The retinoblastoma tumor suppressor protein (pRb) regulates gene transcription by binding E2F transcription factors. pRb can recruit several repressor complexes to E2F bound promoters; however, native pRb repressor complexes have not been isolated. We have purified E2F/RBF repressor complexes from Drosophila embryo extracts and characterized their roles in E2F regulation. These complexes contain RBF, E2F, and Myb-interacting proteins that have previously been shown to control developmentally regulated patterns of DNA replication in follicle cells. The complexes localize to transcriptionally silent sites on polytene chromosomes and mediate stable repression of a specific set of E2F targets that have sex- and differentiation-specific expression patterns. Strikingly, seven of eight complex subunits are structurally and functionally related to C. elegans synMuv class B genes, which cooperate to control vulval differentiation in the worm. These results reveal an extensive evolutionary conservation of specific pRb repressor complexes that physically combine subunits with established roles in the regulation of transcription, DNA replication, and chromatin structure.

No abstract

To determine which E2F/RB-family members are functionally important at E2F-dependent promoters, we used RNA interference (RNAi) to selectively remove each component of the dE2F/dDP/RBF pathway, and we examined the genome-wide changes in gene expression that occur when each element is missing. The results reveal a remarkable division of labor between family members. Classic E2F targets, encoding functions needed for cell cycle progression, are expressed in cycling cells and are primarily dependent on dE2F1 and RBF1 for regulation. Unexpectedly, there is a second program of dE2F/RBF-dependent transcription, in which dE2F2/RBF1 or dE2F2/RBF2 complexes repress gene expression in actively proliferating cells. These new E2F target genes encode differentiation factors that are transcribed in developmentally regulated and gender-specific patterns and not in a cell cycle-regulated manner. We propose that dE2F/RBF complexes should not be viewed simply as a cell cycle regulator of transcription. Instead, dE2F/RBF-mediated repression is exerted on genes that encode an assortment of cellular functions, and these effects are reversed on sets of functionally related genes in particular developmental contexts. As a result, dE2F/RBF regulation is used to link gene expression with cell cycle progression at some targets while simultaneously providing stable repression at others.[Keywords: E2F; retinoblastoma protein; RBF; Drosophila; cell cycle; differentiation] Supplemental material is available at http://www.genesdev.org.

E2F is a heterogenous transcription factor and its role in cell cycle control results from the integrated activities of many different E2F family members. Unlike mammalian cells, that have a large number of E2F-related genes, the Drosophila genome encodes just two E2F genes, de2f1 and de2f2. Here we show that de2f1 and de2f2 provide different elements of E2F regulation and that they have opposing functions during Drosophila development. dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F-regulated genes in vivo. dE2F1 is a potent activator of transcription, and the loss of de2f1 results in the reduced expression of E2F-regulated genes. In contrast, dE2F2 represses the transcription of E2F reporters and the loss of de2f2 function results in increased and expanded patterns of gene expression. The loss of de2f1 function has previously been reported to compromise cell proliferation. de2f1 mutant embryos have reduced expression of E2F-regulated genes, low levels of DNA synthesis, and hatch to give slow-growing larvae. We find that these defects are due in large part to the unchecked activity of dE2F2, since they can be suppressed by mutation of de2f2. Examination of eye discs from de2f1; de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins. This study shows how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions.