In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

Abstract

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB.In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G-or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G-and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.Human T-cell lymphotropic virus type I (HTLV-I) is the prototype of a group of retroviruses (including HTLV-I, HTLV-II, simian T-cell leukemia virus, and bovine leukemia virus [BLV]) that produce regulatory proteins to modulate viral mRNA synthesis and utilization (6). The 40-kDa nuclear protein, Tax, encoded by the 3' region of the HTLV-I genome is a transcriptional activator (6). Tax does not bind DNA directly (13,21). Like many other viral transactivators such as herpes simplex virus VP16 and adenovirus Ela (for a review, see reference 20), Tax forms multiprotein complexes with host cell transcription factors to gain control of the cellular mRNA synthetic machinery for viral replication (1,18,(27)(28)(29). The ability of Tax to alter gene expression in HTLV-I-infected cells appears to be causally linked to HTLV-I pathogenesis, which is manifested clinically as adult T-cell leukemia (5, 16, 24) and tropical spastic paraparesis/HTLV-I-associated myelopathy (2,12,17,22).Tax activates viral transcription from three 21-bp repeats in the U3 region of the HTLV-I long terminal repeat (3,23,25,26). Mutational analyses indicated that two copies of the repeat and the CRE (cyclic AMP [cAMP]-responsive-element)-like sequences, TGACG, in the repeats are crucial for transactivation by Tax (3,13,23,25,26 Tax action (10,19). This is best shown by the inability of HTLV-I Tax to transactivate thre...

The Human T-cell Leukemia Virus-1 Transcriptional Activator Tax Enhances cAMP-responsive Element-binding Protein (CREB) Binding Activity through Interactions with the DNA Minor Groove

“…in methylation protection or methylation interference assays (data not shown). These results are surprising given the dependence of the Tax effect on flanking CG-rich sequences (8,12); however, the approaches utilized address only a limited portion of the DNA available for interaction with DNA-binding proteins. For example, the primary site of modification of DNA by dimethyl sulfate under neutral conditions is N-7 of guanine, in the major groove (30).…”

“…The DNA sequence specificity of the Tax effect, however, argues against the idea that enhanced dimerization accounts for the increased DNA binding activity. Indeed, the ability of Tax to stimulate the DNA binding activity of cross-linked basic region peptides independently of dimerization (15) suggests that Tax might associate with specific DNA sequences, despite the lack of evidence to date for direct Tax-DNA interactions (12,26).…”

“…In addition Tax forms a stable complex with CREB and DNA, also in a sequence-specific manner, dependent on flanking GCrich DNA motifs (12,14,16,17,19). Although Tax does not associate with CREB bound to the somatostatin CRE, it will bind to CREB in the absence of DNA (22)(23)(24).…”

CREB/ATF-Dependent Repression of Cyclin A by Human T-Cell Leukemia Virus Type 1 Tax Protein

“…on May 11, 2018 by guest http://jvi.asm.org/ activate transcription via CREB/ATF sites is context specific since at other CREB binding sites (i.e., those found in cellular promoters), Tax-CREB complex formation may occur (46,59,77), but no activation is seen. The above CycA-luc, DPA-luc, and CycD3-luc results are compatible with an alternative functional interpretation: Tax-CREB interaction at some TATAless promoters manifests as repression.…”

“…The HTLV-1 LTR contains three CREB/ATF binding sites (37). Highly efficient activation of this viral LTR by Tax is, in part, explained by Tax-CREB complex formation at cognate sites in the LTR (23,46,59). This ability of Tax to …”