HTLV-1 21 bp Repeat Sequences Facilitate Stable Association Between Tax and CREB to Increase CREB Binding Affinity

Abstract

The human T-cell leukemia virus type I (HTLV-I) transactivator protein Tax is critical for the activation of viral gene expression and the transformation of T-lymphocytes. Tax activation of HTLV-I gene expression is mediated by three highly homologous regulatory elements known as 21 bp repeats which bind the transcription factor CREB. Questions remain about the mechanism by which Tax can stimulate CREB binding, whether Tax alters CREB binding affinity, what specific sequences in the HTLV-I 21 bp repeat mediate ternary complex formation, and if the ternary complex comprised of Tax and CREB can recruit coactivators such as CBP. To address these points, we used immobilized templates containing either the HTLV-I 21 bp repeats or the somatostatin CRE to assay Tax association with ATF/CREB family members. Tax formed a stable ternary complex on each of the 21 bp repeats with the transcription factor CREB but not related ATF/CREB proteins. In contrast, Tax did not form a similar complex on the CREB binding site in the somatostatin promoter. The formation of this complex was dependent on 3′ sequences flanking the CREB binding site within each of the 21 bp repeats and resulted in marked increases in CREB association and binding affinity. Tax increased the binding of phosphorylated CREB to the 21 bp repeat resulting in increased association of the coactivator CBP. However, Tax did not form a complex on the somatostatin CRE in the presence of either phosphorylated or non-phosphorylated CREB and it did not stimulate CBP association to this element. These studies extend previous work and demonstrate how specific DNA sequences flanking the CREB binding site regulate the formation of a stable ternary complex that is able to more efficiently recruit the coactivator CBP.