif
- Department of Microbiology and Immunology; Dalhousie University; Halifax, Nova Scotia, Canada
| | - Derek Clements
- Department of Pathology; Dalhousie University; Halifax, Nova Scotia, Canada
| | - Shashi A Gujar
- Department of Microbiology and Immunology; Dalhousie University; Halifax, Nova Scotia, Canada; Strategy & Organizational Performance; IWK Health Centre; Halifax, Nova Scotia, Canada
| | - Patrick W K Lee
- Department of Microbiology and Immunology; Dalhousie University; Halifax, Nova Scotia, Canada; Department of Pathology; Dalhousie University; Halifax, Nova Scotia, Canada
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18
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PIAS1-modulated Smad2/4 complex activation is involved in zinc-induced cancer cell apoptosis. Cell Death Dis 2013; 4:e811. [PMID: 24052079 PMCID: PMC3789191 DOI: 10.1038/cddis.2013.333] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2013] [Revised: 08/05/2013] [Accepted: 08/06/2013] [Indexed: 11/27/2022]
Abstract
Prostate cancer is one of the most frequently diagnosed cancers among men. Dietary intake of nutrients is considered crucial for preventing the initiation of events leading to the development of carcinoma. Many dietary compounds have been considered to contribute to cancer prevention including zinc, which has a pivotal role in modulating apoptosis. However, the mechanism for zinc-mediated prostate cancer chemoprevention remains enigmatic. In this study, we investigated the therapeutic effect of zinc in prostate cancer chemoprevention for the first time. Exposure to zinc induced apoptosis and resulted in transactivation of p21WAF1/Cip1 in a Smad-dependent and p53-independent manner in prostate cancer cells. Smad2 and PIAS1 proteins were significantly upregulated resulting in dramatically increased interactions between Smad2/4 and PIAS1 in the presence of zinc in LNCaP cells. Furthermore, it was found that the zinc-induced Smad4/2/PIAS1 transcriptional complex is responsible for Smad4 binding to SBE1 and SBE3 regions within the p21WAF1/Cip1 promoter. Exogenous expression of Smad2/4 and PIAS1 promotes zinc-induced apoptosis concomitant with Smad4 nuclear translocation, whereas endogenous Smad2/4 silencing inhibited zinc-induced apoptosis accompanying apparent p21WAF1/Cip1 reduction. Moreover, the knockdown of PIAS1 expression attenuated the zinc-induced recruitment of Smad4 on the p21WAF1/Cip1 promoter. The colony formation experiments demonstrate that PIAS1 and Smad2/4 silencing could attenuate zinc apoptotic effects, with a proliferation of promoting effects. We further demonstrate the correlation of apoptotic sensitivity to zinc and Smad4 and PIAS1 in multiple cancer cell lines, demonstrating that the important roles of PIAS1, Smad2, and Smad4 in zinc-induced cell death and p21WAF1/Cip1 transactivation were common biological events in different cancer cell lines. Our results suggest a new avenue for regulation of zinc-induced apoptosis, and provide a model that demonstrates zinc endorses the Smad2/4/PIAS1 complex to activate the p21WAF1/Cip1 gene that mediates apoptosis.
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19
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Hill R, Madureira PA, Waisman DM, Lee PWK. DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death. Oncotarget 2012; 2:1094-108. [PMID: 22190353 PMCID: PMC3282069 DOI: 10.18632/oncotarget.378] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
A key determinant of p53-mediated cell fate following various DNA damage modalities is p21WAF1/CIP1 expression, with elevated p21 expression triggering cell cycle arrest and repressed p21 expression promoting apoptosis. We show that under pro-death DNA damage conditions, the DNA-dependent protein kinase (DNA-PKCS) is recruited to the p21 promoter where it forms a protein complex with p53. The DNA-PKCS-associated p53 displays post-translational modifications that are distinct from those under pro-arrest conditions, ablating p21 transcription and inducing cell death. Inhibition of DNA-PK activity prevents DNA-PKCS binding to p53 on the p21 promoter, restores p21 transcription and significantly reduces cell death. These data demonstrate that DNA-PKCS negatively regulates p21 expression by directly interacting with the p21 transcription machinery via p53, driving the cell towards apoptosis.
