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.

Here we report the identification of the LIN complex (LINC), a human multiprotein complex that is required for transcriptional activation of G 2 /M genes. LINC is related to the recently identified dREAM and DRM complexes of Drosophila and C. elegans that contain homologs of the mammalian retinoblastoma tumor suppressor protein. The LINC core complex consists of at least five subunits including the chromatin-associated LIN-9 and RbAp48 proteins. LINC dynamically associates with pocket proteins, E2F and B-MYB during the cell cycle. In quiescent cells, LINC binds to p130 and E2F4. During cell cycle entry, E2F4 and p130 dissociate and LINC switches to B-MYB and p107. Chromatin Immunoprecipitation experiments demonstrate that LINC associates with a large number of E2F-regulated promoters in quiescent cells. However, RNAi experiments reveal that LINC is not required for repression. In S-phase, LINC selectively binds to the promoters of G 2 /M genes whose products are required for mitosis and plays an important role in their cell cycle dependent activation.

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Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo 13 C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H 2 O 2 preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.