In vivo regulation of E2F1 by Polycomb group genes in Drosophila

Abstract

The E2F transcription factors are important regulators of the cell cycle whose function is commonly misregulated in cancer. To identify novel regulators of E2F1 activity in vivo, we used Drosophila to conduct genetic screens. For this, we generated transgenic lines that allow the tissue-specific depletion of dE2F1 by RNAi. Expression of these transgenes using Gal4 drivers in the eyes and wings generated reliable and modifiable phenotypes. We then conducted genetic screens testing the capacity of Exelixis deficiencies to modify these E2F1-RNAi phenotypes. From these screens, we identified mutant alleles of Suppressor of zeste 2 [Su(z)2] and multiple Polycomb group genes as strong suppressors of the E2F1-RNA interference phenotypes. In validation of our genetic data, we find that depleting Su(z)2 in cultured Drosophila cells restores the cell-proliferation defects caused by reduction of dE2F1 by elevating the level of dE2f1. Furthermore, analyses of methylation status of histone H3 lysine 27 (H3K27me) from the published modENCODE data sets suggest that the genomic regions harboring dE2f1 gene and certain dE2f1 target genes display H3K27me during development and in several Drosophila cell lines. These in vivo observations suggest that the Polycomb group may regulate cell proliferation by repressing the transcription of dE2f1 and certain dE2F1 target genes. This mechanism may play an important role in coordinating cellular differentiation and proliferation during Drosophila development.

Keywords: Drosophila; E2F1; PcG; Su(z)2; cell proliferation.

Figure 1

Tissue-specific expression of dE2f1-dsRNA generates phenotypes that can be modified by known factors of the dE2F1 pathway. (A) A normal Drosophila eye (w¹¹¹⁸; GMR-Gal4/+; +/+). (B) Expressing one copy of the UAS-dE2f1-dsRNA (Line #10) generates a slight rough eye phenotype (w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#10/+; +/+), which can be enhanced by reducing the endogenous dE2f1 levels, as shown in (C) (w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#10/+; dE2f1ⁱ²/+), and completely rescued by overexpressing wild-type dE2f1, as shown in (D) (w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#10/+; UAS-dE2f1⁺/+). A stronger rough eye phenotype is generate when multiple copies of UAS-dE2f1dsRNA (line #8) is expressed, as shown in (E) (w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#8/+; +/+). This stronger phenotype can be suppressed by overexpressing wild-type dCycA (F: w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#8/+; UAS-dCycA⁺/+), wild-type dCycE (G: w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#8/UAS-dCycE⁺; +/+), or dCdk4 and dCycD (H: w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#8/+; UAS-dCdk4⁺, UAS-dCycD⁺/+). The scale bar (in H) is 200µm.

Figure 2

Su(z)2 is a strong suppressor of the dE2f1-dsRNA phenotypes in the eye. (A) The design of the dominant modifier genetic screen using deficiency lines. (B-D) shows the modification of the dE2f1-dsRNA eye phenotype by Su(z)2 alleles. The eye phenotype of GMR-Gal4, UAS-dE2f1dsRNA#8/+ (B) flies can be strongly suppressed by the Df(2R)Exel6062 line (C, the genotype is w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#8/Df(2R)Exel6062; +/+) and a null allele of Su(z)2 (D, the genotype is w¹¹¹⁸; GMR-Gal4, UAS-dE2f1dsRNA#8/Su(z)2^1.b7; +/+). (E) Summary of the genetic interactions between Su(z)2 alleles and dE2f1-dsRNA phenotypes in the eye and wing. The suppressive effect was ranked with scores from 1 to 5, with “1” being the weakest and “5” the strongest. “0” means no genetic interaction. The scale bar in (D) is 200µm.

Figure 3

Su(z)2 and additional PcG genes are strong suppressors of the dE2f1-dsRNA phenotypes in the wing. (A) Part of L3-L4 intervein region of a control Drosophila wing (ptc-Gal4/+). Ptc-Gal4 is expressed in the L3-L4 intervein region. At 22∼23°, when dE2f1-dsRNA (line #3) is expressed under control of ptc-Gal4, the L3-L4 intervein region is reduced by ∼50%, as shown in (B) (w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; +/+). This wing phenotype can be strongly suppressed by Df(2R)Exel6062 (C: w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Df(2R)Exel6062; +/+), or the Su(z)2^1.a1 allele (D: w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Su(z)2^1.a1; +/+). The modification of the wing phenotype can be quantified by measuring the width of L3-L4 intervein region (E), and the genotypes of data presented in (E) are as follows: (a) w¹¹¹⁸; ptc-Gal4/+; +; (b) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; +; (c) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Df(2R)Exel6062; +/+; (d) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Su(z)2¹; +/+; (e) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Su(z)2^1.b7; +/+; (f) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Su(z)2^1.a1; +/+; and (g) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/Su(z)2^k06344; +/+; (h) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; Pc^f01890/+; (i) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; Pc³/+; (j) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; His2AvD⁸¹⁰/+; and (k) w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; His2AvD⁰⁵¹⁴⁶/+. At least 15 to 25 wings of each genotype (a∼k) were measured. Each genotype (c∼k) was compared with the control (b: w¹¹¹⁸; ptc-Gal4, UAS-dE2f1dsRNA#3/+; +) and each comparison is highly significant (P < 4.9E-06 based on one-tailed t-test). For simplicity, “ptc-Gal4, UAS-dE2f1dsRNA#3” is referred as “PE3” in (E). The scale bar in (D) is 100µm.

Figure 4

Su(z)2 regulates the transcription of dE2f1 and some of the dE2F1 target genes. (A) Knocking down Su(z)2 (gray bars) leads to up-regulation of dE2f1, and some of the dE2F1 target genes, such as PCNA, dCycE, and to a less extent dE2f2 and no effect of Rbf1, based on qRT-PCR assay. The samples treated with dE2f1-dsRNAs (white bars) serve as a positive control, and T7-dsRNA treated samples are negative controls. (B) Codepletion (gray bars) of Su(z)2 and dE2f1 suppresses the effect of dE2f1-dsRNA treatment and leads to increased expression of dE2f1, PCNA, dCycE, stg, and mcm5. The total dsRNAs are normalized with T7-dsRNA. (C and D) Effect of dsRNA treatment of the growth of SL2 cells: knocking down of Su(z)2 (C), but not Psc (D), suppresses the effect of dE2f1-dsRNA treatment at day 5. For each sample, the total amount of dsRNA is normalized with white-dsRNA and cell viability was determined by using the dimethyltriazoldiphenyl tetrazolium-formazan assay after 1, 3 or 5 days of dsRNA treatment.