Multistep regulation of enhancer activity of the 21-base-pair element of human T-cell leukemia virus type I

Abstract

We examined the regulatory mechanisms of binding and transcriptional enhancement of the 21-bp core element of the enhancer of human T-cell leukemia virus type I (HTLV-I) in response to forskolin, 12-0-tetradecanoylphorbol-13-acetate (TPA), and a viral transactivator, p4laX. The 21-bp core element has been shown to bind to a cyclic AMP-responsive element binding protein (CREB)-like molecule at the site of an imperfect palindrome containing the TGAC motif. Experiments with oligonucleotides with mutations in the imperfect palindrome demonstrated that one TGAC motif is necessary and sufficient for both the binding of the CREB-related factor and transcriptional activity in response to forskolin in a human T-cell line, Jurkat. We also found that binding of the CREB-like factor to the 21-bp core element was enhanced by treatment with TPA, with little effect on transcriptional activity; in contrast, forskolin and p4O"'0 did not facilitate binding, though they enhanced transcription. The combination of forskolin and TPA synergistically induced the transcription activity of the element, showing a hierarchical mechanism of regulation of the HTLV-I core enhancer element to levels sufficient for formation of the factor-enhancer complex and for activation of the complex. Added to those findings, our results indicate that the modes of activation by forskolin and p4O"'0 are different from each other. Enhancers of retroviruses reside in the U3 region of the long terminal repeats which are located at the borders of the integrated viral genome. Human T-cell leukemia virus type I (HTLV-I), which is an etiological agent of adult T-cell leukemia (16, 42, 62) and HTLV-I-associated myelopathy and tropical spastic paraparesis (12, 40), has a unique enhancer. The regulation of activity of the enhancer seems to determine the expression of HTLV-I. Thus, for a better understanding of the replication of HTLV-I, it is important to elucidate the mechanism by which the enhancer is regulated. The activity of the enhancer is induced by several stimuli, including its own viral product, p4Ofax, which is encoded by the pX region between the env gene and the 3' long terminal repeat (48). p4O" has activities not only to activate its own enhancer (6, 49, 54, 55) but also to induce several cellular genes (9, 10, 19, 23, 26-28, 33, 41, 47, 60) involved in cell growth and differentiation. Accordingly, p4Olax belongs to a family of transacting transcriptional activators and is implicated in the leukemogenesis caused by HTLV-I. p4O"' does not seem to have the ability to bind directly to target DNA sequences; rather, host cell factors mediate the effect of p4O'". In addition, forskolin, which increases intracellular cyclic AMP (cAMP) levels (35, 44, 58), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (8, 37, 45), which activates protein kinase C, are known to activate the HTLV-I enhancer.