A Tetrahymena enzyme transcription-associated histone acetyltransferase type A(HAT A) has been cloned that is strikingly homologous to the yeast protein Gcn5, a putative transcriptional adaptor. Recombinant Gcn5p possesses HAT activity. Both the ciliate enzyme and Gcn5p contain potential active site residues found in other acetyltransferases and a highly conserved bromodomain. The presence of this domain in nuclear A-type HATs, but not in cytoplasmic B-type HATs, suggests a mechanism whereby HAT A is directed to chromatin to facilitate transcriptional activation. These findings shed light on the biochemical function of the evolutionarily conserved Gcn5p-Ada complex, directly linking histone acetylation to gene activation, and indicate that histone acetylation is a targeted phenomenon (Brownell, 1996).

Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity that is highly specific for H4 when tested as a free histone. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated); these sites are diagnostic for deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form. These activities are distinct from a macronuclear acetylase, which acetylates H3 and H4 (macro- or micro-nuclear) equally well as free histones and also acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles, which are different from those of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity that acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity that is able to act on exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity that is either modified or altogether distinct from that in macronuclei (Richman, 1998).

Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA-bound transcriptional activators with general transcription factors. GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin. Within GCN5, the minimal catalytic domain necessary to confer HAT activity has been identified. In vivo activity of GCN5 requires this domain. However, complementation of growth and transcriptional activation in gcn5- cells requires not only the HAT domain of GCN5, but also interaction with ADA2. The bromodomain in GCN5 is dispensable for HAT activity and for transcriptional activation by strong activators; however, it is required for full complementation in other assays. Fusion of GCN5 to the bacterial lexA DNA binding domain activates transcription in vivo, and requires both the HAT domain and the ADA2 interaction domain. These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones (Candau, 1997).

The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase that links histone acetylation to gene activation. Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. Lysine 9 is also the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly (Kuo, 1996).

The yeast transcriptional adapter Gcn5p serves as a histone acetyltransferase, directly linking chromatin modification to transcriptional regulation. Two human homologs of Gcn5p have been reported previously: hsGCN5 and hsP/CAF (p300/CREB binding protein [CBP]-associated factor). While hsGCN5 is predicted to be close to the size of the yeast acetyltransferase, hsP/CAF contains an additional 356 amino-terminal residues of unknown function. Both the GCN5 and the P/CAF genes encode proteins containing this extended amino-terminal domain. While a shorter version of GCN5 might be generated upon alternative or incomplete splicing of a longer transcript, mRNAs encoding the longer protein are much more prevalent in both mouse and human cells, and larger proteins are detected by GCN5-specific antisera in both mouse and human cell extracts. Mouse GCN5 (mmGCN5) and mmP/CAF genes are ubiquitously expressed, but maximum expression levels are found in different, complementary sets of tissues. Both mmP/CAF and mmGCN5 interact with CBP/p300. Interestingly, mmGCN5 maps to chromosome 11 and cosegregates with BRCA1: mmP/CAF maps to a central region of chromosome 17. As expected, recombinant mmGCN5 and mmP/CAF both exhibit histone acetyltransferase activity in vitro with similar substrate specificities. However, in contrast to yeast Gcn5p and the previously reported shorter form of hsGCN5, mmGCN5 readily acetylates nucleosomal substrates as well as free core histones. Thus, the unique amino-terminal domains of mammalian P/CAF and GCN5 may provide additional functions important to recognition of chromatin substrates and the regulation of gene expression (Xu, 1998).

Histone acetylation is important in chromatin remodelling and gene activation. Nearly all known histone-acetyltransferase (HAT)-associated transcriptional co-activators contain bromodomains, which are approximately 110-amino-acid modules found in many chromatin-associated proteins. Despite the wide occurrence of these bromodomains, their three-dimensional structure and binding partners remain unknown. The solution structure of the bromodomain of the HAT co-activator P/CAF (p300/CBP-associated factor) is reported in this paper. The structure reveals an unusual left-handed up-and-down four-helix bundle. In addition, it has been shown by a combination of structural and site-directed mutagenesis studies that bromodomains can interact specifically with acetylated lysine, making bromodomains the first known protein modules to do so. The nature of the recognition of acetyl-lysine by the P/CAF bromodomain is similar to that of acetyl-CoA by histone acetyltransferase. Thus, the bromodomain is functionally linked to the HAT activity of co-activators in the regulation of gene transcription (Dhalluin, 1999).

