Pocket Protein Complexes Are Recruited to Distinct Targets in Quiescent and Proliferating Cells

Abstract

Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycledependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.E2F/retinoblastoma tumor suppressor (pRB) complexes play a critical role in the regulation of cell cycle progression. There are at least eight E2F transcription factors expressed in mammals (reviewed in references 3 and 29). pRB and related family members regulate the transcription of E2F-responsive genes by interacting with the E2Fs. Three pRB family members, pRB, p130, and p107 (termed pocket proteins), are expressed in mammalian cells. pRB family members in the hypophosphorylated state bind to E2Fs and inhibit transcription. This inhibition is relieved when the pocket proteins are released from E2F complexes following their phosphorylation by cyclin/CDK activity (15). There are functional and structural differences within the E2F family. E2F-1, E2F-2, and E2F-3 are transcriptional activators and interact with pRB. E2F-4 and E2F-5 are transcriptional repressors and preferentially bind p130 and p107 (17). Although E2F-6 apparently does not have a pocket protein interaction domain, it interacts with Polycomb proteins and thus represses transcription (7,18,32,45). The recently identified E2F-7 and E2F-8 are also believed to repress specific promoters (14, 29).Although p107, p130, and pRB are closely related members of the same family, they have different affinities for E2F family members, and they exhibit distinct temporal regulation during the cell cycle. While E2F4/p130 complexes are the most abundant in quiescent cells, E2F4/p107 and E2F4/pRB complexes accumulate in G 1 cells (31). In addition, while p130 and p107 are bound to a number of promoters in asynchronously growing cells, only p...

Biological reprogramming in acquired resistance to endocrine therapy of breast cancer

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Retinoblastoma tumor-suppressor protein phosphorylation and inactivation depend on direct interaction with Pin1

“…For cell synchronization experiments, T98G cell growth was arrested by contact inhibition and serum starvation for 48 h. For G1/S enrichment, cells were split and grown in DMEM, 10% FBS in 2 mM hydroxyurea for 24 h as described. 35 Drug treatments were carried out with 100 nM Flavopiridol (Sigma-Aldrich, St. Louis, MO, USA) for 16 h. To generate knockdown cells, lentiviral particles were produced as described (http://www.broadinstitute.org/genome_bio/trc/publicProtocols.html). Briefly, 1 Â 10 6 293FT cells (Invitrogen, Carlsbad, CA, USA) were transfected with 2.25 mg of PAX2 packaging plasmid, 0.75 mg of PMD2G envelope plasmid, and 3 mg of pLKO.1 hairpin vector utilizing 30 ml of Fugene HD (Roche, Indianapolis, IN, USA) on 10 cm plates.…”

TGFβ prevents proteasomal degradation of the cyclin-dependent kinase inhibitor p27kip1 for cell cycle arrest

“…A complex temporal regulation of different E2F factors and pocket proteins regulates the cell cycle. 18,19 TGFβ prevents S phase transition in late G 1 phase by directly decreasing synthesis of cyclinA, cyclin E, cdk4, cdk6, cdk2 and by increasing the synthesis of cyclin-dependent kinase inhibitors (CKIs) p15 and p21, which block cdk activity, specifically cyclinD/cdk4,6, and cyclinE/cdk2, respectively. In addition, TGFβ inhibits proliferation by depressing E2F-mediated transcription of the proto-oncogene, c-myc, an inducer of D-type cyclins.…”