Complexes containing the retinoblastoma gene product recognize different DNA motifs related to the E2F binding site

Michel M. Ouellette, Jun Chen, Woodring E. Wright, Jerry W. Shay

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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, TTTTCCCGC-CTTTTTT 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.

Original languageEnglish (US)
Pages (from-to)1075-1081
Number of pages7
JournalOncogene
Volume7
Issue number6
StatePublished - Jun 1992

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Retinoblastoma Genes
Nucleotide Motifs
Binding Sites
Adenoviridae
Retinoblastoma
Phosphoproteins
DNA
Nucleic Acid Databases
Tumor Suppressor Genes
Diploidy
Oncogenes
Oligonucleotides
Genes
Intercellular Signaling Peptides and Proteins
Transcription Factors
Fibroblasts
Polymerase Chain Reaction
Antibodies

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this

Complexes containing the retinoblastoma gene product recognize different DNA motifs related to the E2F binding site. / Ouellette, Michel M.; Chen, Jun; Wright, Woodring E.; Shay, Jerry W.

In: Oncogene, Vol. 7, No. 6, 06.1992, p. 1075-1081.

Research output: Contribution to journalArticle

Ouellette, Michel M. ; Chen, Jun ; Wright, Woodring E. ; Shay, Jerry W. / Complexes containing the retinoblastoma gene product recognize different DNA motifs related to the E2F binding site. In: Oncogene. 1992 ; Vol. 7, No. 6. pp. 1075-1081.
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