Vasily V. Ogryzko

The Transcriptional Coactivators p300 and CBP Are Histone Acetyltransferases

p300/CBP is a transcriptional adaptor that integrates signals from many sequence-specific activators via direct interactions. Various cellular and viral factors target p300/CBP to modulate transcription and/or cell cycle progression. One such factor, the cellular p300/CBP associated factor (PCAF), possesses intrinsic histone acetyltransferase activity. Here, we demonstrate that p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. p300/CBP represents a novel class of acetyltransferases in that it does not have the conserved motif found among various other acetyltransferases. p300/CBP acetylates all four core histones in nucleosomes. These observations suggest that p300/CBP acetylates nucleosomes in concert with PCAF.

Recruitment of p300/CBP in p53-Dependent Signal Pathways

The products of the p53 and CBP/p300 genes have been individually implicated in control of cell growth and regulation of transcription. p53 is known to act as a positive and negative regulator of gene expression. Here we show that p53, in both wild-type and mutant conformation, forms a specific protein complex with p300. However, in its wild-type but not mutant conformation, p53 inhibits a promoter containing the DNA-binding sequences for the transcription factor AP1, in a p300-dependent manner. p300 stimulates the transcriptional activity of p53 on p53-regulated promoters, and it enhances the responsiveness to a physiological upstream modulator of p53 function, ionizing radiation. A dominant negative form of p300 prevents transcriptional activation by p53, and it counteracts p53-mediated G1 arrest and apoptosis. The data implicate p300 as an important component of p53-signaling, thus providing new insight into the mechanisms of cellular proliferation.

Acetylation of general transcription factors by histone acetyltransferases

The acetylation of histones increases the accessibility of nucleosomal DNA to transcription factors [1,2], relieving transcriptional repression [3] and correlating with the potential for transcriptional activity in vivo [4 - 7]. The characterization of several novel histone acetyltransferases - including the human GCN5 homolog PCAF (p300/CBP-associated factor) [8], the transcription coactivator p300/CBP [9], and TAFII250 [10] - has provided a potential explanation for the relationship between histone acetylation and transcriptional activation. In addition to histones, however, other components of the basal transcription machinery might be acetylated by these enzymes and directly affect transcription. Here, we examine the acetylation of the basal transcriptional machinery for RNA polymerase II by PCAF, p300 and TAFII250. We find that all three acetyltransferases can direct the acetylation of TFIIEbetaand TFIIF, and we identify a preferred site of acetylation in TFIIEbeta. Human TFIIE consists of two subunits, alpha(p56) and beta(p34), which form a heterotetramer (alpha2 beta2) in solution ([11], reviewed in [12]). TFIIE enters the preinitiation complex after RNA polymerase II and TFIIF, suggesting that TFIIE may interact directly with RNA polymerase II and/or TFIIF [13,14]. In addition, TFIIE can facilitate promoter melting either in the presence or absence of TFIIH and can stimulate TFIIH-dependent phosphorylation of the carboxy-terminal domain of RNA polymerase II [15-18]. TFIIF has an essential role in both transcription initiation and elongation ([19,20], for review see [21]). We discuss the implications of the acetylation of TFIIEbetaand TFIIF for transcriptional control by PCAF, p300 and TAFII250.

Differential Roles of p300 and PCAF Acetyltransferases in Muscle Differentiation

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Acetylation of MyoD Directed by PCAF Is Necessary for the Execution of the Muscle Program

p300/CBP and PCAF coactivators have acetyltransferase activities and regulate transcription, cell cycle progression, and differentiation. They are both required for MyoD activity and muscle differentiation. Nevertheless, their roles must be different since the acetyltransferase activity of PCAF but not of p300 is involved in controlling myogenic transcription and differentiation. Here, we provide a molecular explanation of this phenomenon and report that MyoD is directly acetylated by PCAF at evolutionarily conserved lysines. Acetylated MyoD displays an increased affinity for its DNA target. Importantly, conservative substitutions of acetylated lysines with nonacetylatable arginines impair the ability of MyoD to stimulate transcription and to induce muscle conversion indicating that acetylation of MyoD is functionally critical.

A viral mechanism for inhibition of P300 and PCAF acetyltransferase activity

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription. p300/CBP and PCAF histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. Here, we show that E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation.

The p400 complex is an essential E1A transformation target.

Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.

A Human RNA Polymerase II Complex Containing Factors That Modify Chromatin Structure

ABSTRACT We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.

Activation of Integrated Provirus Requires Histone Acetyltransferase p300 AND P/CAF ARE COACTIVATORS FOR HIV-1 Tat

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent.

Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.