The crystal structure of the yeast histone acetyltransferase Hat1-acetyl coenzyme A (AcCoA) complex has been solved at 2.3 A resolution. Hat1 has an elongated, curved structure, and the AcCoA molecule is bound in a cleft on the concave surface of the protein, marking the active site of the enzyme. A channel of variable width and depth that runs across the protein is probably the binding site for the histone substrate. A model for histone H4 binding by Hat1 is discussed in terms of possible sources of specific lysine recognition by the enzyme. The structure of Hat1 provides a model for the structures of the catalytic domains of a protein superfamily that includes other histone acetyltransferases such as Gcn5 and CBP (Dutnall, 1998).

If it is assumed that no large conformational change occurs upon H4 binding, then it appears that the Lys-12 side chain can approach the carbonyl group only from one side of the gate over the AcCoA binding cleft. In an extended conformation, the lysine side chain can reach to within 1-2 Angstrom units of the carbonyl group without producing any clashes with the protein structure, particularly from the main chain either side of Lys-12. However, for a peptide with extended conformation, this can only be achieved if it traverses a cleft of varying width and depth that crosses the structure almost perpendicularly to the long axis of the protein. An attractive feature of this model is that it suggests possible explanations for the length of the Hat1 recognition motif and preference for Lys-12 of histone H4. The length of the cleft is long enough to accommodate a 6-7 residue peptide in extended conformation, similar to the length of the proposed recognition motif. When the H4 peptide is oriented in the putative binding cleft, with Lys-12 poised for modification, it brings Leu-10 adjacent to a hydrophobic pocket that forms the deepest part of the cleft. Positioning Leu-10 of H4 in this pocket may contribute to selectivity for Lys-12. It also places Gly-13 and Gly-14 in the shallowest and narrowest part of the cleft, which may explain the preference for glycine residues at these positions of the recognition motif. One last additional source of preference for Lys-12 of histone H4 may arise from interactions with the nonmodified lysine residues on either side of this site (Lys-8 and Lys-16). In the current model, these side chains are adjacent to acidic patches on the surface of the protein, which could add binding energy through electrostatic interactions (Dutnall, 1998).

The structure of Hat1 sheds light on its relationship to other histone acetyltransferases and improves the basis for comparison among those proteins that have only a low degree of sequence similarity. This leads to a modification of the sequence alignments proposed in earlier publications. Most of the known HAT enzymes share conserved sequence motifs with one another and with a more extended superfamily, called the GCN5-related N-acetyltransferase (GNAT) superfamily, which includes protein N-acetyltransferases (NATs), metabolic enzymes, detoxification and drug resistance enzymes, and other proteins whose function is unknown. Among the HAT enzymes included in this superfamily are Hat1 and its homologs, the Gcn5 family of proteins (which includes P/CAF and T. thermophila Hat-A. Four conserved sequence motifs (A-D) have been described to characterize the GNAT superfamily. Of these, motif A is the longest and most highly conserved. According to this scheme, the HAT families lack motif C. The location of these motifs in the Hat1 structure is shown in this paper. It has been previously suggested that these conserved motifs are involved in AcCoA binding. The structure of Hat1 shows that, indeed, these structural motifs make up a large part of the AcCoA binding region (Dutnall, 1998 and references).

Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo; they may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, a series of hGCN5-yGCN5 chimeras were constructed to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complements gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduces both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation (Wang, 1997).

The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. These findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin (Mizzen, 1996).

The CBP protein acts as a transcriptional adaptor for many different transcription factors by directly contacting DNA-bound activators. One mechanism by which CBP is thought to stimulate transcription is by recruiting the histone acetyltransferase (HAT) P/CAF to the promoter. CBP has intrinsic HAT activity. The HAT domain of CBP is adjacent to the binding site for the transcriptional activator E1A. Although E1A displaces P/CAF from CBP, it does not disrupt the CBP-associated HAT activity. Thus E1A carries HAT activity when complexed with CBP. Targeting CBP-associated HAT activity to specific promoters may therefore be a mechanism by which E1A acts as a transcriptional activator (Bannister, 1996).

The yeast transcriptional activator ADR1, which is required for ADH2 and peroxisomal gene expression, contains four separable and partially redundant activation domains (TADs). Mutations in ADA2 or GCN5, encoding components of the ADA coactivator complex involved in histone acetylation, severely reduce LexA-ADR1-TAD activation of a LexA-lacZ reporter gene. Similarly, the ability of the wild-type ADR1 gene to activate an ADH2-driven promoter is compromised in strains deleted for ADA2 or GCN5. In contrast, defects in other general transcription cofactors such as CCR4, CAF1/POP2, and SNF/SWI display much less or no effect on LexA-ADR1-TAD activation. Using an in vitro protein binding assay, ADA2 and GCN5 were found to specifically contact individual ADR1 TADs. ADA2 can bind TAD II, and GCN5 physically interacts with all four TADs. Both TADs I and IV were also shown to make specific contacts to the C-terminal segment of TFIIB. In contrast, no significant binding to TBP was observed. TAD IV deletion analysis indicates that its ability to bind GCN5 and TFIIB is directly correlated with its ability to activate transcription in vivo. ADR1 TADs appear to make several contacts, which may help explain both their partial redundancy and their varying requirements at different promoters. The contact to and dependence on GCN5, a histone acetyltransferase, suggests that rearrangement of nucleosomes may be one important means by which ADR1 activates transcription (Chiang, 1996).

