Association of the circadian factor Period 2 to p53 influences p53's function in DNA-damage signaling

Circadian period proteins influence cell division and death by associating with checkpoint components, although their mode of regulation has not been firmly established. hPer2 forms a trimeric complex with hp53 and its negative regulator Mdm2. In unstressed cells, this association leads to increased hp53 stability by blocking Mdm2-dependent ubiquitination and transcription of hp53 target genes. Because of the relevance of hp53 in checkpoint signaling, we hypothesize that hPer2 association with hp53 acts as a regulatory module that influences hp53's downstream response to genotoxic stress. Unlike the trimeric complex, whose distribution was confined to the nuclear compartment, hPer2/hp53 was identified in both cytosol and nucleus. At the transcriptional level, a reporter containing the hp21WAF1/CIP1 promoter, a target of hp53, remained inactive in cells expressing a stable form of the hPer2/hp53 complex even when treated with γ-radiation. Finally, we established that hPer2 directly acts on the hp53 node, as checkpoint components upstream of hp53 remained active in response to DNA damage. Quantitative transcriptional analyses of hp53 target genes demonstrated that unbound hp53 was absolutely required for activation of the DNA-damage response. Our results provide evidence of the mode by which the circadian tumor suppressor hPer2 modulates hp53 signaling in response to genotoxic stress.

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The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells

Molecular Biology of the Cell ◽

10.1091/mbc.e14-05-0993 ◽

2014 ◽

Vol 25 (19) ◽

pp. 3081-3093 ◽

Cited By ~ 40

Author(s):

Tetsuya Gotoh ◽

Marian Vila-Caballer ◽

Carlo S. Santos ◽

Jingjing Liu ◽

Jianhua Yang ◽

...

Keyword(s):

Feedback Loop ◽

Transcriptional Response ◽

Genotoxic Stress ◽

Negative Regulator ◽

Trimeric Complex ◽

Physiological Processes ◽

Period 2 ◽

Damage Response ◽

Stress Signals ◽

Clock Mechanism

Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell's susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

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Regulation of p53 by Hypoxia: Dissociation of Transcriptional Repression and Apoptosis from p53-Dependent Transactivation

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.1297-1310.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 1297-1310 ◽

Cited By ~ 232

Author(s):

Constantinos Koumenis ◽

Rodolfo Alarcon ◽

Ester Hammond ◽

Patrick Sutphin ◽

William Hoffman ◽

...

Keyword(s):

Dna Damage ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Target Genes ◽

P53 Protein ◽

Genotoxic Stress ◽

Protein Accumulation ◽

Dependent Cell ◽

Apoptotic Activity ◽

Induced Apoptosis

ABSTRACT Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

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Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.23.4.1441-1452.2003 ◽

2003 ◽

Vol 23 (4) ◽

pp. 1441-1452 ◽

Cited By ~ 62

Author(s):

Vladimir I. Bashkirov ◽

Elena V. Bashkirova ◽

Edwin Haghnazari ◽

Wolf-Dietrich Heyer

Keyword(s):

Dna Damage ◽

Target Genes ◽

Genotoxic Stress ◽

Dna Damage Checkpoint ◽

Kinase Signaling ◽

M Cell ◽

Fha Domain ◽

Checkpoint Pathway

ABSTRACT The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G2/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G2/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

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Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis

Cell Death and Disease ◽

10.1038/s41419-021-03902-6 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Jun Wang ◽

Holly R. Thomas ◽

Zhang Li ◽

Nan Cher Yeo ◽

Hannah E. Scott ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Responses ◽

Target Genes ◽

Genotoxic Stress ◽

Negative Regulator ◽

Null Alleles ◽

Secondary Wave ◽

Apoptotic Response ◽

Induced Apoptosis

AbstractCellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

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Protein Kinase C δ Induces Transcription of the TP53 Tumor Suppressor Gene by Controlling Death-Promoting Factor Btf in the Apoptotic Response to DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.01126-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8480-8491 ◽

Cited By ~ 62

Author(s):

Hanshao Liu ◽

Zheng-Guang Lu ◽

Yoshio Miki ◽

Kiyotsugu Yoshida

Keyword(s):

Dna Damage ◽

Protein Kinase C ◽

Protein Kinase ◽

Tumor Suppressor ◽

Gene Transcription ◽

Core Promoter ◽

Genotoxic Stress ◽

Tp53 Gene ◽

Transcriptional Level ◽

Kinase C

ABSTRACT Expression of the TP53 tumor suppressor is tightly controlled for its ability to function as a critical regulator of cell growth, proliferation, and death in response to DNA damage. However, little is known about the mechanisms and contributions of the transcriptional regulation of TP53. Here we report that protein kinase C δ (PKCδ), a ubiquitously expressed member of the novel subfamily of PKC isoforms, transactivates TP53 expression at the transcriptional level. Reporter assays demonstrated that PKCδ induces the promoter activity of TP53 through the TP53 core promoter element (CPE-TP53) and that such induction is enhanced in response to DNA damage. The results also demonstrate that, upon exposure to genotoxic stress, PKCδ activates and interacts with the death-promoting transcription factor Btf to co-occupy CPE-TP53. Inhibition of PKCδ activity decreases the affinity of Btf for CPE-TP53, thereby reducing TP53 expression at both the mRNA and the protein levels. In concert with these results, we show that disruption of Btf-mediated TP53 gene transcription by RNA interference leads to suppression of TP53-mediated apoptosis following genotoxic stress. These findings provide evidence that activation of TP53 gene transcription by PKCδ triggers TP53-dependent apoptosis in response to DNA damage.

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UV-Induced Stabilization of c-fos and Other Short-Lived mRNAs

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3616-3625.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3616-3625 ◽

Cited By ~ 70

Author(s):

Christine Blattner ◽

Patricia Kannouche ◽

Margarethe Litfin ◽

Klaus Bender ◽

Hans J. Rahmsdorf ◽

...

Keyword(s):

Dna Damage ◽

Target Genes ◽

Rna Stability ◽

Growth Factor Receptor ◽

Receptor Activation ◽

Transcriptional Level ◽

Delayed Response ◽

Wild Type ◽

Mrna Accumulation ◽

Uv Dose

ABSTRACT Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IκB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

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F-box protein FBXO31 directs degradation of MDM2 to facilitate p53-mediated growth arrest following genotoxic stress

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510929112 ◽

2015 ◽

Vol 112 (28) ◽

pp. 8632-8637 ◽

Cited By ~ 31

Author(s):

Sunil K. Malonia ◽

Parul Dutta ◽

Manas Kumar Santra ◽

Michael R. Green

Keyword(s):

Dna Damage ◽

Tumor Suppressor ◽

Critical Role ◽

Growth Arrest ◽

Genotoxic Stress ◽

26S Proteasome ◽

Negative Regulator ◽

Induce Cell Cycle Arrest ◽

Cycle Arrest ◽

F Box Protein

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.

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The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress

eLife ◽

10.7554/elife.29953 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 7

Author(s):

Takeshi Wakida ◽

Masae Ikura ◽

Kenji Kuriya ◽

Shinji Ito ◽

Yoshiharu Shiroiwa ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Cell Cycle Progression ◽

Genotoxic Stress ◽

Dna Damage Checkpoint ◽

Proliferating Cells ◽

Checkpoint Activation ◽

Checkpoint Signaling ◽

Checkpoint Response ◽

Promote Cell Proliferation

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

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Increased DNA Damage Sensitivity and Apoptosis in Cells Lacking the Snf5/Ini1 Subunit of the SWI/SNF Chromatin Remodeling Complex

Molecular and Cellular Biology ◽

10.1128/mcb.26.7.2661-2674.2006 ◽

2006 ◽

Vol 26 (7) ◽

pp. 2661-2674 ◽

Cited By ~ 87

Author(s):

Agnes Klochendler-Yeivin ◽

Eli Picarsky ◽

Moshe Yaniv

Keyword(s):

Dna Damage ◽

Chromatin Remodeling ◽

Target Genes ◽

Genotoxic Stress ◽

Tumor Formation ◽

Gene Encoding ◽

Chromatin Remodeling Complex ◽

Specific Subset ◽

Elevated Expression ◽

Primary Fibroblasts

ABSTRACT The gene encoding the SNF5/Ini1 core subunit of the SWI/SNF chromatin remodeling complex is a tumor suppressor in humans and mice, with an essential role in early embryonic development. To investigate further the function of this gene, we have generated a Cre/lox-conditional mouse line. We demonstrate that Snf5 deletion in primary fibroblasts impairs cell proliferation and survival without the expected derepression of most retinoblastoma protein-controlled, E2F-responsive genes. Furthermore, Snf5-deficient cells are hypersensitive to genotoxic stress, display increased aberrant mitotic features, and accumulate phosphorylated p53, leading to elevated expression of a specific subset of p53 target genes, suggesting a role for Snf5 in the DNA damage response. p53 inactivation does not rescue the proliferation defect caused by Snf5 deficiency but reduces apoptosis and strongly accelerates tumor formation in Snf5-heterozygous mice.

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The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020185118 ◽

2021 ◽

Vol 118 (8) ◽

pp. e2020185118

Author(s):

Nalini Dhingra ◽

Sahiti Kuppa ◽

Lei Wei ◽

Nilisha Pokhrel ◽

Silva Baburyan ◽

...

Keyword(s):

Dna Damage ◽

Genotoxic Stress ◽

Dna Helicase ◽

Dna Damage Checkpoint ◽

Recombinational Repair ◽

Checkpoint Signaling ◽

Cellular Survival ◽

Moderate Reduction ◽

Srs2 Helicase ◽

Harmful Effects

The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin insrs2mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.

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