E2f3 is critical for normal cellular proliferation - PubMed (original) (raw)

. 2000 Mar 15;14(6):690-703.

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E2f3 is critical for normal cellular proliferation

P O Humbert et al. Genes Dev. 2000.

Abstract

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, we generated an E2f3 mutant mouse strain. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, we analyzed the properties of embryonic fibroblasts derived from E2f3 mutant mice. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. We have been able to identify a number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, whose expression is dependent on the presence of E2F3 but not E2F1. We further show that a critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G(1)/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. Finally, we show that E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. We conclude that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells.

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Figures

Figure 1

Figure 1

E2F3 does not affect the expression of other E2F species. (A) The E2f3 gene was inactivated in ES cells using the targeting construct shown. This strategy introduces an in-frame termination codon upstream of the NLS of E2F3 and replaces the exons encoding the NLS, DNA-binding domain (DBD), and leucine zipper (LZ) domain (shown in black) with a neomycin resistance gene. (B) Western blot analysis was performed on whole cell extracts generated from MEFs derived from the progeny of E2f3+/− crosses. E2F3 is indicated (arrow). (C) To examine how E2F3 loss affects other E2F species, gel shift analysis was performed in the absence or presence of sodium deoxycholate (DOC), which releases the free E2F · DP complexes from the associated pRB family members. The free DP · E2F3 complex is indicated (arrow).

Figure 2

Figure 2

_E2f3_−/− cells have a proliferation defect. Passage 4 MEFs derived from wild-type and E2f3 mutant littermates were cultured under high (A) or low (B) density conditions as described in the methods. (● E2f3+/+; ▵ E2f3+/−; ○ _E2f3_−/−).

Figure 3

Figure 3

_E2f3_−/− cells have defects in the initiation and progression of DNA synthesis. (A) Wild-type and E2f3 mutant MEFs from two different MEF preparations (H and E) were synchronized by serum starvation and their cell cycle re-entry properties assayed using [3H]thymidine incorporation. (B) MEFs derived from a wild-type, an E2f1+/−, and three _E2f1_−/− littermate embryos were analyzed as in A. (C,D) Wild-type and _E2f3_−/− cells were plated on coverslips and synchronized by serum starvation. At each time point after serum addition, DNA synthesis was monitored by assaying for BrdU incorporation.

Figure 4

Figure 4

E2F3 is required for the mitogen-induced, transcriptional activation of most known E2F-responsive genes. Wild-type, E2f3 mutant MEFs were synchronized by serum starvation. At each time point, RNA was isolated and equal amounts of RNA were subjected to Northern blot analysis to determine the pattern of expression of E2F-responsive genes (A,C). (B) The expression level of selected genes shown in A was quantitated by PhosphorImager analysis and normalized to the ARPP PO control.

Figure 5

Figure 5

The loss of E2F1 does not affect the expression of most E2F-responsive genes. Wild-type, heterozygous, or homozygous E2f1 mutant MEFs were synchronized by serum starvation/readdition and Northern blot analysis was performed at various cell cycle stages. The expression levels of selected genes was quantitated by PhosphorImager analysis and normalized to the ARPP PO control.

Figure 6

Figure 6

The proliferation defect of the _E2f3_−/− cells can be rescued by the ectopic expression of E2F3 or E2F1. Wild-type or E2f3 mutant MEFs were infected with either control or E2F3- or E2F1-expressing retroviruses. (●) E2f3+/+; (○) _E2f3_−/−. After 4 days in selection, the cells were plated at equal densities and their growth rates monitored.

Figure 7

Figure 7

E2F3 deficiency impairs the proliferation of transformed cell lines. Wild-type or _E2f3_−/− MEFs were infected with retroviruses expressing an activated ras allele (H-rasV12) and either E1A or a dominant-negative p53 allele, p53 R175H. After selection, cells were plated at equal densities and their proliferation rates were monitored by daily counting. To assess anchorage-independent growth, equal numbers of transformed cells were plated in 0.3% low melting point agarose. Representative wild-type and E2f3 mutant fields are shown.

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