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. 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 (Ogryzko, 1996).

PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, it is found that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases (Herrera, 1997).

Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear. Efforts to clarify these relationships have revealed two novel properties of nuclear receptors (Blanco, 1998).

(1) First, the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and increased expression of PCAF leads to enhanced retinoid-responsive transcription. Of the two domains present in PCAF, the carboxy-terminal domain (from amino acid position 352 to 832) represents the region homologous to the yeast GCN5 and contains histone acetylase activity. The amino-terminal region (composed of amino acids 1-351) shares little homology with known genes, and its function has not been fully elucidated. To determine a region of PCAF involved in binding to the heterodimer, truncated recombinant PCAFs lacking either the amino-terminal or carboxy-terminal domain were examined. Recombinant deltaN1 or deltaN2, lacking either the amino-terminal domain alone or the amino-terminal region plus the additional 113 amino acids of the carboxy-terminal region, binds to the heterodimer-RARE complex, although the binding of deltaN2 is slightly weaker than that of deltaN1 and full-length PCAF. In contrast to this, deltaC shows little binding to the complex. Ligand has no effect on the binding activity of the truncated PCAF. These results indicate that the conserved carboxy-terminal domain is required for binding to the heterodimer-RARE complex (Blanco, 1998).

(2) With respect to the second novel property of nuclear receptors, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, and therefore defines a novel cofactor interaction surface. These results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors. This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself. To evaluate whether PCAF enhances ligand-dependent promoter activity through its histone acetylase activity, two additional deletion constructs were examined in which the recently identified catalytic domain of PCAF was deleted (deltaHAT1 and deltaHAT2). Histone acetylase activity is found to be completely abrogated in these deletion constructs in vitro. deltaHAT1 and deltaHAT2, similar to deltaC and deltaN2, fails to give full enhancement in promoter activity attained by the intact PCAF. These results support the idea that PCAF potentiates retinoid-dependent transcription at least partly through its histone acetylase activity. Immunoblot analysis performed with transfected cells shows that exogenous PCAF is expressed in a dose-dependent manner, while the expression of the endogenous p300 remains unchanged. On the basis of these observations, it is suggested that PCAF may play a more central role in nuclear receptor function than previously anticipated (Blanco, 1998).

It was found that corepressors from the N-CoR-SMRT family inhibit binding of recombinant PCAF to the nuclear receptor heterodimer in the absence of ligand, but that this binding is restored upon addition of ligand, concomitant with repressor release. These results suggest that corepressors, by virtue of their dissociation from the receptor, confer ligand dependence on PCAF binding. It has been shown that N-CoR and SMRT bind to the hinge region of receptors. Because the hinge region present in the ligand-binding domain is only ~30 amino acids away from the DNA-binding domain, corepressors could cause either a steric block of PCAF binding or induce a local conformational change that precludes PCAF binding. Adding to this passive regulation of activity exclusion, corepressors have recently been shown to be associated with the histone deacetylase HDAC-1 and mSin3, which are thought to establish transcriptional repression via modification of chromatin. A model depicting how ligand reverses this process in two steps is presented: first the ligand promotes the dissociation of the repressor complex, which in turn enables the second step of PCAF recruitment. Like the repressors, PCAF itself also functions in at least two ways: (1) as a histone acetylase it has the direct capacity to modify chromatin to reverse repression, and (2) via its p300/CBP- and SRC-interaction domains, it serves to recruit additional activators (Blanco, 1998).

P/CAF is shown to be able to regulate transcription: this function is independent of its binding to CBP. The HAT domain of P/CAF has transcriptional activation potential in yeast. In mammalian cells P/CAF can stimulate transcription of the RSV promoter, using the activity of its HAT domain. The adenovirus protein E1A is shown to target P/CAF and sequester its transcriptional activity. Binding of E1A to P/CAF is direct, independent of CBP and requires residues within E1A conserved region 1. The P/CAF binding residues in E1A are within a motif shown to be essential for efficient disruption of myogenesis by E1A. The fact that E1A can directly bind and regulate the activity of P/CAF, independently of its regulation of CBP, highlights an important role for P/CAF in the process of cell differentiation (Reid, 1998).

The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly. Acetylation takes place at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. Acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. The acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene (Munshi, 1998).