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Affiliation(s)
- Richard Hill
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
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20
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Sullivan KD, Gallant-Behm CL, Henry RE, Fraikin JL, Espinosa JM. The p53 circuit board. Biochim Biophys Acta Rev Cancer 2012; 1825:229-44. [PMID: 22333261 DOI: 10.1016/j.bbcan.2012.01.004] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2011] [Revised: 01/27/2012] [Accepted: 01/28/2012] [Indexed: 12/17/2022]
Abstract
The p53 tumor suppressor is embedded in a large gene network controlling diverse cellular and organismal phenotypes. Multiple signaling pathways converge onto p53 activation, mostly by relieving the inhibitory effects of its repressors, MDM2 and MDM4. In turn, signals originating from increased p53 activity diverge into distinct effector pathways to deliver a specific cellular response to the activating stimuli. Much attention has been devoted to dissecting how the various input pathways trigger p53 activation and how the activity of the p53 protein itself can be modulated by a plethora of co-factors and post-translational modifications. In this review we will focus instead on the multiple configurations of the effector pathways. We will discuss how p53-generated signals are transmitted, amplified, resisted and eventually integrated by downstream gene circuits operating at the transcriptional, post-transcriptional and post-translational levels. We will also discuss how context-dependent variations in these gene circuits define the cellular response to p53 activation and how they may impact the clinical efficacy of p53-based targeted therapies.
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Affiliation(s)
- Kelly D Sullivan
- Howard Hughes Medical Institute & Department of Molecular, Cellular and Developmental Biology, The University of Colorado at Boulder, Boulder, CO 80309-0347, USA
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21
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Vigneron AM, Vousden KH. An indirect role for ASPP1 in limiting p53-dependent p21 expression and cellular senescence. EMBO J 2011; 31:471-80. [PMID: 22068052 DOI: 10.1038/emboj.2011.402] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Accepted: 10/17/2011] [Indexed: 12/31/2022] Open
Abstract
In addition to acting as a transcriptional cofactor for p53, ASPP1 has been shown to function in the cytoplasm to regulate the nuclear localization and activity of YAP/TAZ. We show here that the ability of ASPP1 to activate YAP results in the decreased expression of LATS2, which lowers the ability of p53 to induce p21, cell-cycle arrest and senescence. ASPP1 expression peaks in S-phase, and down-regulation of ASPP1 leads to a reduction in DNA synthesis and enhanced senescence in response to drugs that impede DNA replication. These activities of cytoplasmic ASPP1 in opposing p53-mediated p21 expression are in contrast to the role of nuclear ASPP1 in cooperating with p53 to induce the expression of apoptotic target genes, and may help to dampen p53 activity in normal cells.
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Affiliation(s)
- Arnaud M Vigneron
- The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK
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22
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Lake RJ, Basheer A, Fan HY. Reciprocally regulated chromatin association of Cockayne syndrome protein B and p53 protein. J Biol Chem 2011; 286:34951-8. [PMID: 21852235 DOI: 10.1074/jbc.m111.252643] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The Cockayne syndrome complementation group B (CSB) protein is an ATP-dependent chromatin remodeler with an essential function in transcription-coupled DNA repair, and mutations in the CSB gene are associated with Cockayne syndrome. The p53 tumor suppressor has been known to interact with CSB, and both proteins have been implicated in overlapping biological processes, such as DNA repair and aging. The significance of the interaction between CSB and p53 has remained unclear, however. Here, we show that the chromatin association of CSB and p53 is inversely related. Using in vitro binding and chromatin immunoprecipitation approaches, we demonstrate that CSB facilitates the sequence-independent association of p53 with chromatin when p53 concentrations are low and that this is achieved by the interaction of CSB with the C-terminal region of p53. Remarkably, p53 prevents CSB from binding to nucleosomes when p53 concentrations are elevated. Examining the enzymatic properties of CSB revealed that p53 excludes CSB from nucleosomes by occluding a nucleosome interaction surface on CSB. Together, our results suggest that the reciprocal regulation of chromatin access by CSB and p53 could be part of a mechanism by which these two proteins coordinate their activities to regulate DNA repair, cell survival, and aging.
