IN-VITRO SELECTION OF DNA ELEMENTS HIGHLY RESPONSIVE TO THE HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-I TRANSCRIPTIONAL ACTIVATOR, TAX

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Ta x, the ubiquitous transcriptional factor cyclic AMP (cAMP) response el ement-binding protein (CREB protein), and the 21-bp repeats in the HTL V-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 a long with purified Tax and CREB proteins, we selected DNA elements bou nd specifically by the Tax-CREB complex in vitro. Two distinct but rel ated groups of sequences containing the cAMP response element (CRE) fl anked by long runs of G and C residues in the 5' and 3' regions, respe ctively, 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 r esemblance 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 t he 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 recogni zes the viral 21-bp repeats. The expanded DNA binding specificity of T ax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transc riptional activity of leucine zipper proteins like CREB.