Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics

Abstract

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16(Ink4a)-cycD/cdk4-pRB-E2F and p19(Arf)-mdm2-p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21(Cip1). Thus, E2F3 is a key repressor of the p19(Arf)-p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21(Cip1) in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3(-/-) cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation.

Figure 1.

E2f3^-/- MEFs have increased levels of p19^Arf, leading to activation of p53. (A) Wild-type MEFs (solid line) and E2f3^-/- MEFs (dotted line) were synchronized by serum starvation and cell cycle re-entry was monitored by [³H]thymidine incorporation. (B,C) Total protein extracts were prepared at the indicated times after stimulation with 10% serum and subjected to Western blotting for cyclin A, p107, p19^Arf, p16^Ink4a,orp21^Cip1 (B,C), or electrophoretic mobility shift assay for active p53 (C).

Figure 2.

Elimination of p19^Arf rescues cell cycle re-entry and p53 activation defects of E2f3-deficient MEFs. (A) Wild-type MEFs (solid black line), E2f3^-/- MEFs (dotted black line), Arf^-/- MEFs (solid gray line), and E2f3^-/-;Arf^-/- DKO MEFs (dotted gray line) were synchronized by serum starvation and cell cycle re-entry was monitored by [³H]thymidine incorporation. (B) Total protein extracts were prepared at the indicated times after serum stimulation and subjected to Western blotting for cyclin A, p107, and p21^Cip1, or electrophoretic mobility shift assay for active p53. (C) Wild-type MEFs (solid black line), E2f3^-/- MEFs (dotted black line), p53^-/- MEFs (solid gray line), and E2f3^-/-;p53^-/- DKO MEFs (dashed gray line) were analyzed exactly as described in A.

Figure 3.

E2F3 is a direct repressor of Arf. (A) Wild-type (solid lines) or E2f3^-/- (dotted lines) MEFs were synchronized by serum starvation, and RNA was extracted at the indicated times after stimulation. Quantitative real-time RT–PCR analysis of p107 or Arf mRNA is shown. (B) ChIP analysis of asynchronous wild-type (WT) or E2f3^-/- MEFs. Sonicated, cross-linked chromatin was immunoprecipitated with the indicated antibodies, and the purified DNA was analyzed by PCR with primers specific for the p107 or Arf promoters, or a control sequence lacking E2F sites (1 kb upstream of the E2f1 promoter). Input, 0.5% of chromatin in IP reactions was analyzed by PCR. (C) Schematic of the E2F3a and E2F3b proteins, and the antibodies used in ChIP analysis. (Black box) DNA-binding domain; (light gray box) dimerization domain; (dark gray box) transactivation domain. (D) Cell lysates of asynchronous (AS) wild-type MEFs or wild-type MEFs incubated in 0.1% serum for 3 d (SS) were subjected to Western blotting for the two E2F3 isoforms. ChIP assays for the indicated E2F and pocket protein family members were performed on the serum-starved wild-type MEFs.

Figure 4.

The activating E2Fs bind to the Arf promoter during oncogenic challenge. (A) Schematic of the mouse Ink4a/Arf locus, indicating the exon structure, and the region deleted in the Ink4a/Arf^-/- MEFs (bracket). Shaded regions in the exons indicate regions coding for the p16^Ink4a (light gray) and p19^Arf (black) proteins. The small black boxes indicate two consensus E2F-binding sites in the Arf promoter, and the two small arrows indicate the location of the primers used in ChIP analysis. (B) Ink4a/Arf^-/- MEFs were infected with retrovirus overexpressing E2F1, E1A, or an empty virus (vector) and subjected to ChIP assays with the indicated antisera.

Figure 5.

Arf is regulated by E2F in a distinct manner from classic E2F-responsive genes.