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Affiliation(s)
- Robert J Lake
- Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6145, USA
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23
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Chen Y, Zhang L, Jones KA. SKIP counteracts p53-mediated apoptosis via selective regulation of p21Cip1 mRNA splicing. Genes Dev 2011; 25:701-16. [PMID: 21460037 DOI: 10.1101/gad.2002611] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The Ski-interacting protein SKIP/SNW1 functions as both a splicing factor and a transcriptional coactivator for induced genes. We showed previously that transcription elongation factors such as SKIP are dispensable in cells subjected to DNA damage stress. However, we report here that SKIP is critical for both basal and stress-induced expression of the cell cycle arrest factor p21(Cip1). RNAi chromatin immunoprecipitation (RNAi-ChIP) and RNA immunoprecipitation (RNA-IP) experiments indicate that SKIP is not required for transcription elongation of the gene under stress, but instead is critical for splicing and p21(Cip1) protein expression. SKIP interacts with the 3' splice site recognition factor U2AF65 and recruits it to the p21(Cip1) gene and mRNA. Remarkably, SKIP is not required for splicing or loading of U2AF65 at other investigated p53-induced targets, including the proapoptotic gene PUMA. Consequently, depletion of SKIP induces a rapid down-regulation of p21(Cip1) and predisposes cells to undergo p53-mediated apoptosis, which is greatly enhanced by chemotherapeutic DNA damage agents. ChIP experiments reveal that SKIP is recruited to the p21(Cip1), and not PUMA, gene promoters, indicating that p21(Cip1) gene-specific splicing is predominantly cotranscriptional. The SKIP-associated factors DHX8 and Prp19 are also selectively required for p21(Cip1) expression under stress. Together, these studies define a new step that controls cancer cell apoptosis.
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Affiliation(s)
- Yupeng Chen
- Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA
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24
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Mukerjee R, Claudio PP, Chang JR, Del Valle L, Sawaya BE. Transcriptional regulation of HIV-1 gene expression by p53. Cell Cycle 2010; 9:4569-78. [PMID: 21088492 DOI: 10.4161/cc.9.22.13836] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). However, the mechanisms of this negative effect remain unclear. In here, we showed that over expression of p53 wild type prevented the phosphorylation of serine 2 in the carboxyl terminal domain (CTD) of RNA polymerase II. As a result of this inhibition, p53 stalled transcriptional elongation on the HIV-1 LTR leading to a significant reduction of HIV-1 replication in primary microglia and astrocytes. However, despite the delay/pause caused by p53, viral transcription and replication decreased and then salvaged. These studies suggest that the negative effect of p53 is alleviated by a third factor. In this regard, our Preliminary Data point to the involvement of the Pirh2 protein in p53 inhibition. Therefore, we suggest that p53 may be a novel therapeutic target for the inhibition of HIV-1 gene expression and replication and the treatment of AIDS.
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Affiliation(s)
- Ruma Mukerjee
- Molecular Virology Lab, Department of Neurology, Temple University School of Medicine, Philadelphia, PA, USA
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25
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Gomes NP, Espinosa JM. Disparate chromatin landscapes and kinetics of inactivation impact differential regulation of p53 target genes. Cell Cycle 2010; 9:3428-37. [PMID: 20818159 PMCID: PMC3047614 DOI: 10.4161/cc.9.17.12998] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2010] [Accepted: 07/13/2010] [Indexed: 11/19/2022] Open
Abstract
The p53 transcription factor regulates the expression of genes involved in cellular responses to stress, including cell cycle arrest and apoptosis. The p53 transcriptional program is extremely malleable, with target gene expression varying in a stress- and cell type-specific fashion. The molecular mechanisms underlying differential p53 target gene expression remain elusive. Here we provide evidence for gene-specific mechanisms affecting expression of three important p53 target genes. First we show that transcription of the apoptotic gene PUMA is regulated through intragenic chromatin boundaries, as revealed by distinct histone modification territories that correlate with binding of the insulator factors CTCF, Cohesins and USF1/2. Interestingly, this mode of regulation produces an evolutionary conserved long non-coding RNA of unknown function. Second, we demonstrate that the kinetics of transcriptional competence of the cell cycle arrest gene p21 and the apoptotic gene FAS are markedly different in vivo, as predicted by recent biochemical dissection of their core promoter elements in vitro. After a pulse of p53 activity in cells, assembly of the transcriptional apparatus on p21 is rapidly reversed, while FAS transcriptional activation is more sustained. Collectively these data add to a growing list of p53-autonomous mechanisms that impact differential regulation of p53 target genes.
