The p400 E1A-associated protein is a novel component of the p53 --> p21 senescence pathway

Abstract

Adenovirus E1A-associated p400 belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Here, we report that p400 is a component of the p53-p21(WAF1/CIP1/sid1) pathway, regulating the p21 transcription and senescence induction program. Acute depletion of p400 expression by shRNA (short hairpin RNA) synthesis led to premature senescence of untransformed human fibroblasts, whose features include G1 arrest, p21 induction, senescence-associated heterochromatic foci (SAHF), and beta-gal staining. Importantly, p400shRNA-induced premature senescence phenotypes were rescued by coexpression of p53-shRNA or p21-shRNA. Furthermore, p400 complex colocalized with p53 on the p21 promoter. These data suggest that the p400 complex inhibits p53 --> p21 transcription and the development of premature senescence.

Figure 1.

shRNA-p400-mediated cell cycle arrest in IMR90. (A) Experimental design and time frame. (B) Immunoblot of p400 from IMR90. (Super) The empty retroviral backbone control. Total protein loaded was standardized by Bradford assay (Bio-Rad). (C) Cell growth assay performed in IMR90. (D) FACS analysis and BrdU incorporation assay from two independent experiments.

Figure 2.

shRNA-p400 induces SAHF and premature senescence in IMR90. (A) DNA staining by DAPI (panels 1,3) and H3K9-Me staining (panels 2,4) of cells as indicated. The cells in panels 2 and 4 correspond to the cells in panels 1 and 3, respectively. (B) Senescent β-gal staining of IMR90 following the indicated perturbations. Ras was included as a positive control, and shRNA-c-myc and pSuper were included as negative controls. (C) IB and IP-IB of the indicated proteins from IMR90 exposed to the indicated perturbations. (D) Quantitative RT–PCR measuring p21 mRNA in control IMR90 or IMR90 exposed to shRNA against p400. Unnormalized actin and p21 mRNA levels are presented in graph i. (Graph ii) After normalization against actin mRNA, the relative p21 mRNA abundance was calculated. The data presented are average values obtained from quadruplicate samples.

Figure 3.

Inactivation of p53/p21, but not pocket family proteins, rescues shRNA-p400-mediated premature senescence. (A) Summary of SAHF and senescent β-gal staining in IMR90 exposed to the indicated perturbations. (B,C) IB and IP-IB of the indicated IMR90 proteins after the indicated perturbations.

Figure 4.

p400 colocalizes with p53 on the p21 promoter. (A) Diagrammatic presentation of the p21 promoter, indicating the primers used for ChIP (#1, #2, and #3). (B–D) ChIP analysis of p400 complexes bound at the p21 promoter using the indicated primer sets. Antibodies used for ChIP are indicated. Minus sign (–) indicates a control reaction mixture with no Ab added. p53 and max bound to regions #1 and #2, respectively, were as reported (Kaeser and Iggo 2002; Seoane et al. 2002; Wu et al. 2003). The presence of p53 in #2 could be consistent with the report of a second p53-binding site ∼1 kbp upstream of the transcription start site in the p21 promoter (Resnick-Silverman et al. 1998). The arrows indicate the specific PCR products, and the asterisk indicates the position of a primer dimmer band. (E) Quantitative analysis of p53 binding to the p21 promoter in control IMR90 and IMR90 depleted of p400. Results from three different ChIP experiments are presented. In each ChIP experiment, triplicate samples were subjected to quantitative PCR, and the average value was used to calculate percent of total input chromatin bound. In three experiments, there were, respectively, 1.69, 1.60, and 1.64 times more p53 binding to #1 in IMR90-shRNA-p400 cells compared with control cells.

Figure 5.

A model of p400 regulation of p53 → p21 expression and its related senescence program. p400–p53/p21 and Ras–p16/pRb are two distinct pathways leading to SAHF. p400 represses p53 → p21 transcription to prevent premature senescence. It remains to be determined whether p400 complex function is perturbed in replicative senescence, and if so, whether its perturbation contributes to the biological outcome.