Enhancement of the p300 HAT Activity by HIV-1 Tat on Chromatin DNA

Abstract

HIV-1 Tat is able to form a ternary complex with P/CAF and p300 and increase the affinity for CDK9/P-TEFb CTD kinase complex. Our previous study demonstrated that Tat binds to p300/CBP in the minimal HAT domain (aa 1253-1790) and that the interaction results in a change of conformation on p300/CBP. Here, we show that the Tat-p300 interaction increases the HAT activity of p300 on histone H4 that is associated with nucleosomal DNA and not with free histones. Nucleosomal histone H4 was acetylated on lysines 8, 12, and 16. Acetylation of H4 was inhibited by Lys-coenzyme A (CoA), a selective inhibitor of p300 acetyltransferase activity. Unexpectedly, we also found that Tat could autoacetylate itself, which was specific to lysine residues 41 and 71. Peptides lacking these two lysines could not enhance the HAT activity of p300. Comparison of the sequences of Tat with other HIV-1 clades and HAT containing transcription factors indicated sequence identity in the acetyl-CoA binding motif A, KGXG. Furthermore, when utilizing an in vitro transcription assay, as well as a Tat mutant virus, we found that ectopic expression of only wild-type Tat in the presence of p300, and not a lysine 41 Tat mutant, could activate HIV-1 chromatin DNA, as evidenced by the absence of HIV-1 virion antigen. Therefore, transcription of integrated viral DNA in vivo requires the HAT activity of coactivators that are modulated by Tat to derepress the HIV-1 chromatin structure and aid in activated transcription.

Probing Lysine Acetylation in Proteins

“…In the example of Tat, the reactivity of cysteines with AcCoA is probably the real cause for the reported enzymatic acetylation of the Tat CRR and the so called autoacetylation of the Tat core region. Disregarding the non-enzymatic cysteine acetylation it was concluded from radioactive acetylation assays that Lys 28 in the Tat CRR is enzymatically acetylated by PCAF (44) and that Lys 41 is autoacetylated (55). By resolving the number of attached acetyl groups to the wild-type and to various mutant peptides by MS it became evident that the acetylation of these regions was exclusively due to the non-enzymatic acetylation of the cysteine residues present in the sequence (50).…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…Although it remains debated (258), the p300-mediated acetylation of HIV-1 integrase might increase the binding affinity of integrase for nucleosomal DNA and promote the integrase strand transfer reaction (255). In addition to p300, other cellular cofactors (e.g., CREB and GCN5) are recruited by Tat to promote HIV-1 transactivation (260)(261)(262)(263). Overall, p300 acts as an acetyltransferase to alter the activities of HIV-1 Tat and integrase.…”

The Regulation of HIV-1 mRNA Biogenesis

“…In addition to histones, Tat itself is a substrate for acetylation by HATs. Lysines at positions 50 and 51 are major substrates for acetylation by p300 and hGCN5 (Col, et al, 2001, Deng, et al, 2001, Kiernan, et al, 1999. Acetylation of lysine 50 of Tat promotes the dissociation of Tat from TAR RNA during early transcription elongation and recruitment of the SWI/SNF chromatin-remodeling complex (Treand, et al, 2006), which synergize with p300 acetyltransferase and acetylated Tat to remodel the nucleosome at the HIV promoter in order to activate transcription (Mahmoudi, et al, 2006).…”