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Affiliation(s)
- Nathan P Gomes
- Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA
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Jung YS, Qian Y, Chen X. Examination of the expanding pathways for the regulation of p21 expression and activity. Cell Signal 2010; 22:1003-12. [PMID: 20100570 PMCID: PMC2860671 DOI: 10.1016/j.cellsig.2010.01.013] [Citation(s) in RCA: 333] [Impact Index Per Article: 22.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2010] [Accepted: 01/16/2010] [Indexed: 02/06/2023]
Abstract
p21(Waf1/Cip1/Sdi1) was originally identified as an inhibitor of cyclin-dependent kinases, a mediator of p53 in growth suppression and a marker of cellular senescence. p21 is required for proper cell cycle progression and plays a role in cell death, DNA repair, senescence and aging, and induced pluripotent stem cell reprogramming. Although transcriptional regulation is considered to be the initial control point for p21 expression, there is growing evidence that post-transcriptional and post-translational regulations play a critical role in p21 expression and activity. This review will briefly discuss the activity of p21 and focus on current knowledge of the determinants that control p21 transcription, mRNA stability and translation, and protein stability and activity.
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Affiliation(s)
- Yong-Sam Jung
- Center for Comparative Oncology, University of California, Davis, California 95616, USA
| | - Yingjuan Qian
- Center for Comparative Oncology, University of California, Davis, California 95616, USA
| | - Xinbin Chen
- Center for Comparative Oncology, University of California, Davis, California 95616, USA
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Chiu A, Shi XL, Lee WKP, Hill R, Wakeman TP, Katz A, Xu B, Dalal NS, Robertson JD, Chen C, Chiu N, Donehower L. Review of chromium (VI) apoptosis, cell-cycle-arrest, and carcinogenesis. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH. PART C, ENVIRONMENTAL CARCINOGENESIS & ECOTOXICOLOGY REVIEWS 2010; 28:188-230. [PMID: 20859824 PMCID: PMC4330561 DOI: 10.1080/10590501.2010.504980] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
Hexavalent chromium combines with glutathione in chloride intracellular channel carrier to form tetravalent and pentavalent chromium in plasma and organelle membranes. It also combines with NADH/NADPH to form pentavalent chromium in mitochondria. Tetravalent- and pentavalent- chromium (directly and indirectly) mediated DNA double strand breaks activate DNA damage signaling sensors: DNA-dependent-protein-kinase signals p53-dependent intrinsic mitochondrial apoptosis, and ataxia-telangiectasia-mutated and ataxia-telangiectasia-Rad3-related signal cell-arrest for DNA repair. Tetravalent chromium may be the most potent species since it causes DNA breaks and somatic recombination, but not apoptosis. Upon further failure of apoptosis and senescence/DNA-repair, damaged cells may become immortal with loss-of-heterozygosity and genetic plasticity.
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Affiliation(s)
- A Chiu
- National Center for Environmental Assessment DC, US Environmental Protection Agency, Washington, DC 20460, USA.
