DNA binding by c‐Ets‐1, but not v‐Ets, is repressed by an intramolecular mechanism

• Published in: The EMBO journal Vol. 11; no. 2; pp. 643 - 652
• Main Authors: Lim, F., Kraut, N., Framptom, J., Graf, T.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.02.1992
• Subjects: Animals; Avian Leukosis Virus - genetics; Base Sequence; Biological and medical sciences; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Cells, Cultured; Chick Embryo; Chromosome Deletion; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Molecular Sequence Data; Oligodeoxyribonucleotides; Oncogenes; Plasmids; Polymerase Chain Reaction; Protein Biosynthesis; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-ets; Proto-Oncogenes; Recombinant Proteins - metabolism; Restriction Mapping; Retroviridae Proteins, Oncogenic - genetics; Retroviridae Proteins, Oncogenic - metabolism; Templates, Genetic; Transcription Factors - metabolism; Transcription, Genetic; Transcriptional Activation; Transfection; Oncovirinae; Transcription; Gene product; Transcription promoter; Retroviridae; V-Onc gene; Molecular interaction; Cell transformation; Host virus relation; Biological activity; Mechanism; Virus; Type C oncovirus; C-Onc gene; Avian leukosis virus; Comparative study; Protooncogene
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1992.tb05096.x

The E26 avian retrovirus causes an acute leukemia in chickens and transforms both myeloid and erythroid cells. The virus encodes a 135 kDa fusion protein which contains amino acid sequences derived from the viral Gag protein and the two cellular transcription factors c‐Myb and c‐Ets‐1p68. Previously we have shown that like v‐myb, v‐ets on its own is also active in transformation, but only within the erythroid lineage. To understand better the mechanisms involved in the oncogenic activation of c‐Ets‐1p68, we used the polyoma PEA3 element, a known Ets binding site, to compare the sequence‐specific DNA binding and transactivating properties of v‐Ets and c‐Ets‐1p68. Using Ets protein synthesized in rabbit reticulocyte lysate in gel retardation assays, we detected little binding of c‐Ets‐1p68 to an oligonucleotide containing the PEA3 motif whereas v‐Ets bound strongly. However, in transient cotransfection assays in chicken embryo fibroblasts both c‐Ets‐1p68 and v‐Ets transactivated transcription from a heterologous promoter linked to PEA3 elements. Interestingly, fragments of c‐Ets‐1p68 with strong DNA binding activity could be produced by limited proteolysis, indicating that the DNA binding domain is repressed within the full‐length molecule. By deletion mapping the DNA binding domain was localized to the most highly conserved region of the Ets‐related proteins known as the ETS domain. The C‐terminus as well as a region in the middle of the polypeptide chain are involved in repression of DNA binding in c‐Ets‐1p68. Significantly, v‐Ets contains a 16 amino acid substitution at the C‐terminus. Our results suggest that intramolecular repression of DNA binding is a regulatory mechanism in c‐Ets‐1p68 which is lost in v‐Ets.