Both LyF-1 and an Ets protein interact with a critical promoter element in the murine terminal transferase gene.
Terminal deoxynucleotidyltransferase (TdT) is a template-independent DNA polymerase that is expressed transiently during the earliest stages of B- and T-cell ontogeny. Previously, we characterized the promoter for the murine TdT gene and identified a novel DNA-binding protein, called LyF-1, that interacts with a DNA sequence element found to be critical for transcriptional activity in lymphoid cell lines. Here, we present a more detailed analysis of this 30-bp control element, called the TdT D' element, which is centered approximately 60 bp upstream of the transcription start site. We found that both the murine and human D' elements are recognized by multiple proteins, including LyF-1 and at least two Ets family proteins, Ets-1 and Fli-1. Additional protein-DNA interactions were identified through studies using unfractionated nuclear extracts, in which the D' element was apparently incorporated into a multiprotein complex, possibly containing an Ets protein as a core component. By analyzing a series of substitution mutations, two adjacent binding sites for LyF-1 were identified in the murine D' element, with the Ets protein binding site closely coinciding with the proximal, lower-affinity LyF-1 site. Transient transfection analysis with these mutations revealed that only a 10-bp region, containing precisely the Ets and proximal LyF-1 binding sites, was needed for D' activity. These results suggest an important role for an Ets family protein in the expression of the TdT gene. The role of LyF-1 is less clear; it might act in conjunction with the Ets protein bound at the D' element or it might be unnecessary for D' activity.
Ernst
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Howard Hughes Medical Institute, Department of Microbiology and Immunology, UCLA School of Medicine 90024-1662.
Hahm
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Smale
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D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004253:DNA Nucleotidylexotransferase / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000201:enzymology; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
C494041:ETS1 protein, human; C494042:Ets1 protein, mouse; D009838:Oligodeoxyribonucleotides; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase; D004253:DNA Nucleotidylexotransferase