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Abstract
Inactivation of p53 is critical for the formation of most tumors. Illumination of the key function(s) of p53 protein in protecting cells from becoming cancerous is therefore a worthy goal. Arguably p53's most important function is to act as a transcription factor that directly regulates perhaps several hundred of the cell's RNA polymerase II (RNAP II)-transcribed genes, and indirectly regulates thousands of others. Indeed p53 is the most well studied mammalian transcription factor. The p53 tetramer binds to its response element where it can recruit diverse transcriptional coregulators such as histone modifying enzymes, chromatin remodeling factors, subunits of the mediator complex, and components of general transcription machinery and preinitiation complex (PIC) to modulate RNAPII activity at target loci (Laptenko and Prives 2006). The p53 transcriptional program is regulated in a stimulus-specific fashion (Murray-Zmijewski et al. 2008; Vousden and Prives 2009), whereby distinct subsets of p53 target genes are induced in response to different p53-activating agents, likely allowing cells to tailor their response to different types of stress. How p53 is able to discriminate between these different loci is the subject of intense research. Here, we describe key aspects of the fundamentals of p53-mediated transcriptional regulation and target gene promoter selectivity.
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Affiliation(s)
- Rachel Beckerman
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
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Gomes NP, Espinosa JM. Differential regulation of p53 target genes: it's (core promoter) elementary. Genes Dev 2010; 24:111-4. [PMID: 20080948 DOI: 10.1101/gad.1893610] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
p53 is a pleiotropic transcription factor driving a flexible transcriptional program that mediates disparate cellular responses to stress, including cell cycle arrest and apoptosis. The mechanisms by which p53 differentially regulates its diverse target genes remain poorly understood. In this issue of Genes & Development, Morachis and colleagues (pp. 135-147) demonstrate the critical role of core promoter elements at p53 target loci, in that they dictate differential RNA polymerase II recruitment and activity in a p53-autonomous fashion.
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Affiliation(s)
- Nathan P Gomes
- Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology, The University of Colorado at Boulder, Boulder, Colorado 80309, USA
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Soria G, Gottifredi V. PCNA-coupled p21 degradation after DNA damage: The exception that confirms the rule? DNA Repair (Amst) 2010; 9:358-64. [PMID: 20060369 DOI: 10.1016/j.dnarep.2009.12.003] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/15/2009] [Indexed: 10/20/2022]
Abstract
While many are the examples of DNA damaging treatments that induce p21 accumulation, the conception of p21 upregulation as the universal response to genotoxic stress has come to an end. Compelling evidences have demonstrated the existence of converging signals that negatively regulate p21 bellow basal levels when replication forks are blocked. Moreover, conclusive reports identified the E3-ligase CRL4(CDT2) (CUL4-DDB1-CDT2) as the enzymatic complex that promotes p21 proteolysis when treatments such as UV irradiation trigger replication fork stress. A pre-requisite for CRL4(CDT2)-driven proteolysis is the interaction of p21 with PCNA. Interestingly as well, CRL4(CDT2)-dependent proteolysis is not limited to p21 and affects other PCNA partners, including the specialized DNA polymerase eta (pol eta). These recent discoveries are particularly intriguing since the UV-induced degradation of p21 has been shown to be required for efficient pol eta recruitment to DNA lesions. Herein we review the findings that lead to the identification of the molecular mechanism that triggers damage-induced PCNA-coupled protein proteolysis. We propose a novel model in which CRL4(CDT2)-dependent protein degradation facilitates a sequential and dynamic exchange between PIP box bearing proteins at stall forks during Translesion DNA synthesis (TLS). Moreover, given the tight spatiotemporal control that CRL4(CDT2)-driven proteolysis is able to confer to PCNA-regulated processes, we discuss the impact that this degradation mechanism might have in other molecular switches associated with the repair of damaged DNA.
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Affiliation(s)
- Gastón Soria
- Fundación Instituto Leloir - CONICET, Universidad de Buenos Aires, Argentina.
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Beckerman R, Donner AJ, Mattia M, Peart MJ, Manley JL, Espinosa JM, Prives C. A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint. Genes Dev 2009; 23:1364-77. [PMID: 19487575 DOI: 10.1101/gad.1795709] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
We reported previously that when cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. Here we show that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3' end of the p21 gene. Recruitment of specific elongation and 3' processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/3' processing machinery.
