Studies on coactivator function during Tax-mediated HTLV-I transcription.

摘要

Expression of human T-cell leukemia virus type I (HTLV-I) genes is regulated by the virally encoded transcriptional activator Tax and cellular factors. It has been shown that Tax interacts with coactivators CBP, p300 and PCAF. One key property of the coactivators is the presence of histone acetyltransferase (HAT) activity, which endows p300/CBP and PCAF with the capacity to modify chromatin structure. In this dissertation, evidence is presented for a HAT-independent PCAF transcription function in the presence of the HTLV-I Tax protein. In vitro GST-Tax pull-down and in vivo co-immunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-I TRE site in the presence of Tax and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-I LTR. Point mutations at Tax amino acid 318 (TaxS318A) or 319–320 (Tax M47), which have decreased or no activity on the HTLV-I promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent upon the PCAF HAT domain. In contrast, p300 stimulation of Tax transactivation is HAT-dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral LTR. In the second study, a biochemical approach was utilized to examine the function of p300/CBP HAT in Tax transactivation using a reconstituted HTLV-I LTR chromatin template. The data presented in this dissertation demonstrate that p300 and CBP facilitate transcription of a chromatin template in the presence of Tax and CREB. The ability of p300 and CBP to activate transcription from the chromatin template is dependent upon the HAT activity. p300 acetylates histories H3 and H4 within nucleosomes located in the promoter and 5 ′ proximal regions of the template. Nucleosome acetylation is accompanied by an increase in the level of binding of TFIID and RNA polymerase II to the promoter. Finally, this dissertation provides the first direct experimental evidence that p300 and CBP are associated with the HTLV-I LTR in vivo .