Abstract
Tax-1, the transcriptional activation protein of human T-cell leukemia virus-1, increases transcription from the human T-cell leukemia virus-1 long terminal repeat and specific cellular promoters through interactions with cellular DNA-binding proteins. The Tax response elements (TxREs) of the long terminal repeat resemble cAMP response elements (CREs), the target of cAMP-responsive element-binding protein (CREB). CREB binds the TxRE with reduced affinity; however, the interaction is specifically enhanced by Tax. Using a fluorescence quenching method, we determined that CREB dimerizes in the absence of DNA, and that Tax does not enhance dimerization. DNA footprinting of the TxRE with 1, 10-phenanthroline-copper complex demonstrates that Tax contacts DNA and extends the footprint of CREB to GC-rich sequences flanking the core CRE-like element. The minor groove-binding drug chromomycin A3, but not distamycin A, disrupted Tax-enhanced CREB binding to the TxRE. Substitution of the guanine-rich sequences flanking the core of the TxRE with inosine residues also blocked the Tax effect. Finally, the IC-substituted TxRE binds CREB with increased affinity, suggesting flanking DNA influences the binding of CREB to the core CRE-like element. These data indicate that Tax does not regulate DNA binding of CREB by altering dimerization, but rather enhances DNA binding by additionally interacting with the minor groove of flanking DNA sequences.
Highlights
Viruses typically exploit host cellular processes to promote their own replication
We and others have shown that Tax augmentation of cAMPresponsive element-binding protein (CREB) DNA binding activity is sequence-specific; Tax enhances binding of CREB to the Tax response elements (TxREs) but not to the perfect palindromic cAMP response elements (CREs) derived from the somatostatin promoter (12–14, 16, 17, 19 –21)
By DNA footprinting with 1,10-phenanthroline-copper complex, a minor groove-selective cleavage reagent, we demonstrate that Tax interacts with the minor groove of the DNA flanking the core CRE-like motif of the TxRE, in the context of the CREB-Tax-DNA ternary complex
Summary
Plasmids and Expression Vectors—The bacterial expression vector for HTLV-1 Tax has been described previously [28]. HTLV1 Tax expressed as a C-terminally histidine-tagged protein in E. coli HB101 was prepared essentially as described previously [28], except that Tax partially purified by nickel-nitriloacetic acid resin (Qiagen Inc.) was further purified by Q-Sepharose anion exchange column chromatography. Modified proteins were dialyzed extensively against 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1 mM DTT. All binding measurements were determined in 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 5% glycerol, 50 g/ml BSA, at 20 °C in a 1-ml binding reaction. Binding reactions consisted of a buffer containing 25 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2, 50 g/ml poly(dI1⁄7dC), 200 g/ml BSA (New England Biolabs), 1 nM 32P-labeled duplex oligodeoxyribonucleotide, and Tax and CREB concentrations as indicated in the figure legends. Chromomycin A3 (100 mM) was prepared in dimethyl sulfoxide and stored at Ϫ20 °C
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