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Affiliation(s)
- Rachel Beckerman
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
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Toyoshima M. Analysis of p53 dependent damage response in sperm-irradiated mouse embryos. JOURNAL OF RADIATION RESEARCH 2009; 50:11-17. [PMID: 19218778 DOI: 10.1269/jrr.08099] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Ionizing radiation activates a series of DNA damage response, cell cycle checkpoints to arrest cells at G1/S, S and G2/M, DNA repair, and apoptosis. The DNA damage response is thought to be the major determinant of cellular radiosensitivity and thought to operate in all higher eukaryotic cells. However, the radiosensitivity is known to differ considerably during ontogeny of mammals and early embryos of mouse for example are much more sensitive to radiation than adults. We have focused on the radiation-induced damage response during pre-implantation stage of mouse embryo. Our study demonstrates a hierarchy of damage responses to assure the genomic integrity in early embryonic development. In the sperm-irradiated zygotes, p53 dependent S-phase checkpoint functions to suppress erroneous replication of damaged DNA. The transcription-dependent function is not required and the DNA-binging domain of the protein is essential for this p53 dependent S-phase checkpoint. p21 mediated cleavage arrest comes next during early embryogenesis to prevent delayed chromosome damage at morula/ blastocyst stages. Apoptosis operates even later only in the cells of ICM at the blastocyst stage to eliminate deleterious cells. Thus, early development of sperm-irradiated embryos is protected at least by three mechanisms regulated by p53 and by p21.
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Affiliation(s)
- Megumi Toyoshima
- Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan.
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33
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Martinez A, Hinz JM, Gómez L, Molina B, Acuña H, Jones IM, Frias S, Coleman MA. Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2008; 656:1-7. [DOI: 10.1016/j.mrgentox.2008.06.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/15/2007] [Revised: 06/13/2008] [Accepted: 06/17/2008] [Indexed: 12/15/2022]
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Li P, Yao H, Zhang Z, Li M, Luo Y, Thompson PR, Gilmour DS, Wang Y. Regulation of p53 target gene expression by peptidylarginine deiminase 4. Mol Cell Biol 2008; 28:4745-58. [PMID: 18505818 PMCID: PMC2493360 DOI: 10.1128/mcb.01747-07] [Citation(s) in RCA: 172] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2007] [Revised: 11/27/2007] [Accepted: 05/20/2008] [Indexed: 11/20/2022] Open
Abstract
Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.
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Affiliation(s)
- Pingxin Li
- Center for Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA
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Singer GAC, Wu J, Yan P, Plass C, Huang THM, Davuluri RV. Genome-wide analysis of alternative promoters of human genes using a custom promoter tiling array. BMC Genomics 2008; 9:349. [PMID: 18655706 PMCID: PMC2527337 DOI: 10.1186/1471-2164-9-349] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2007] [Accepted: 07/25/2008] [Indexed: 11/23/2022] Open
Abstract
BACKGROUND Independent lines of evidence suggested that a large fraction of human genes possess multiple promoters driving gene expression from distinct transcription start sites. Understanding which promoter is employed in which cellular context is required to unravel gene regulatory networks within the cell. RESULTS We have developed a custom microarray platform that tiles roughly 35,000 alternative putative promoters from nearly 7,000 genes in the human genome. To demonstrate the utility of this array platform, we have analyzed the patterns of promoter usage in 17beta-estradiol (E2)-treated and untreated MCF7 cells and show widespread usage of alternative promoters. Most intriguingly, we show that the downstream promoter in E2-sensitive multiple promoter genes tends to be very close to the 3'-terminus of the gene, suggesting exotic mechanisms of expression regulation in these genes. CONCLUSION The usage of alternative promoters greatly multiplies the transcriptional complexity available within the human genome. The fact that many of these promoters are incapable of driving the synthesis of a meaningful protein-encoding transcript further complicates the story.
