dDP is needed for normal cell proliferation - PubMed (original) (raw)

dDP is needed for normal cell proliferation

Maxim V Frolov et al. Mol Cell Biol. 2005 Apr.

Abstract

To gain insight into the essential functions of E2F, we have examined the phenotypes caused by complete inactivation of E2F and DP family members in Drosophila. Our results show that dDP requires dE2F1 and dE2F2 for DNA-binding activity in vitro and in vivo. In tissue culture cells and in mutant animals, the levels of dE2F and dDP proteins are strongly interdependent. In the absence of dDP, the levels of dE2F1 and dE2F2 decline dramatically, and vice versa. Accordingly, the cell cycle and transcriptional phenotypes caused by targeting dDP mimic the effects of targeting both dE2F1 and dE2F2 and are indistinguishable from the effects of inactivating all three proteins. Although trans-heterozygous dDP mutant animals develop to late pupal stages, the analysis of somatic mutant clones shows that dDP mutant cells are at a severe proliferative disadvantage when compared directly with wild-type neighbors. Strikingly, the timing of S-phase entry or exit is not delayed in dDP mutant clones, nor is the accumulation of cyclin A or cyclin B. However, the maximal level of bromodeoxyuridine incorporation is reduced in dDP mutant clones, and RNA interference experiments show that dDP-depleted cells are prone to stall in S phase. In addition, dDP mutant clones contain reduced numbers of mitotic cells, indicating that dDP mutant cells have a defect in G2/M-phase progression. Thus, dDP is not essential for developmental control of the G1-to-S transition, but it is required for normal cell proliferation, for optimal DNA synthesis, and for efficient G2/M progression.

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Figures

FIG. 1.

FIG. 1.

E2F-specific DNA-binding activity is dependent upon dDP in vitro. (A) Total extracts of SL2 cells were prepared and analyzed in EMSA with oligonucleotides containing an E2F-binding site (underlined) (ATTTAAG

TTTCGCGC

CCTTTCTCAAATTT) (left lane). The specificity of the retarded complex was verified by preincubating the extracts with a 100-fold molar excess of competing unlabeled oligonucleotides containing either a wild-type (WT) or a mutant (MT) E2F-binding site. End-labeled oligonucleotide alone was also migrated in the gel (right lane). The nonspecific band is designated by an asterisk. (B) Cells were treated with nonspecific dsRNA used as a control (NS) or with dE2F1 (E1), dE2F2 (E2), and dDP (dDP) dsRNAs. Note the lack of E2F-specific DNA-binding activity in SL2 cells following depletion of dDP.

FIG.2.

FIG.2.

dE2F2 colocalizes with dDP on polytene chromosomes. (A to F) Wild-type polytene chromosomes were stained with anti-dE2F2 and anti-dDP antibodies and counterstained with YOYO to visualize DNA. (G to I) Higher magnification of a fragment of wild-type polytene chromosome that was costained with anti-dE2F2 and anti-dDP antibodies. The arrows indicate one of the sites that stains for dDP but not for dE2F2. (J to L) dDP mutant chromosomes stained with anti-dE2F2 antibody. (M to O) de2f2 mutant chromosomes stained with anti-dDP antibody. Note the reduction in the number of dDP binding sites in the de2f2 mutant compared to the wild type. (P to R) de2f2 de2f1 double-mutant chromosomes stained with anti-dDP antibody.

FIG. 3.

FIG. 3.

Proliferative properties of SL2 cells depleted of dE2F1, dE2F2, or dDP. (A) Cells depleted of dE2F1, dE2F2, and dDP or their combinations were labeled with BrdU, and the percentage of BrdU-positive cells were assayed by FACS. The number of BrdU-positive cells is dramatically reduced in the absence of dE2F1. Removal of dE2F2 does not significantly affect the number of BrdU-positive cells but restores BrdU incorporation lost in cells depleted of dE2F1. Note that dE2F1/dE2F2-, dDP-, dE2F1/dDP-, and dE2F1/dE2F2/dDP-depleted cells show similar values of BrdU incorporation. The error bars indicate standard deviations. NS, nonspecific dsRNA. (B) Total RNA was isolated from dsRNA-treated cells, and the levels of PCNA, cyclin E, RNR2, and MCM3 transcripts were followed by Northern blot analysis. (C) Northern analysis of total RNAs isolated from eye disks of different genotypes shows that E2F-regulated genes are expressed at similar levels in dDP mutant and de2f1 de2f2 double-mutant animals.

FIG. 4.

FIG. 4.

S-phase defects of SL2 cells depleted of dE2F1, dE2F2, or dDP. (A and B) Cells depleted of dE2F1, dE2F2, and dDP were labeled with [3H]thymidine to measure DNA synthesis or with BrdU for 16 h and subjected to two-dimensional FACS analysis. The error bars indicate standard deviations. (C) Proliferation indices of cells following RNAi treatment. Loss of dE2F1 results in a significant reduction of S-phase cells and an increased G1 population. Removal of dE2F2 restores S-phase entry defects of dE2F1-depleted cells but does not rescue S-phase progression defects. Despite containing BrdU-positive cells, populations of dDP, dE2F1/dE2F2, dE2F1/dDP, and dE2F1/dE2F2/dDP RNAi-treated cells have low rates of DNA synthesis and proliferate very slowly. In populations of dDP- or dE2F1/dE2F2-depleted cells, a substantial number of BrdU-negative cells have intermediate DNA contents, indicating that these cells are stalled within S phase.

