In this study, we identified four novel isoforms of hGli2 α, β, γ, and δ, that bind to a Tax response element, TRE2S, in the LTR of HTLV-1. The novel isoforms had the same sequence at the 5′ region as THP previously described (30
), except for two deletions, which allowed further extension of the translation. Careful reexamination indicated that the sequence of the previous THP was a cloning artifact. hGli2 α showed 80% homology of amino acid sequence to mGli2 (14
). The isoforms were formed by combinations of two independent alternative splicings in the coding sequence, and the larger isoforms, α and β, were abundantly expressed in HTLV-1-infected T-cell lines and also in other cell lines so far tested. All these isoforms were found to bind to TRE2S with conservation of the zinc finger motifs. Comparing the sequence of mGli2 reported by Hughes et al. (14
) with the isoforms of hGli2 described in this paper, mGli2 appeared to correspond to the β form. It underwent splicing at the first site but not at the second site, although the site showed very high homology, as shown in Fig. .
Fusion proteins of hGli2 with the DNA binding domain of Gal4 enhanced the Tax-dependent activation of gene expression when the reporter contained the Gal4 binding site and the 21-bp sequence. From these observations, we concluded that binding of hGli2 to the TRE2S sequence enhanced Tax-dependent transactivation of transcription. However, the free form of hGli2 did not enhance but, instead, rather efficiently inhibited Tax-dependent transcription, probably because the endogenous Gli2 gene is abundantly expressed in the cell line used and the expression of the reporter gene had been activated by Tax alone at the maximum level. The mechanism of this inhibition is not well understood, but two possibilities can be assumed: (i) overexpressed hGli2 is squelching Tax-mediated transactivation, or (ii) it should be modified to be active, but, it might not be sufficiently modified in transfected cells.
A unique property of the enhancement was that binding of hGli2 alone to TRE2S had almost no effect on transcription. Two additional factors were required for the enhancement; these were the 21-bp sequence as a cis
element and Tax protein as a trans
-acting factor. These requirements for transcriptional activation suggest that hGli2 might function through a unique mechanism, although the mechanism is not well understood. A possible mechanism, however, could be as follows: hGli2 and CREB bind to each cis
element in the vicinity and interact with each other on the DNA. Such an interaction could be enhanced by the Tax protein, since Tax binds to CREB to activate the enhancer activity of the 21-bp repeats (29
). A putative interaction of hGli2 and CREB might be supported by the previous observations on YY1, a human Gli-Kruppel-related protein (26
). YY1 interacts with CREB/ATF and exerts repression of specific transcription (36
). Furthermore, the repression exerted by YY1 is relieved by adenovirus E1A (26
). Another factor, UCRBP, a zinc finger protein of the Gli family, bound to the upstream conserved region of the murine leukemia virus LTR and down-regulated the MuLV promoter activity (5
). These observations on other factors related to Gli proteins allow speculation that hGli2 is a transcriptional repressor and that HTLV-1 Tax can prevent the repression. However, this mechanism seems unlikely, because hGli2 did not show any inhibitory activity on transcription in the absence of Tax.
Whatever the mechanism, the presence of the TRE2S sequence adjacent to the 3′ proximal 21-bp sequence in the LTR (1
) and our observations reported in this paper suggest that the binding of hGli2 and CREB to TRE2S and 21-bp sequences, respectively, in the LTR would be an alternative pathway to augment the capacity of Tax to transactivate viral gene expression.