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Affiliation(s)
- Gregory AC Singer
- Human Cancer Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA
| | - Jiejun Wu
- Human Cancer Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA
| | - Pearlly Yan
- Human Cancer Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA
| | - Christoph Plass
- Human Cancer Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA
- German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Tim HM Huang
- Human Cancer Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA
| | - Ramana V Davuluri
- Human Cancer Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA
- Center for Systems and Computational Biology, Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA
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36
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Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis along with reduced endoreduplication in acute myelogenous leukemia. Blood 2008; 112:2886-95. [PMID: 18633130 DOI: 10.1182/blood-2008-01-128611] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Aberrant expression of Aurora kinases and inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events in acute myelogenous leukemia (AML), and preclinical data for inhibition of Aurora kinases or Mdm2 are promising. However, it remains largely unknown whether the viability of cells exposed to Aurora kinase inhibitors depends on the p53 status. We investigated the interaction of Aurora kinases and p53 pathways after their simultaneous blockades using a small-molecule pan-Aurora kinase inhibitor, MK-0457, and a selective small-molecule antagonist of Mdm2, Nutlin-3. We found that MK-0457, which itself activates p53 signaling, acts synergistically with Nutlin-3 to induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13 but not in p53-null HL-60 cells. MK-0457 and Nutlin-3 showed synergism in inducing p53, conformational change of Bax and Deltapsi(m) loss, suggesting an involvement of p53-mediated mitochondrial apoptosis. Nutlin-3 constrained endoreduplication after Aurora inhibition via activation of a p53-dependent postmitotic checkpoint and p21 induction in pseudo-G1 cells. Our findings provide the molecular rationale for concomitant targeting of Aurora kinases and Mdm2 in AML where TP53 mutations are rare and downstream p53 signaling is mostly intact.
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37
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Espinosa JM. Mechanisms of regulatory diversity within the p53 transcriptional network. Oncogene 2008; 27:4013-23. [PMID: 18278067 PMCID: PMC2914794 DOI: 10.1038/onc.2008.37] [Citation(s) in RCA: 87] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2007] [Revised: 01/28/2008] [Accepted: 01/28/2008] [Indexed: 01/18/2023]
Abstract
p53 is arguably the most intensively studied protein to date, yet there is much that we ignore about its function as a transcription factor. The p53-dependent transcriptional program is remarkably flexible, as it varies with the nature of p53-activating stimuli, the cell type and the duration of the activation signal. This flexibility may allow cells to mount alternative responses to p53 activation, such as cell cycle arrest or apoptosis. Here, I organize the available data into two alternative models to explain how this regulatory diversity is achieved.
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Affiliation(s)
- J M Espinosa
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA.
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38
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Kan Q, Jinno S, Kobayashi K, Yamamoto H, Okayama H. Cdc6 Determines Utilization of p21WAF1/CIP1-dependent Damage Checkpoint in S Phase Cells. J Biol Chem 2008; 283:17864-72. [DOI: 10.1074/jbc.m802055200] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
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Abstract
When cells progressing in mid-S phase are damaged with a base-modifying chemical, they arrest in S phase long after the CHK1 checkpoint signal fades out, partly because of p53-mediated long-lasting induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). We have recently found that enforced expression of Cdc6, the assembler of prereplicative complexes, markedly advances recovery from the prolonged S-phase arrest and reactivation of Cdk2 despite the presence of a high level of induced p21. Here, we report that Cdc6 protein can activate p21-associated Cdk2 in an ATP-dependent manner in vitro. Consistently, Cdc6 mutated for ATPase or a putative cyclin binding motif is no longer able to activate the Cdk2 in vitro or promote reinitiation of S-phase progression and reactivation of Cdk2 in vivo. These results reveal the never anticipated function of Cdc6 and redefine its role in the control of S-phase progression in mammalian cells.
