This study was funded in part by the Howard Hughes Medical Institute. growth. genome encodes two E2F factors (dE2F1 and dE2F2), a single dDP protein as the heterodimeric partner for dE2Fs, and two Rb homologs (Rbf1 and Rbf2). The highly effective RNAi-mediated disruption of gene function in cultured cells provides a loss-of-function approach to examining the requirement of potential E2F regulators. However, such a study suggested that the HDACs and SWI/SNF chromatin-remodeling complex were dispensable for E2F inhibition (10), raising questions about the mechanisms underlying actions of Rb, and more generally, repression of E2F, which is important for tumor suppression. In the present study, we carried out a genome-wide RNAi screen in cultured fly cells CNQX disodium salt (11) to systematically search for essential E2F repressors. Here we describe genes identified from the screen that negatively regulate E2F. These factors are likely to be modulators of chromatin structure, and their mutations cause abnormal cell proliferation promoter and tandem copies of an E2F-binding site, which can bind E2Fs and Rb/E2F complexes (12). A luciferase reporter driven by the same minimal promoter was included as a normalization control. Comparison between the two reporters allowed us to detect E2F-specific activity. S2* cells were transiently transfected with both reporter constructs and then cultured in the presence of synthesized long pieces of dsRNA. S2* cells can take up dsRNAs from the medium and process them into numerous siRNAs to efficiently degrade mRNAs of specific target genes. We tested the reporter system by RNAi of known positive and negative regulators. As expected, RNAi knockdown of dE2F1 or dDP decreased the E2F-dependent reporter activity, whereas RNAi depletion of Rbf1 substantially up-regulated the reporter (Fig. 1cell culture. (control reporter contains the same HSP and the luciferase gene (RLu). The two constructs share the same plasmid backbone. (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 4C8. Similar results were also obtained by using several other cell lines (e.g., S2, Kc167, CNQX disodium salt Dl2, Cl8). We next examined a panel of chromatin-modulating proteins, most of which were previously implicated in Rb-E2F regulation. However, RNAi-mediated removal of HMTs, including Suv4C20; Suv3C9; and fly homologs of G9a, ESET, and arginine methyltransferase PRMT5, failed to activate the E2F reporter [supporting information (SI) Fig. 6]. In addition, depletion of HDAC1, or Brm and Mi2, the ATPase subunits of two chromatin-remodeling complexes SWI/SNF and Mi2, respectively, did not increase the reporter activity (SI Fig. 6). Combined RNAi of some of these genes produced similar results (not shown). Therefore, even though Rb represses E2F in this assay, none of these chromatin CNQX disodium salt repressors is required for inhibition of E2F. This conclusion is in agreement with a previous report using endogenous E2F targets as a readout (10). Although this result might Rabbit Polyclonal to TBX3 reflect the redundant or context-dependent activities of these regulators, it prompted us to systematically screen the whole genome by RNAi for additional chromatin-related factors that are essential for E2F repression. In initial control experiments and pilot screens involving hundreds of genes tested, Rbf1 was detected as the only predominant negative regulator of E2F, and RNAi of the rest of the genes did not display any significant reporter activation (generally a 10% increase) above background. From a high-throughput screen of the dsRNA library containing 21,000 dsRNA species and covering 90% of the annotated genome (11), only 18 dsRNAs CNQX disodium salt up-regulated the reporter to 50% (SI Table 1), which was set as an arbitrary cutoff. Identification of Rbf1 provided validation for the screen. Repression of E2F Activity by the Dom and Malignant Brain Tumor (MBT) Proteins. Among the identified genes are several potential chromatin regulators, including Domino (Dom) (14) and two uncharacterized fly genes, CG14514 and CG4621, all of which are homologous to the yeast SWR1 chromatin-remodeling complex subunits Swr1, Bdf1, and Vps72, respectively (15C17). Dom is a member of the SNF2 family of ATPases (14). The three factors were previously found to be copurified in a fly chromatin-modulating complex (18). To verify whether these factors repress E2F, we synthesized new dsRNAs from distinct sets of gene-specific primers and repeated the RNAi/reporter assay. Consistent with the original screen, RNAi depletion of Dom, CG14514 or CG4621, resulted in activation of the.