The transcriptional coactivator CREB binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity that is important for gene regulation. CBP binds to and cooperates with numerous nuclear factors to stimulate transcription, but it is unclear if these factors modulate CBP HAT activity. Our previous work showed that CBP interacts with the Epstein-Barr virus-encoded basic region zipper (b-zip) protein, Zta, and augments its transcriptional activity. Here we report that Zta strongly enhances CBP-mediated acetylation of nucleosomal histones. Zta stimulated the HAT activity of CBP that had been partially purified or immunoprecipitated from mammalian cells as well as from affinity-purified, baculovirus expressed CBP. Stimulation of nucleosome acetylation required the CBP HAT domain, the Zta DNA binding and transcription activation domain, and nucleosomal DNA. In addition to Zta, we found that two other b-zip proteins, NF-E2 and C/EBP␣, strongly stimulated nucleosomal HAT activity. In contrast, several CBP-binding proteins, including phospho-CREB, JUN/FOS, GATA-1, Pit-1, and EKLF, failed to stimulate HAT activity. These results demonstrate that a subset of transcriptional activators enhance the nucleosome-directed HAT activity of CBP and suggest that nuclear factors may regulate transcription by altering substrate recognition and/or the enzymatic activity of chromatin modifying coactivators.Eukaryotic gene expression is inhibited by higher order chromatin structures that limit the access of transcription factors to regulatory DNA sequences (reviewed in references 43, 45, and 67). These higher order structures are thought to be regulated by posttranslational modification of chromatin components (66). The acetylation of lysine residues in the core histone amino terminal tails is one chromatin modification that correlates with increased transcription activity (reviewed in references 36, 40, 56, 66, and 70). Several transcriptional coactivators possess intrinsic histone acetyltransferase (HAT) activity, including the CREB binding protein (CBP), its close relative p300, the SAGA-associated GCN5, the GCN5-related P/CAF, the MYST family protein Tip60, and the TATA-binding protein-associated factor TAF II 250 (7,15,35,39,59,61). Numerous transcriptional activators bind HAT-containing coactivators through interaction domains that are essential for transcription function (68). One important question is whether the coactivator HAT activity is constitutive or whether it can be modulated by interaction with transcriptional activators.CBP was isolated by virtue of its ability to bind the transcriptionally active phosphorylated form of CREB (34). Subsequently, CBP has been shown to function as a transcriptional coactivator for numerous cellular transcription factors, including the tumor suppressor p53, proto-oncoproteins c-JUN and c-MYB, nuclear hormone receptors, the hematopoietic transcription factors EKLF, GATA-1, and NF-E2, and the viral proteins, such as the adenovirus E1A, the simian virus 40 T antigen, the ...