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40
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Kan Q, Jinno S, Yamamoto H, Okayama H. Chemical DNA damage activates p21 WAF1/CIP1-dependent intra-S checkpoint. FEBS Lett 2007; 581:5879-84. [PMID: 18067863 DOI: 10.1016/j.febslet.2007.11.075] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2007] [Revised: 11/27/2007] [Accepted: 11/27/2007] [Indexed: 11/17/2022]
Abstract
When cells progressing in G(1) phase are irradiated with UV light, two damage checkpoint pathways are activated: CHK1-Cdc25A and p53-p21WAF1/CIP1, both targeting Cdk2 but the latter inducing long lasting inactivation. In similarly irradiated S phase cells, however, p21WAF1/CIP1-dependent checkpoint is largely inactive. We report here that p21-dependent checkpoint can effectively be activated and induce a prolonged S phase arrest with similarly extended inactivation of Cdk2 by association of p21 if mid-S phase cells are damaged with a base-modifying agent instead of UV light, indicating that the poor utilization of p21-dependent checkpoint is not an innate property of S phase cells.
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Affiliation(s)
- Qiuming Kan
- Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Balakrishnan SK, Gross DS. The tumor suppressor p53 associates with gene coding regions and co-traverses with elongating RNA polymerase II in an in vivo model. Oncogene 2007; 27:2661-72. [PMID: 18026140 DOI: 10.1038/sj.onc.1210935] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Sequence-specific transcriptional regulators function by stably binding cognate DNA sequences followed by recruitment of both general and specialized factors to target gene promoters. The tumor suppressor p53 mediates its anti-oncogenic effect on cells by functioning as a sequence-specific regulator. p53 employs a secondary mechanism to suppress tumor formation by permeabilizing the outer mitochondrial membrane, thereby releasing pro-apoptotic factors. Here, we report a potential third biological function of p53: as a transcriptional elongation factor. Using chromatin immunoprecipitation, we demonstrate that human p53 robustly associates with RNA polymerase II (Pol II), but neither Pol I- nor Pol III-transcribed regions in the budding yeast, Saccharomyces cerevisiae. p53's association with open reading frames is mediated by its physical interaction with elongating Pol II, with which p53 travels in vivo and co-immunoprecipitates in vitro. When similarly expressed, the potent acidic activator VP16 cannot be cross-linked to Pol II coding regions. p53 levels comparable to those found in induced mammalian cells confer synthetic sickness or lethality in combination with deletions in genes encoding transcription elongation factors; p53 likewise confers hypersensitivity to the anti-elongation drug 6-azauracil. Collectively, our results indicate that p53 can physically interact with the transcription elongation complex and influence transcription elongation, and open up new avenues of investigation in mammalian cells.
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Affiliation(s)
- S K Balakrishnan
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center and The Feist-Weiller Cancer Center, Shreveport, LA 71130-3932, USA
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42
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Donner AJ, Szostek S, Hoover JM, Espinosa JM. CDK8 is a stimulus-specific positive coregulator of p53 target genes. Mol Cell 2007; 27:121-33. [PMID: 17612495 PMCID: PMC2936241 DOI: 10.1016/j.molcel.2007.05.026] [Citation(s) in RCA: 208] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2006] [Revised: 03/07/2007] [Accepted: 05/25/2007] [Indexed: 10/23/2022]
Abstract
The p53 transcriptional network orchestrates alternative stress responses such as cell-cycle arrest and apoptosis. Here we investigate the mechanism of differential expression of p21, a key mediator of p53-dependent cell-cycle arrest. We demonstrate that the transcriptional activity of the p21 promoter varies greatly in response to distinct p53-activating stimuli. Chromatin immunoprecipitation analysis of the p21 locus indicates that histone acetyltransferases, general transcription factors, and Mediator subunits are assembled into alternative transcriptional complexes of different activity. Interestingly, core Mediator subunits MED1 and MED17 are recruited to the p21 locus regardless of the p53-activating stimuli utilized. In contrast, three subunits of the CDK module of Mediator (CDK8, MED12, and cyclin C) are exclusively recruited during conditions of strong p21 transcriptional activation. Furthermore, increased binding of CDK8 to p53 target genes correlates positively with transcriptional strength. RNAi experiments demonstrate that CDK8 functions as a coactivator within the p53 transcriptional program.
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Affiliation(s)
- Aaron Joseph Donner
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA
| | - Stephanie Szostek
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA
| | - Jennifer Michelle Hoover
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA
| | - Joaquin Maximiliano Espinosa
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA
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