Drosophila RB Proteins Repress Differentiation-Specific Genes via Two Different Mechanisms

Abstract

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...

The Complex Containing Drosophila Myb and RB/E2F2 Regulates Cytokinesis in a Histone H2Av-Dependent Manner

“…Despite reported interactions with the dREAM complex or mammalian Rb, the Rpd3, l(3)mbt, Su(var)3-9, and Polycomb group (PcG) genes failed to genetically interact with Myb in our screen (Table 2) (8,(45)(46)(47). Therefore, if these proteins do play a role in repression by the dREAM complex, halving their dose is not sufficient to alter the binucleate cell phenotype.…”

“…The purified dREAM complex also deacetylated histones in vitro and interacted with Rpd3/ HDAC1, a histone deacetylase (8). Moreover, in Drosophila S2 cells, repression of dREAM-regulated differentiation genes required both histone deacetylase activity and the Polycomb group protein E(z) (45). Many epigenetic regulators, including the Rpd3/HDAC1 and PcG proteins, act in a dose-dependent fashion (42,43).…”

A Dual Role for the dREAM/MMB Complex in the Regulation of Differentiation-Specific E2F/RB Target Genes

“…Group D/E genes are not regulated by the activator dE2F1, and dE2F1 does not bind to their promoters. We have previously shown that dREAM/MMB plays a role in two different mechanisms of repression of group D/E genes: the deacetylation of upstream promoter regions and the dimethylation of histone H3 K27 at sequences that are downstream of the transcription start site (21). While the first mechanism of repression is shared with cell cycle-regulated genes, the second is unique for group D/E genes.…”

“…The initial identi- fication and characterization of the dREAM/MMB complexes has led to the speculation that dREAM is a repression complex that functions by affecting chromatin structure (20,22). The complex is not stably associated with any enzymatic activity, yet we have demonstrated that group D/E genes are repressed via two distinct mechanisms in a dREAM-dependent manner (21). Is the observed dependence on dREAM the result of its ability to affect chromatin structure or is it an indirect consequence of its role in dE2F2/RBF binding?…”

Loss of RBF1 changes glutamine catabolism

“…Also like pRB, most of the literature on RBF1 centers on its ability to limit dE2F-dependent proliferation. However, RBF1 has recently been shown to possess additional functions in the immune response (Longworth et al 2012), in cell differentiation (Lee et al 2010(Lee et al , 2012Nicolay et al 2010;Popova et al 2012), and in chromatin organization (Longworth et al 2008;Manning et al 2010). Thus, like its mammalian counterpart, the loss of RBF1 has ramifications that extend beyond the cell cycle.…”