IntroductionThe retinoblastoma tumor suppressor protein (pRB) and its relatives, p107 and p130, are negative regulators of cell proliferation that figure prominently in most models of cellcycle control. Cell-cycle progression is driven by mitogenic growth signals. These signals result in the synthesis of G1 cyclins, the positive-regulatory partners of cyclin-dependent kinases (CDKs), and eventually the accumulating pool of CDK activity overrides the constraining effects of CDK inhibitors. Once active, G1-specific CDKs target multiple substrates, including the pRB family. By phosphorylating pRB-family members, CDKs relieve the constraints on cell proliferation that this group of proteins maintain. Although pRB-family members are periodically inactivated during the normal cell cycle, these proteins are also inappropriately inactivated in several other situations. pRB-family proteins are targeted by DNA tumor virus proteins during oncogenic transformation, and pRB is thought to be functionally inactivated in most tumor cells, either through mutation of the RB1 gene itself or through dysregulation of the kinases that control its activity.Precisely how pRB-family proteins control cell proliferation is not completely understood. There is a broad range of possibilities given that pRB-family members associate with a wide variety of transcription factors and chromatin-associated complexes. Nevertheless, pRB-family members are generally believed to function through their effects on the transcription of genes regulated by the E2F proteins. E2F-binding sites are found in the promoters of many genes whose functions are needed for cell proliferation or whose products drive cell-cycle progression. The best-studied forms of E2F are heterodimeric complexes that contain one subunit encoded by the E2F family together with a subunit encoded by the DP family. Mammalian cells contain at least seven E2F-family members ( Fig. 1) and two DP-family members. Although subunits E2F-1 to E2F-6 act as heterodimers with a DP subunit, the recently described E2F-7 subunit binds to DNA in a DP-independent manner. E2F-1 to E2F-5 associate with pRB-family members, whereas E2F-6 and E2F-7 appear to act independently of pRB-family proteins.For simplicity, the E2F family is often subdivided into activator E2Fs (E2F-1, E2F-2 and E2F-3a) and repressor E2Fs (E2F-4, E2F-5 and E2F-6). This classification is based upon differences in the ability of these overexpressed proteins to activate transcription, or to drive quiescent cells into the cell cycle, as well as on the phases of the cell cycle where the E2F proteins can be shown to be present at E2F-regulated promoters. However, the distinction between these two groups is not cut and dried. For example, 'repressor' E2Fs can activate transcription when overexpressed, and 'activator' E2Fs have the potential to form complexes with repressor proteins. Nevertheless, a separation of activator and repressor E2Fs is useful in concept, and the distinction is reinforced by the pattern of interactions between E2F-and ...

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.

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. Here, we describe the properties of RBF2, a second family member. RBF2 represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. dE2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to dE2F2, a form of E2F that functions as a transcriptional repressor. We find that RBF2-mediated repression requires dE2F2. More over, RBF2 and dE2F2 act synergistically to antagonize dE2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and dE2F1 or dE2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in DROSOPHILA.