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. 2012 Apr 13;336(6078):225-8.
doi: 10.1126/science.1218395.

Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress

In Hye Lee  1 , Yoshichika Kawai, Maria M Fergusson, Ilsa I Rovira, Alexander J R Bishop, Noboru Motoyama, Liu Cao, Toren Finkel
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"VSports注册入口" Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress

In Hye Lee et al. Science. .

"VSports手机版" Erratum in

  • Science. 2012 Aug 24;337(6097):911
  • Science. 2013 Aug 2;341(6145):457

Abstract

Withdrawal of nutrients triggers an exit from the cell division cycle, the induction of autophagy, and eventually the activation of cell death pathways. The relation, if any, among these events is not well characterized. We found that starved mouse embryonic fibroblasts lacking the essential autophagy gene product Atg7 failed to undergo cell cycle arrest VSports手机版. Independent of its E1-like enzymatic activity, Atg7 could bind to the tumor suppressor p53 to regulate the transcription of the gene encoding the cell cycle inhibitor p21(CDKN1A). With prolonged metabolic stress, the absence of Atg7 resulted in augmented DNA damage with increased p53-dependent apoptosis. Inhibition of the DNA damage response by deletion of the protein kinase Chk2 partially rescued postnatal lethality in Atg7(-/-) mice. Thus, when nutrients are limited, Atg7 regulates p53-dependent cell cycle and cell death pathways. .

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Figures

Fig. 1
Fig. 1
Requirement of Atg7 for p21CDKN1A expression and for cell cycle arrest. (A) Percentage reduction in S-phase entry as measured by BrdU incorporation during starved versus fed conditions for early-passage primary wild-type (WT) or Atg7−/− MEFs. Shown is one representative experiment performed in triplicate with greater than 200 individual cells assessed per condition. (B) Protein immunoblot assessment of p21CDKN1A and p27CDKN1B expression in WT and Atg7−/− MEFs after withdrawal of nutrients. Actin is used as a loading control. (C) Abundance of p21CDKN1A mRNA after starvation in WT and Atg7−/− MEFs. (D) Activity of the p21CDKN1A promoter containing a p53 binding element in WT or Atg7−/− MEFs under fed and starved conditions. (E) ChIP of the p21CDKN1A promoter prepared from starved HCT116 cells with antibodies to endogenous p53, Atg7, or an irrelevant immunoglobulin G (IgG) control. (F) Starvation-induced p21CDKN1A protein expression in the human HCT116 cell line after transfection with a scrambled control siRNA or after depletion of Atg7. (G) p21CDKN1A protein after depletion of Beclin 1 (Atg6). (H) Confluent density achieved by either WT or Atg7−/− primary MEFs seeded initially at a high but equal density. (I) Corresponding expression of p21CDKN1A as a function of confluent density in WT or Atg7−/− MEFs. *P < 0.01 (Student t test between WT and Atg7−/− MEFs), n ≥ 3.
Fig. 2
Fig. 2
Interaction of Atg7 with p53. (A) Protein-protein interaction in transfected HCT116 cells assessed by protein immunoblot (WB) analysis with myc-tagged Atg7 and Flag-tagged p53. Immunoprecipitation (IP) was performed using an antibody to the myc epitope or, where indicated (−), an irrelevant IgG iso-type control serum. IP was performed using 2 mg of protein lysate; the input represents 50 μg of lysate. (B) Interaction between endogenous Atg7 and p53 in HCT116 cells under fed conditions (t=0) and after withdrawal of nutrients. (C) In vitro interaction between full-length myc-tagged Atg7 and various GST full-length (FL) or truncation mutants of p53. (D) A GST-p53 construct (ΔC) consisting of the first 300 amino acids of p53 does not bind Atg7. (E) Autophagy is stimulated by full-length Atg7 (1–673), whereas constructs lacking either the active cysteine (Cys571), the C terminus (ΔC), or both of these alterations lack E1-like enzymatic activity. A minimum of 300 cells per sample were counted using quadruplicate samples per construct (**P < 0.01, full-length Atg7 compared to other conditions). (F) In vitro binding of hemagglutinin (HA)–tagged Atg7 constructs to the GST-p53 tetramerization domain. (G) p21CDKN1A promoter activity in fed Atg7−/− MEFs transfected with WT Atg7 or Atg7 constructs with and without E1-like enzymatic activity (n = 4 per condition, **P < 0.01 compared to vector control).
Fig. 3
Fig. 3
The absence of Atg7 increases mitochondrial ROS, augments DNA damage, and activates p53. (A) Relative mRNA expression in starved WT or Atg7−/− MEFs for a panel of proapoptotic p53-responsive genes (**P < 0.01 compared to WT MEFs). (B) ChIP assay 3 hours after the induction of nutrient stress, demonstrating increased p53 binding to the Noxa promoter in HCT116 cells depleted of Atg7. (C) Ser20 phosphorylation in control or Atg7-depleted cells. (D) Images of DNA damage in WT and Atg7−/− MEFs as assessed by γ-H2AX staining. (E) Fluorescence-activated cell sorter analysis of WT and Atg7−/− MEFs under fed (black) or starved (red) conditions, using the redox-dependent fluorophore dichlorodihydrofluorescein diacetate (DCFDA). A rightward shift indicates higher intracellular ROS levels. (F) γ-H2AX foci formation in fed or starved WT and Atg7−/− MEFs. Cells were treated as indicated with the antioxidant NAC (**P < 0.01 compared to starved cells not treated with NAC). (G) NAC treatment inhibits starvation-induced p53 Ser20 phosphorylation in Atg7-depleted cells. (H) Puma expression in WT and Atg7−/− MEFs under fed (−) or starved (+) conditions, in the presence or absence of the cell permeant antioxidant N-acetylcysteine (NAC). (I) Cell death during fed or starved conditions in WT or Atg7−/− MEFs in the presence or absence of NAC. Unless specified, all results are from at least triplicate determinations.
Fig. 4
Fig. 4
Rescue of Atg7 deficiency after Chk2 deletion. (A) p53 Ser20 phosphorylation in HCT116 cells with stable short hairpin RNA depletion of Atg7 with and without additional siRNA-mediated depletion of Chk2. (B) Starvation-induced Puma expression in primary MEFs with the indicated genotype (n ≥ 3 per genotype per condition). (C) Cell viability of MEFs with the indicated genotype under fed or starved conditions (**P < 0.01 between starved WT and Atg7−/− cells, n = 4 per genotype per condition). (D) Expression of proapoptotic gene products in the liver of pups about 8 hours after birth (**P < 0.01, n = 9 determinations per genotype). (E) Survival of consecutive births of Atg7−/− pups with the indicated Chk2 status.
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