Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus

• Published in: Genes & development Vol. 4; no. 4; pp. 667 - 679
• Main Authors: Gunther, C V, Nye, J A, Bryner, R S, Graves, B J
• Format: Journal Article
• Language: English
• Published: United States 01.04.1990
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cattle; Cells, Cultured; DNA, Viral - metabolism; Gene Library; Humans; Mice; Molecular Sequence Data; Moloney murine sarcoma virus - genetics; Multigene Family; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-ets; Proto-Oncogenes; Repetitive Sequences, Nucleic Acid; Sarcoma Viruses, Murine - genetics; Sequence Homology, Nucleic Acid; T-Lymphocytes - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.4.4.667

The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function is unknown. In a study of cellular proteins involved in the transcriptional regulation of murine retroviruses in T lymphocytes, we have discovered that a member of the ets gene family encodes a sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening a mouse thymus cDNA expression library with a double-stranded oligonucleotide probe representing 20 bp of the Moloney murine sarcoma virus (MSV) long terminal repeat (LTR). The cDNA sequence has an 813-bp open reading frame (ORF) whose predicted amino acid sequence is 97.6% identical to the 272 carboxy-terminal amino acids of the human ets-1 protein. The ORF was expressed in bacteria, and the 30-kD protein product was shown to bind DNA in a sequence-specific manner by mobility-shift assays, Southwestern blot analysis, and methylation interference. A mutant LTR containing four base pair substitutions in the ets-1 binding site was constructed and was shown to have reduced binding in vitro. Transcriptional efficiency of the MSV LTR promoter containing this disrupted ets-1 binding site was compared to the activity of a wild-type promoter in mouse T lymphocytes in culture, and 15- to 20-fold reduction in expression of a reporter gene was observed. We propose that ets-1 functions as a transcriptional activator of mammalian type-C retroviruses and speculate that ets-related genes constitute a new group of eukaryotic DNA-binding proteins.