dE2F2-independent rescue of proliferation in cells lacking an activator dE2F1

Abstract

In Drosophila melanogaster, the loss of activator de2f1 leads to a severe reduction in cell proliferation and repression of E2F targets. To date, the only known way to rescue the proliferation block in de2f1 mutants was through the inactivation of dE2F2. This suggests that dE2F2 provides a major contribution to the de2f1 mutant phenotype. Here, we report that in mosaic animals, in addition to de2f2, the loss of a DEAD box protein Belle (Bel) also rescues proliferation of de2f1 mutant cells. Surprisingly, the rescue occurs in a dE2F2-independent manner since the loss of Bel does not relieve dE2F2-mediated repression. In the eye disc, bel mutant cells fail to undergo a G1 arrest in the morphogenetic furrow, delay photoreceptor recruitment and differentiation, and show a reduction of the transcription factor Ci155. The down-regulation of Ci155 is important since it is sufficient to partially rescue proliferation of de2f1 mutant cells. Thus, mutation of bel relieves the dE2F2-mediated cell cycle arrest in de2f1 mutant cells through a novel Ci155-dependent mechanism without functional inactivation of the dE2F2 repressor.

FIG. 1.

Rescue of proliferation in clones of de2f1 mutant cells by mutation of de2f2. Eye imaginal discs were dissected from third-instar larva. Clones of mutant cells were induced with ey-FLP and are identified by the lack of GFP (green). Posterior is to the right. Genotypes are shown. (A) Control experiment showing clones of homozygous tissue with the wild-type chromosome. In the absence of mutations, there are approximately equal amounts of tissue marked by GFP and by the lack of GFP. (B) Clones of de2f2^c03344 mutant cells (GFP negative) do not express dE2F2 protein. (C, C*, D, and D*) Induction of clones of cells that are homozygously mutant for de2f2^c03344 and de2f1⁷²⁹. Double-mutant cells fail to express GFP. Cells that are homozygously mutant for the de2f1⁷²⁹ allele express β-Gal (red) from the PZ[lacZ] element inserted into the de2f1 gene. Note that large clones of de2f1 homozygous mutant cells (red) can be recovered only in de2f2 homozygous mutant cells (lack of GFP), while, in the presence of de2f2 (GFP positive), clones of de2f1 mutant cells do not exceed one to three cells (yellow). (C*) GFP clone marker alone of the same image as in panel C. (D and D*) Higher magnification of the same image as in panels C and C*. Two clones of de2f1 de2f2 double-mutant cells are outlined. Arrows point to de2f1 single-mutant cells that carry wild-type de2f2. Staining with DNA dye 4′,6′-diamidino-2-phenylindole confirms that these are individual cells. (D*) GFP clone marker of the same image as in panel D.

FIG. 2.

The loss of bel rescues proliferation of de2f1 mutant cells. Wild-type cells are marked by the presence of GFP (green), while mutant tissue lacks GFP. Clones of mutant cells were induced with ey-FLP and are identified by the lack of GFP (green). (A) Clones of bel^7d19 de2f1⁷²⁹ double-mutant cells. (B and B*) The loss-of-function bel^L4740 mutant allele makes no Bel protein (red), as revealed by the absence of staining with anti-Bel antibody in clones of bel^L4740 mutant cells (absence of green). (B*) The same image as in panel B, showing expression of Bel (red) without GFP. (C) The known loss-of-function bel^L4740 mutant allele rescues proliferation of de2f1 mutant cells. Note the appearance of patches of double-mutant, GFP-negative tissue. (D) The eye discs were labeled with BrdU (red) to visualize S phases. The MF is marked by the arrowhead. Wild-type cells asynchronously cycle anterior to the MF, are arrested in G₁ within the MF, and synchronously enter S phase in the SMW posterior to the MF. The loss of bel partially rescues the SMW in de2f1 mutant cells, as evident by the appearance of bel de2f1 double-mutant BrdU-positive cells in the SMW. (E) Distribution of Bel protein in a wild-type eye disc. Bel is shown in red, and DAPI staining is shown in blue. Bel exhibits a predominantly cytoplasmic distribution.

FIG. 3.

Levels of dE2F2 and RBF1 are normal in bel mutant cells. Clones of mutant cells were induced with ey-FLP and are identified by the lack of GFP (green). (A, A*, B, and B*) Clones of bel mutant cells were induced in the eye discs, and the levels of RBF1 and dE2F2 were determined by immunofluorescence. Clones of mutant cells are distinguished by the lack of GFP. (A and A*) RBF1 (red) is expressed ubiquitously in the eye disc, with a slight increase anterior to the MF. Staining with anti-phospho-H3 antibody (blue in panel A) marks the positions of the MF (arrowhead) and the SMW. (A*) The same image as in panel A, showing phospho-H3 (white) and RBF1 (red) expression. (B and B*) dE2F2 (red) is expressed normally in clones of bel mutant cells (lack of GFP). (B*) The same image as in panel B, showing dE2F2 expression (red). (C) Bel was depleted by RNAi in S2 cells, and the levels of RBF1, cyclin A (CycA), CycB, and dE2F2 were determined by Western blot analysis. A nonspecific band for dE2F2 is marked by an arrow. Tubulin was used as a loading control.

FIG. 4.

Effect of Bel on dE2F2-mediated repression. (A) The loss of bel fails to restore the normal level of PCNA-GFP reporter (green) in the SMW inhibited in de2f1 mutant cells. bel de2f1 double-mutant cells are marked by the presence of β-Gal (red), and wild-type cells are distinguished by the lack of β-Gal. The position of the MF is shown by the arrowhead. (B) A well-known dE2F2-specific target, Arp53D, remains fully repressed in the eye discs containing clones of bel single-mutant and bel de2f1 double-mutant cells. Arp53D is strongly derepressed in dDP mutant eye discs (DP) and in the eye discs containing clones of dDP mutant cells (FLP DP). The level of Arp53D was determined by real-time reverse transcription-PCR. The means of the three experiments are shown above the bars. (C) Depletion of Bel in tissue culture cells has no effect on the ability of dE2F2 to repress a PCNA-luc reporter in transient transfections. Cells were incubated with control (solid bars) or Bel (open bars) double-stranded RNA (dsRNA). After 4 days, cells were transfected with a PCNA-luc reporter and with either an empty vector or pIE4-dE2F2 expression plasmids in triplicate. To normalize for the efficiency of transfection, a β-Gal expression plasmid was included. Depletion of Bel was verified by Western blotting. (D) Bel does not repress the E2F reporter in transient transfections. Cells were cotransfected with a PCNA-luc reporter; a β-Gal expression plasmid; and vector alone or a pIE-dE2F2, pIE4-dE2F1, or pIE-S-Bel expression construct. As expected, dE2F2 represses the E2F reporter while dE2F1 activates it. In contrast, overexpression of Bel does not repress the PCNA-luc reporter. Expression of exogenous Bel was verified by Western blotting as shown below.

FIG. 5.

bel mutant cells fail to arrest in G₁ in the MF. Clones of mutant cells are identified by the lack of GFP (green). (A and B) In wild-type discs, cells are arrested in G₁ in the MF (arrowhead). This is evident by the lack of cells that are BrdU positive (red) and express mitotic marker phospho-H3 (blue) (A) and the absence of cells that express the G₂ marker cyclin B (CycB; red) (B) within the MF. The image shown in panel A is at a higher magnification than the image in panel B. Anterior to the MF, the number of mitotic cells is increased. In the SMW, a synchronous wave of S phases is followed by a wave of mitoses. (C, D, E, and E*) bel mutant cells (GFP negative) fail to arrest in the anterior region of the MF and continue cycling, as evident by the appearance of mutant cells that show ectopic markers of S phase (BrdU), G₂ (CycB), and mitosis (phospho-H3). (C) bel mutant cells ectopically incorporate BrdU (red) in the MF. (D) bel mutant cells ectopically express CycB (red; arrow) in the MF. (E) bel mutant cells show ectopic CycB (red) and phospho-H3 (blue) in the MF. Double staining with CycB and phospho-H3 reveals cells that express only CycB (red; arrowhead), CycB, and phospho-H3 (magenta; thick arrow) and cells that degraded CycB but still express phospho-H3 (blue; thin arrow). Random distribution of the cell cycle markers indicates that bel mutant cells are asynchronously cycling. (E*) The same image as in panel E, showing the GFP clone marker (green) and CycB (red). The position of a clone is outlined. (F and G) bel mutant cells arrest normally following gamma irradiation. Clones of cells homozygous for wild-type (F) and bel^L4740 mutant (G) chromosomes were induced with ey-FLP. Larvae were irradiated and kept at 25°C for 1 h, and mitotic cells were visualized with anti-phospho-H3 antibody.

FIG. 6.

Mutation of bel specifically reduces the level of Ci155. Clones of bel mutant cells were induced with ey-FLP are marked by the absence of GFP (green). Panels marked with an asterisk lack the GFP signal, and clones of bel mutant cells are outlined. (A and A*) A marker of the Dpp pathway, pMad (red in panel A and white in panel A*), is expressed in bel mutant cells. (B and B*) Expression of Ci155 (red in panel A and white in panel A*) is strongly reduced in bel mutant cells. (C and C*) Ptc is expressed in differentiating ommatidia and at a low level in the MF (marked with a bar under the image). Ptc (red in panel C and white in panel C*) is strongly down-regulated in the MF in bel mutant cells. (D and D*) Expression of Notch (N) (red in panel D and white in panel D*) is not altered in bel mutant cells. (E and E*) Expression of the Notch ligand, Dl (red in panel E and white in panel E*), is normal in bel mutant cells. (F and F*) Expression of p-ERK, a marker of activated EGFR (red in panel F and white in panel F*), is unaffected by a mutation in bel. (G) Mutation of smo partially rescues the small clone size of de2f1 mutant cells. smo de2f1 double-mutant cells are distinguished by the lack of GFP (green). (H and I) The effect of Bel on transcriptional activation of a Ptc reporter gene in Drosophila S2 cells. (H) Ci155 fails to efficiently activate the reporter in Bel-deficient cells. Control cells (solid bars) or cells treated with Bel double-stranded RNA (dsRNA) (open bars) were transfected with an empty vector or pDA-Ci plasmid (Ci) together with a luciferase reporter plasmid under the control of either a wild-type (ptcΔ136-Luc) or mutant (ptcΔ136-mut) Ptc promoter. To determine transfection efficiency, a β-Gal expression plasmid was cotransfected in each well. The experiments were done in triplicate to calculate the averages and standard errors. (I) Bel potentiates Ci155-dependent activation of the Ptc reporter construct. Transfections were done essentially as in panel H but without an RNAi treatment, and an S-Tag-Bel expression plasmid was included. Expression of S-Tag-Bel was confirmed by Western blotting.

FIG. 7.

The loss of bel delays the onset of differentiation. Clones of bel mutant cells were induced with ey-FLP and are distinguished by the lack of GFP (green). Cells are stained with an R8 marker anti-Ato (A), neuronal marker anti-Elav (B), and the cone cell markers anti-Prospero (C), and anti-Cut (D), shown in red. Panels marked with an asterisk lack the GFP signal.