Isolation of two novel cDNAs whose products associate with the amino terminus of the E2F1 transcription factor.
The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.
Jordan
K L
KL
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541, USA.
Evans
D L
DL
Steelman
S
S
Hall
D J
DJ
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002455:Cell Division; D002460:Cell Line; D002846:Chromatography, Affinity; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; C432687:EBNA1BP2 protein, human; C499008:EBP2 protein, S cerevisiae; C499009:Ebna1bp2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C101633:PA2G4 protein, human; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D056465:Retinoblastoma-Binding Protein 1; D029701:Saccharomyces cerevisiae Proteins; D050680:Transcription Factor DP1; D014157:Transcription Factors