Interaction of Gli2 with CREB protein on DNA elements in the long terminalrepeat of human T-cell leukemia virus type 1 is responsible for transcriptional activation by Tax protein

The long terminal repeat (LTR) of human T-cell leukemia virus type I (HTLV- 1) has two distinct DNA elements, one copy of TRE2S and three copies of a 2 1-bp sequence that respond to the viral trans-activator protein, Tax. Eithe r multiple copies of the 21-bp sequence or a combination of one copy each o f TRE2S and 21-bp sequence is required for efficient trans activation by Ta x. In the trans activation of multiple copies of 21-bp sequence, CREB/ATF p rotein plays an essential role in forming a complex with Tax. To understand the role of TRE2S in trans activation of one copy of 21-bp sequence, we ex amined protein binding to the DNA elements by DNA affinity precipitation as say including Gli2 protein binding to TRE2S and CREB protein binding to 21- bp sequence. Binding of CREE to a DNA probe containing both elements, TRE2S -21bp probe, was dependent on Gli2 protein under restricted conditions and was enhanced in a dose-dependent fashion by the binding of Gli2 protein to the same probe. Mutation in either element abolished the efficient binding of CREB, A glutathione S-transferase fusion protein of a fragment of Gli2 w as able to bind to CREB. Therefore, Gli2-CREB interaction on the DNA probe is proposed to stabilize CREB binding to DNA, Tax can bind to CREB protein on the DNA; therefore, stabilization of DNA binding of CREB results in more recruitment of Tax onto DNA. Conversely, Tax increased the DNA binding of CREB, although it had almost no effect on the binding of Gli2. These result s suggest that Gli2 binds to the DNA element and interacts with CREB, resul ting in more recruitment of Tax, which in turn stabilizes DNA binding of CR EB. Similar cooperation of the protein binding to TRE2S-21bp probe was also observed in nuclear extract of an HTLV-1-infected T-cell line. Consistent, vith the Gli2-CREB interaction on the DNA elements, Tax-mediated trans acti vation was dependent on the size of the spacer between TRE2S and 21-bp sequ ence. The effective sizes of the spacer suggest that TRE2S in the LTR would cooperate with the second and third copies of the 21-bp sequence and contr ibute to trans activation of the viral gene transcription.