Tax interacts with the cellular CREB protein and facilitates the binding of the coactivator CBP, forming a multimeric complex on the CRE-like sites in the HTLV-I promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I LTR. Studies have shown that the p300/CBP binding protein-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In a recent report, we provide evidence for a HAT-independent transcription function that is activated in the presence of the HTLV-I Tax protein. In vitro and in vivo GST-Tax pull down and 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 over 10-fold. 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. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. 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. Most recently, we have developed an in vitro transcription assay using a reconstituted chromatin template. Our data demonstrate that p300 and CBP facilitate transcription of a reconstituted 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. Moreover, the coactivator HAT activity must be tethered to the template by Tax and CREB, since a p300 mutant that fails to interact with Tax did not facilitate transcription or acetylate histones. p300 acetylates histones 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. Interestingly, we found distinct transcriptional activities between CBP and p300. CBP, but not p300, possesses an N-terminal activation domain which directly activates Tax-mediated HTLV-I transcription from a naked DNA template. Finally, using the chromatin immunoprecipitation assay, we provide the first direct experimental evidence that p300 and CBP are associated with the HTLV-I LTR in vivo.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC005254-21
Application #
6761445
Study Section
(BRL)
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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