Complexes containing the retinoblastoma gene product recognize different DNA motifs related to the E2F binding site.
The retinoblastoma tumor-suppressor gene encodes a 105-kDa nuclear phosphoprotein (RB) that can associate with DRTF-1 and E2F. These two transcription factors can recognize the same DNA motif in the adenovirus E2A promoter and can bind to it by themselves or in association with RB. In the present report, we describe the use of CASTing (cyclic amplification and selection of targets) to determine the consensus binding site of RB-containing complexes. An anti-human RB antibody was used to isolate RB-containing complexes formed after mixing nuclear extracts obtained from human diploid fibroblasts with a pool of random oligonucleotides flanked with polymerase chain reaction (PCR) primers. After the immunoselection, the DNA was isolated, amplified, mixed with fresh nuclear extract and reselected. After six CASTing cycles, the DNA was cloned and sequenced. We found that the highest affinity motifs recognized by RB-containing complexes are related to the E2F/DRTF-1 binding site and fall into three classes: TTTTCCCGCCAAAA, TTTTCCCGCCTTTTTT or TTTTCCCGCGCTTTTTT. Competition experiments revealed that these three classes are functionally equivalent to each other and to the E2F/DRTF-1 binding site in the adenovirus E2A promoter. Screening these sequences against a DNA database identified their presence in non-coding regions of many oncogenes, growth factor genes and in the RB gene itself.
Ouellette
M M
MM
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9039.
Chen
J
J
Wright
W E
WE
Shay
J W
JW
D000255:Adenosine Triphosphate / Q000378:metabolism; D017456:Adenovirus Early Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016161:Genes, Retinoblastoma; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D009857:Oncogenes; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011519:Proto-Oncogenes; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*
D017456:Adenovirus Early Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D000255:Adenosine Triphosphate