A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression

Abstract

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, we have conducted a genome-wide RNAi screen in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related CG16975/dSfmbt. These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth.

Fig. 1.

RNAi analysis of the E2F pathway in Drosophila cell culture. (A) Schematics of the reporter constructs. The E2F-dependent reporter (E2F-Luc) carries four copies of an artificial E2F site (4xE2F), a minimal HSP70 promoter (HSP), and the firefly luciferase gene (Luc). The Renilla control reporter contains the same HSP and the Renilla luciferase gene (RLu). The two constructs share the same plasmid backbone. (B) Effects of RNAi depletion of known E2F pathway components on the E2F-reponsive reporter. Drosophila S2* cells were transiently transfected with E2F-Luc and the control reporter and incubated with dsRNAs in 24-well plates. RNAi of dE2F1/dDP and Rbf1 considerably decreased and increased the E2F-dependent reporter, respectively. RNAi of Rbf2 or dE2F2 had little effect. The reporter responses remain constant during incubation days 4–8. Similar results were also obtained by using several other Drosophila cell lines (e.g., S2, Kc167, Dl2, Cl8).

Fig. 2.

Regulation of E2F by the Dom complex and MBT proteins. (A) Effects of RNAi of the Dom complex components and their epistatic effect relative to Rbf1. (B) Epistasis analysis of the MBT proteins (L3mbt and CG16975) relative to Dom and Rbf1. (C) Epistasis analysis of known and potential E2F regulators including Rbf1, Dom, and MBTs relative to dE2F1 and Drosophila cdk4. (D) A model for Dom and MBT-mediated regulation of E2F. Dom and MBT confer E2F inhibitory activities that are independent of Rb. They may act downstream of or in parallel to cdk. Absence or small scale of error bar indicates that results from duplicate experiments are nearly identical.

Fig. 3.

The Dom and MBT proteins form complexes with E2F and are present on E2F target gene promoters. (A) The Dom and MBT proteins associate with E2F. Cellular extracts from regular S2* cells or S2* cells transiently transfected with a Flag-L3mbt expression vector were subjected to affinity purification with antibodies for Dom, Rbf1, dE2F1, and Flag and Western blot analysis with anti-dE2F1. No signal was observed in control precipitation in which cellular lysates from untransfected cells were incubated with the anti-Flag antibody. (B) The Dom, H2Av, and MBT proteins are present at the promoters of E2F target genes. Chromatin from cross-linked regular S2* cells was immunoprecipitated with anti-Dom (Dom), anti-H2Av (H2Av), and anti-Flag (control) antibodies. In a parallel experiment (for the right three columns), extracts from S2* cells expressing Flag-L3mbt were precipitated with anti-Flag (F-L3mbt) and nonspecific IgG (control) antibodies. The pelleted chromatin fragments were analyzed by PCR with primers specific for PCNA, DNA polα (Pol), CG14545, CG8399, CG3105 (belonging to the groups A to E, respectively), and RP49 promoters. (C) Recruitment of Dom and MBT proteins to E2F targets depends on E2F. Flag-L3mbt transfected S2* cells were treated with RNAi of dE2Fs (dE2F1, or combined dE2F1 and dE2F2), and then subjected to ChIP with Dom and Flag antibodies, respectively. Several E2F targets were selected for promoter-binding assay. (D) Deposition of H2Av at selected E2F target promoters depends on Dom and dE2F1. S2* cells were incubated with Dom or dE2F1 dsRNAs and subsequently subjected to ChIP assays with an anti-H2Av antibody.

Fig. 4.

Changes in gene expression and histone modification induced by depletion of MBT. (A) Northern blot analysis of MBT-repressed E2F target genes. S2* cells were treated with RNAi of indicated E2F regulators. Total RNA was extracted from cells incubated with dsRNAs for 6 days, and the blots were probed for expression of several E2F target genes. The ethidium bromide staining of rRNA showed comparable loadings for each sample. Depletion of MBTs resulted in a strong induction in RNA expression of the selected E2F targets, pointing out their essential role in repression of endogenous E2F-regulated genes. (B) Epigenetic changes at E2F target promoters after RNAi of MBT. S2* cells were treated with RNAi of L3mbt and CG16975 for 6 days and then subjected to ChIP with antibodies recognizing dimethylated H3K9 or acetylated H4. Several E2F targets were chosen for promoter-binding analysis.

Fig. 5.

dom mutation genetically suppresses cycE and enhances the phenotype of transgenic dE2F1. (A) Homozygotic cycE^JP/cycE^JP flies display a small eye phenotype because of reduced cell proliferation during eye development. (B) In a dom heterozygotic background, the cycE^JP eye phenotype is rescued in the cycE^JP, dom¹/cycE^JP flies. (C) Flies heterozygous for both cycE^JP and dom (cycE^JP, dom¹/+) are normal and do not exhibit any obvious phenotype. (D) Mutation in his2Av also suppresses the cycE^JP eye phenotype as shown in the cycE^JP/cycE^JP; his2Av/+ flies. (E) Eyes of the cycE and his2Av double heterozygous flies (cycE^JP/+; his2Av/+) are normal. (F) Transgenic flies (GMR-dE2FdDPp35/+) with overexpression of dE2F1, its dimerization partner dDP, and p35 (a baculovirus antiapoptotic protein) in the eye imaginal disc using the GMR promoter causes an increase in the number of cells in the adult eye and disruption of the regular pattern of ommatidia. (G) Reducing the dosage of dom using the dom¹⁴ heterozygotes (GMR-dE2FdDPp35/dom) resulted in strong enhancement of the E2F rough eye phenotype. All pictures were taken at ×100 magnification.