title: Dual mechanisms of repression of E2F1 activity by the retinoblastoma gene product.
abstract: The retinoblastoma gene product, pRb, negatively regulates cell proliferation by modulating the activity of the transcription factor E2F1 that controls expression of S-phase genes. To dissect transcriptional regulation of E2F1 by pRb, we developed a means to control the subcellular localization of pRb by exchanging its constitutive nuclear localization signal (NLS) with an inducible nuclear targeting domain from the glucocorticoid receptor (GR). In co-transfection experiments in hormone-free media, pRb delta NLS-GR sequestered E2F1 in the cytoplasm; addition of steroid hormones induced co-translocation of pRb delta NLS-GR and E2F1 to the nucleus. A pRb allele lacking a NLS, pRb delta NLS, also sequestered E2F1 in the cytoplasm. Both nuclear and cytoplasmic pRb delta NLS-GR repressed transcription from a simple, E2F1-activated, promoter equally well. pRb delta NLS-GR exerted differential effects on complex promoters containing an activator and E2F sites that acted as either positive or negative elements. We propose a dual mechanism of transcriptional repression by pRb which allows tight control of E2F1-responsive genes: a pRb-E2F1 repressor unit is assembled off DNA to pre-empt transcriptional activation by E2F1; recruitment of this repressor unit to cognate binding sites on promoters allows silencing of adjacent promoter elements.
authors.affiliation: Division of Immunology and Cancer Research, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Zacksenhaus
authors.forename: Z
authors.initials: Z
authors.lastname: Jiang
authors.forename: R A
authors.initials: RA
authors.lastname: Phillips
authors.forename: B L
authors.initials: BL
authors.lastname: Gallie
mesh_terms: D016475:3T3 Cells; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016161:Genes, Retinoblastoma; D051379:Mice; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors