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, 31 (19), 1933-1938

Genetic Interrogation of Replicative Senescence Uncovers a Dual Role for USP28 in Coordinating the p53 and GATA4 Branches of the Senescence Program

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Genetic Interrogation of Replicative Senescence Uncovers a Dual Role for USP28 in Coordinating the p53 and GATA4 Branches of the Senescence Program

Anna E Mazzucco et al. Genes Dev.

Erratum in

Abstract

Senescence is a terminal differentiation program that halts the growth of damaged cells and must be circumvented for cancer to arise. Here we describe a panel of genetic screens to identify genes required for replicative senescence. We uncover a role in senescence for the potent tumor suppressor and ATM substrate USP28. USP28 controls activation of both the TP53 branch and the GATA4/NFkB branch that controls the senescence-associated secretory phenotype (SASP). These results suggest a role for ubiquitination in senescence and imply a common node downstream from ATM that links the TP53 and GATA4 branches of the senescence response.

Keywords: GATA4; USP28; senescence.

Figures

Figure 1.
Figure 1.
Loss-of-function screens identify high-confidence candidate genes. (A) Schematic showing screen design and library pool deconvolution for the senescence bypass and nutlin resistance screens. (Bottom left) Venn diagram showing the overlap between the senescence bypass screen candidate gene list in E7 BJ fibroblasts and the nutlin resistance screen performed in human mammary epithelial cells (HMECs) for the genome-scale library. The probability of overlap was calculated by hypergeometric probability distribution. (Right) Venn diagram showing the gene overlap between the senescence resistance validation screens in E6, E7, and naïve BJs. The numbers shown refer to the number of genes that were enriched twofold or greater and targeted by three or more shRNAs. (B) Representative validation data from nutlin resistance screens as shown by multicolor competition assays in HMECs in the presence or absence of nutlin-3a for three PDs. Cells expressing either Luc shRNA or mock-infected served as negative controls in HMECs expressing GFP (green fluorescent protein). Cells expressing shRNAs against p21 served as positive controls. Data are plotted as the difference between the log2 ratios of untreated and nutlin-treated cells. (C) Validation rescreen data indicating the number of selected shRNAs that scored to twofold or greater above the average of 100 negative control shRNAs for the indicated subset of genes for the sublibrary validation screen in E7 BJ fibroblasts (left) and naïve BJs (right). (D) Components of the NFκB pathway that scored in our screens in E6 BJ fibroblasts. (E) Table showing the number of selected shRNAs that scored twofold or greater with three or more shRNAs. (F) Table showing the number of shRNAs that scored above each threshold relative to the average of the negative control shRNAs in the validation screen performed in naïve BJ fibroblasts.
Figure 2.
Figure 2.
USP28 is required for p53, p21, and GATA4 induction during senescence. (A) Growth of late passage BJ cells expressing the indicated shRNAs. (B) Quantitation of SA-β-Gal staining in BJ cells expressing the USP28 shRNAs in A. (C) Western blot analysis of USP28 levels in BJ cells expressing the indicated shRNAs in A. (D) Quantitation of IR-induced SA-β-Gal staining in BJ cells expressing the indicated shRNAs. Data are mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA). Data are representative of four independent experiments. (E) Western blot analysis of late passage BJ cells expressing the indicated shRNAs. (Below) Densitometric analysis to determine the USP28/GAPDH and p53/GAPDH ratios. (F) RT-qPCR for p21 expression in mid-passage BJ fibroblasts expressing the indicated shRNAs with and without 8 µM nutlin treatment at 6 h. (G) Western blot analysis of IMR90 cells expressing the indicated shRNAs 7 d after 12 Gy of IR treatment. (H) Western blot analysis of IMR90 cells expressing the indicated shRNAs 7 d after treatment with 12 Gy of IR. The arrow indicates GATA4 protein.
Figure 3.
Figure 3.
USP28 is a TSG that controls p53 and GATA4 in a catalytic activity-dependent manner. (A) Growth of mid-passage BJ fibroblasts expressing the indicated protein or empty vector. (B) Quantitation of SA-β-Gal staining in mid-passage BJ fibroblasts expressing the indicated protein or empty vector. (C) Western blotting of BJ cells expressing USP28 or an empty vector for 10 d. (D) SA-β-Gal staining on day 7 of doxycycline (Dox) treatment of BJ cells carrying a Dox-inducible vector (Tet-on) expressing either wild-type USP28 or the USP28C171A mutant and the indicated shRNAs. Data are mean ± SEM. Statistical significance was calculated by one-way ANOVA. Data are representative of three independent experiments. (E) Levels of BrdU-positive cells on day 7 of Dox treatment of the BJ cells described in F. Data are mean ± SEM. Statistical significance was calculated by ANOVA. Data are representative of three independent experiments. (F) The distribution of different classes of USP28 mutations in a collection of 8200 tumors compared with a typical neutral gene (Davoli et al. 2013). Polyphen2 was used to predict missense mutations as benign or damaging. A loss-of-function mutation is defined as either a stop codon or frameshift. (G) A schematic diagram indicating major senescence pathways driving cell cycle arrest and the formation of senescence-associated heterochromatic foci (SAHFs) and the SASP as well as points where USP28 acts within these pathways, with genes identified in this study indicated in red.

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