FIG. 5.

FIG. 5.

dE2F1 is not sufficient to induce E2F-dependent transcription. (A and B) Eye disks were stained with anti-dE2F1 antibody. (A) In a wild-type disk, dE2F1 is expressed in the morphogenetic furrow and in the cells anterior and posterior to the furrow. (B) No staining with anti-dE2F1 antibody is observed in the eye disks _trans-_heterozygous for de2f1 mutant alleles. (C) In situ hybridization of eye disks from wild-type larvae with PCNA probe. PCNA transcripts are detected in cells posterior to the furrow. (D) Direct comparison of dE2F1 (red) and PCNA (green) expression patterns using a transgene producing a GFP-PCNA fusion protein. Arrow, position of the morphogenetic furrow.

FIG. 6.

FIG. 6.

Levels of dE2F1 and dE2F2 are reduced in dDP mutant clones. Clones were induced with ey-FLP, and eye disks were stained with anti-dE2F2 (A to D) or with anti-dE2F1 (E to G) antibody. Mutant clones were identified by the absence of GFP marker (green). (A and B) dE2F2 protein is at a low level in dDP mutant clones. (C and D) The loss of dE2F2 staining is rescued by a dDP transgene. (E to G) dE2F1 staining is reduced and diffuse in a dDP mutant clone. Arrow, position of the morphogenetic furrow.

FIG. 7.

FIG. 7.

dDP and dE2F2 levels are interdependent in mutant animals and in cells. (A) Cells were treated with nonspecific (NS), dE2F1 (E1), dDP, and dE2F2 (E2) dsRNAs, and cell extracts were analyzed on Western blots using antibodies specific for dE2F1, dE2F2, and dDP. Note that depletion of dDP affects the levels of both dE2F1 and dE2F2, and conversely, dDP is reduced following depletion of dE2F1 and dE2F2. Anti-tubulin mouse monoclonal antibody was used as a loading control. (B) Western blot analysis of larval extracts from Canton S (wild-type), de2f2 76Q1/de2f2 G5.1 (de2f2 −), de2f2 76Q1/de2f2 G5.1, de2f1 91/de2f1 rm729 (de2f2 −; de2f1 −), and dDP a2/Df(2R)vg-B (dDP −) animals. (C) A wild-type level of dDP can be restored in de2f2 mutants by the reexpression of dE2F2. A heat shock-inducible de2f2 transgene, hs-de2f2, was introduced into de2f2 mutant animals. The level of dDP in larvae was monitored by Western blot analysis before heat shock (no HS) and at 30 min (30′ AHS) and 2 h (2 h AHS) following a 1-h heat shock (de2f2 − hs-de2f2). The rescue was not due to the heat shock treatment, because the level of dDP was unchanged in de2f2 mutants that lack an hs-de2f2 transgene (de2f2 −). (D) Northern blot analysis of larvae of the same genotypes used in panel B. 32P-labeled RNA probes for de2f1, de2f2, and dDP reveal that there is no decrease in the steady-state level of dDP mRNA in the de2f1 de2f2 double mutant or of de2f1 and de2f2 mRNAs in the dDP mutant.

FIG. 8.

FIG. 8.

dDP mutant cells have a severe proliferative disadvantage. Clones of dDP mutant cells were induced at the first-instar larval stage by mitotic recombination. Examples of the clones observed in the eye (A) and wing (B) imaginal disks are presented. Homozygous mutant clones were visualized by the absence of GFP, while homozygous wild-type twin spots (+/+ in panel B) show a doubled GFP signal on a heterozygous background. (C) Clones were induced as described for panel B, wing imaginal disks were dissected and fixed at the early third-instar larval stage, and the sizes of the mutant clones and the corresponding twin spots were determined. The bars are ordered according to the size of the wild-type twin spots. Values for average clone areas of dDP mutant cells are indicated.

FIG. 9.

FIG. 9.

Cell cycle characteristics of dDP mutant cells. Clones of dDP mutant cells were induced with ey-FLP, and eye imaginal disks were labeled with BrdU to visualize cells in S phase (A to D, J, and K) or stained with anti-cyclin A antibody (E and F), anti-cyclin B antibody (G and H), and anti-phos-H3 antibody (I to L). Mutant cells lack GFP signal. (A and B) Cells in dDP mutant clones incorporate BrdU at the same time as wild-type cells but with less efficiency. (C and D) Reduced efficiency of BrdU labeling in dDP mutant clones is fully rescued by a dDP genomic rescue construct. The timing of induction of cyclin A (E and F) and cyclin B (G and H) is normal in a dDP mutant clone. The number of cells in mitosis is reduced in dDP mutant cells, as visualized by anti-phos-H3 staining (I). Ectopic expression of cyclin E restores the efficiency of BrdU incorporation (J and K) but not the reduced number of cells entering mitosis (L) in dDP mutant clones. Arrow, position of the morphogenetic furrow.

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