The RB and E2F proteins play important roles in the regulation of cell division, cell death, and development by controlling the expression of genes involved in these processes. The mechanisms of repression by the retinoblastoma protein (pRB) have been extensively studied at cell cycle-regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here, we have taken advantage of the simplicity of the E2F/RB pathway in flies to inspect the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex, dREAM/MMB, in a cell type-and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle-regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells, both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional mechanism of repression by the Polycomb group (PcG) protein Enhancer of zeste [E(Z)], which is involved in silencing of these genes through the dimethylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).The retinoblastoma protein (pRB) is a critical regulator of cell division, cell death, and differentiation in metazoans, and its activity is altered in most human tumors (9,22,47,48,60). The best understood property of pRB is its ability to modulate the action of the E2F family of transcription factors and to regulate cell cycle progression (11,13,56). pRB and the related proteins p107 and p130, collectively referred to as "pocket proteins," or RB family proteins (RB), bind to the heterodimeric E2F/DP factors and provide a module of transcriptional regulation that couples the expression of many genes with cell cycle progression. In quiescent cells, E2F and pocket proteins form repressive complexes that prevent the transcription of genes required for S-phase entry. This repression is then relieved at the G 1 -to-S transition by the activity of cyclin-dependent kinases (Cdk). At the promoters of cell cycleregulated genes, repressive E2F/RB complexes are replaced by activating E2Fs, and this allows for the coordinated expression of many genes required for cell division (13, 56).The biological activities of pRB extend beyond cell cycle regulation. Work in the past several years has greatly expanded the spectrum of genes regulated by E2F and RB. In addition to genes required for DNA replication and cell cycle progression, these now include a number of genes involved in sex determination, differentiation, and development (6,12,25,36,40,50,61,62,64). While pRB-dependent control of differentiation has been implicated in tumor suppression, the regulation of differentiation by pRB remains poorly understood (7,27,31).Despite extensive studies of the mechanism of repression by pRB at cell cycle target genes, little is known a...

E2F and RB proteins regulate the expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. Recent studies indicate that they function as part of an evolutionarily conserved multiprotein complex termed dREAM/ DREAM/LINC. Here we characterize the role of the Drosophila complex, dREAM, in the regulation of differentiation-specific E2F target genes in actively proliferating cells. These genes are regulated differently from cell cycle-related E2F targets, they do not depend on E2F activation, and E2F/RB repression is maintained throughout the cell cycle. In proliferating cells, their repression is dependent on dREAM. We find that dREAM plays a dual role in their regulation. First, it is required for the stability of the repressive dE2F2/RBF complexes at their promoters during S phase. Second, we find that dREAM is indispensable for both transcriptional repression mechanisms employed at these genes. Both cell division and exit from active proliferation cycles are integral parts of the development of multicellular organisms and are orchestrated by transcription factors that regulate spatiotemporal expression of specific sets of target genes. The E2F and RB families of transcription factors are critical regulators of these processes, and E2F/RB activity is altered in many human tumors (5,7,9,31,40).The E2F/RB pathway was first studied in the context of G 1 /S control (6, 9, 37). E2F proteins can be subdivided into repressors and activators of transcription. RB family members, also known as pocket proteins, negatively regulate E2F activity in two ways. Binding to activator E2Fs inhibits E2F transactivation. When bound to repressor E2Fs, they recruit transcriptional corepressors such as histone deacetylases (HDACs) or histone methyltransferases (HMTs) to inhibit expression of genes required for cell cycle progression (11). Cyclin-dependent kinases (CDKs) phosphorylate pocket proteins during cell cycle progression, thereby disrupting E2F-RB interactions. This results in both the relief of E2F/RB-mediated repression and the release of transcriptionally active E2F that triggers entry into S phase.It is now clear that E2F/RB functions extend beyond controlling S-phase entry. Biological activities for the pathway are inferred from both functional studies in mammals and other model organisms and through the identification of a vast network of target genes (2,9,19,34,39), including the control of the G 1 /S and G 2 /M transitions of the cell cycle, checkpoint control, DNA repair and recombination, apoptosis, differentiation, and development. How are all these diverse activities regulated? One answer may lie in the composite nature of the E2F/RB network; there are eight E2F factors in mammals, five of which can associate with three different pocket proteins. Individual E2F/RB complexes could perform distinct tasks and regulate different sets of genes in response to various signals. This idea is supported by several lines of evidence (9) and references therein.Another answer might be prov...