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Eukaryotic transcription is a highly regulated process, and acetylation is now known to play a major role in this regulation. Specifically, acetyltransferase enzymes that act on particular lysine side chains of histones and other proteins are intimately involved in transcriptional activation. By modifying chromatin proteins and transcription-related factors, these acetylases are believed to regulate the transcription of many genes.

Chromatin structure, the way in which DNA is packaged in the eukaryotic cell, is known to have a major impact on levels of transcription. In eukaryotes, DNA typically exists in vivo as a repeating array of nucleosomes (271), in which 146 bp of DNA are wound around a histone octamer (consisting of two each of histone proteins H2A, H2B, H3, and H4). Nucleosomes are the first level of chromatin organization, although they in turn are organized into higher-order structures of increasing complexity (129), an extreme example being the condensed metaphase chromosome during cell division. A number of studies have demonstrated that nucleosomal DNA is generally repressive to transcription (91, 183); thus, nucleosome structure and DNA-histone interactions typically make the DNA of genes and their regulatory regions unavailable for the binding of the transcriptional machinery and other factors involved in activation. The direct connection between chromatin alteration and transcriptional activation has been increasingly demonstrated in recent years.

Certain enzymes and protein complexes are now known to bring about changes in the state of chromatin by numerous mechanisms, with resultant effects on gene expression. One class of complexes alter the DNA packaging (remodel chromatin) in an ATP-dependent manner; these include the Swi-Snf complex and a number of others from various organisms (114, 126). Another class of chromatin-altering factors act by covalently modifying histone proteins. These modifications can include phosphorylation, ubiquitination, ADP-ribosylation, and methylation (25), but the best-characterized mechanism is acetylation, catalyzed by histone acetyltransferase (HAT) enzymes.

HATs function enzymatically by transferring an acetyl group from acetyl-coenzyme A (acetyl-CoA) to the -amino group of certain lysine side chains within a histone's basic N-terminal tail region (149). Within a histone octamer, these regions extend out from the associated globular domains, and in the context of a nucleosome, they are believed to bind the DNA through charge interactions (positively charged histone tails associated with negatively charged DNA) or mediate interactions between nucleosomes (67, 151). Lysine acetylation, which neutralizes part of a tail region's positive charge, is postulated to weaken histone-DNA (107, 221) or nucleosome-nucleosome interactions (68, 152) and/or signal a conformational change (175), thereby destabilizing nucleosome structure or arrangement and giving other nuclear factors, such as the transcription complex, more access to a genetic locus. In agreement with this is the fact that acetylated chromatin has long been associated with states of transcriptional activation (99, 244). Recently, some of the proteins and complexes that carry out these acetylation functions have been characterized, and they will be discussed in this review. Interestingly, certain HATs have also recently been shown to specifically acetylate lysine residues within transcription-related proteins other than histones; these events and their regulatory potential will be discussed as well.

Finally, histone acetylation is a reversible process, and deacetylases are also integral to cycles of transcription. Acetylation is generally associated with activation, whereas lack of acetylation tends to correlate with repressiontwo regulatory processes working in harmony to achieve appropriate levels of transcription (135). While outside the scope of this review, it should be noted that a number of deacetylase proteins and complexes have been characterized in the last several years. This has provided a further conceptual linkage between acetylation and transcriptional activity, since some of the histone deacetylases (HDACs) and the proteins with which they associate are previously known DNA-binding repressors or corepressors (reviewed in reference 186).

The phenomenon of histone acetylation in the eukaryotic cell has been known for many years, and since the early 1970s various HAT activities have been isolated and partially characterized. Each of these enzymes generally belongs to one of two categories (30, 74): type A, located in the nucleus, or type B, located in the cytoplasm, although recent evidence indicates that some HAT proteins may function in multiple complexes or locations and thus not precisely fit these historical classifications (200). B-type HATs are believed to have somewhat of a housekeeping role in the cell, acetylating newly synthesized free histones in the cytoplasm for transport into the nucleus, where they may be deacetylated and incorporated into chromatin (4, 199). The A-type HATs, on the other hand, acetylate nucleosomal histones within chromatin in the nucleus; these HATs are potentially linked to transcription and thus are the main focus of this review. A summary of known HAT proteins is presented in Table 1, and these are discussed further in the text.

The best-understood set of acetyltransferases is the GNAT (Gcn5-related N-acetyltransferase) superfamily (174), which have been grouped together on the basis of their similarity in several homology regions and acetylation-related motifs (Fig. 1A). This group includes the HAT Gcn5, its close relatives, and at least three more distantly related HATs, Hat1, Elp3, and Hpa2. It also contains a variety of other eukaryotic and prokaryotic acetyltransferases with different substrates, indicating the conservation and wide application of this type of acetylation mechanism throughout evolution. Four sequence motifs whose functions are not yet fully understoodC, D, A, and B, in N-terminal to C-terminal orderdefine this superfamily. The C motif is found in most of the GNAT family acetyltransferases but not in the majority of known HATs. Motif A is the most highly conserved region, and it is shared with another HAT family, the MYST proteins, described later in this review. Furthermore, it contains an Arg/Gln-X-X-Gly-X-Gly/Ala segment that has been specifically implicated in acetyl-CoA substrate recognition and binding (59, 270).

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Similarities of GNAT (Gcn5-related N-acetyltransferase) superfamily members. (A) Alignment of GNAT homology motifs A, B, C, and D for HATs and representatives of other types of acetyltransferases. Reversed type indicates consensus sequence residues, as determined by Neuwald and Landsman (174), and solid circles mark residues that are particularly conserved throughout the superfamily. The asterisk indicates the glutamate residue known to be critical for HAT catalysis in yeast Gcn5. At the bottom are several members of another acetyltransferase family, the MYST proteins, that share just the A motif. (B) Alignment of proteins from the Gcn5 subgroup of the GNAT superfamily, showing the location of the HAT domain and bromodomain. Shown at the top is the overall homology region shared by all four proteins, with the similarity between the yeast and human proteins indicated; Tetrahymena Gcn5 has 62% similarity with the three others over this same region. The A2 label designates a region in yeast Gcn5 known to interact with the adaptor protein Ada2 (37). In addition, the N-terminal region of PCAF has been generally defined as its site of interaction with p300/CBP and nuclear receptor coactivator SRC-1 (130, 276); this region and the HAT domain also interact with viral protein E1A (40, 193).

Gcn5. The first protein identified as an A-type, transcription-related HAT was discovered in the ciliate Tetrahymena thermophila (31). By way of an in-gel assay of nuclear extract chromatographic fractions run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 55-kDa polypeptide (p55) was found to have acetylation activity on free histones (29). Subsequent protein sequencing revealed that it was a homolog of Saccharomyces cerevisiae (yeast) Gcn5 (77), previously identified as a transcriptional adaptor (or coactivator) involved in the interaction between certain activators and the transcription complex (17, 154, 213). Homologs of Gcn5 have more recently been cloned and sequenced from numerous divergent organismssuch as human (36), mouse (276), Schizosaccharomyces pombe, Drosophila melanogaster (215), Arabidopsis thalania, and Toxoplasma gondii (102)suggesting that its function is highly conserved throughout the eukaryotes.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Primary histone acetylation specifities of some of the known HAT proteins in vitro. Shown are the amino acid sequences for the N-terminal tail regions of human histones H2A, H2B, H3, and H4, with lysine residues numbered and arrows indicating the predominant sites used by various HATs in in vitro experiments. TAFII230 is the Drosophila homolog of human TAFII250 used in site specificity determinations. The above H3 and H4 sequences are nearly identical to those of S. cerevisiae and Drosophila. Specific but relatively nonpreferred or minor sites for certain HATs are not indicated, for example, H4 lysine-8 for Gcn5 and PCAF. It should be noted that lysine specificities may be somewhat expanded or restricted with native HAT complexes and/or on nucleosomal substrates.

PCAF. The gene for PCAF (also referred to as P/CAF) was originally identified from a human cDNA database on the basis of its homology to Gcn5. Because of functional similarities between the yeast activator Gcn4 (which interacts with the adaptor complex) and the activator c-Jun in higher eukaryotes (which interacts with coactivators p300 and CREB-binding protein [CBP]), it was postulated that a human counterpart of Gcn5 may participate in p300/CBP-mediated activation. When PCAF was cloned and investigated, in vitro and in vivo studies revealed that it interacts with p300 and CBP (279), hence its name. p300 and CBP are very closely related coactivators that mediate the transcription of many genes and are also HATs, as described below. PCAF HAT activity, like full-length GCN5, in recombinant form acetylates either free histones or nucleosomes (279), primarily on lysine-14 of histone H3, and more weakly on lysine-8 of histone H4 (207).

Hat1, Elp3, Hpa2, and other acetyltransferases. Gcn5, its homologs, and PCAF have high sequence similarity, but as members of the GNAT superfamily, they are also related by sequence motifs to other HATs and numerous nonhistone acetyltransferases, even prokaryotic ones (174). As shown by the abbreviated list in Fig. 1A, these include the yeast HATs Hat1, Elp3, and Hpa2, protein N-acetyltransferases (which modify N-termini), metabolic enzymes, acetylases involved in drug resistance and detoxification, and a variety of other proteins with unknown specific functions. In addition, GNAT homology is seen in several known transcriptional regulators for which acetylase activity has not yet been describedthe yeast Spt10 protein (173), for example, which affects the expression of various genes (172, 278), including certain histone genes (55).

Structure and mechanism. Along with mutant studies of yeast GCN5, structure determination of several Gcn5-related proteins has added to our knowledge of the mechanisms of acetylation by these enzymes. The first two GNAT superfamily members to have the crystal structures of their acetyltransferase domains solved were yeast Hat1 (59) and Serratia marcescens aminoglycoside N-acetyltransferase (270), a bacterial enzyme that inactivates certain antibiotics by acetylation. In each case, a truncated, catalytically active fragment of the protein bound to CoA or acetyl-CoA substrate was crystallized. Subsequently, HAT domain structures from the Gcn5 subgroupTetrahymena (146, 197) and yeast (241) Gcn5 and human PCAF (48)and HAT protein Hpa2 (5) were also determined.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Stereo diagram of the structure of a HAT domain bound to its substrates. Shown is the GNAT superfamily protein Tetrahymena Gcn5 (blue) with a histone H3 N-terminal tail peptide (red) and CoA (green) bound to its upper and lower clefts, respectively. At the active site, the glutamate-122 residue (aqua), analogous to yeast Gcn5 glutamate-173, catalyzes the transfer of an acetyl group from acetyl-CoA to the lysine-14 sidechain (orange) of H3 peptide. The N termini of both the Gcn5 protein and the H3 peptide are to the left in diagram, and C-termini are to the right.

Another group of evolutionarily related proteins that are known or hypothesized to be HATs is the MYST family, named for its founding members: MOZ, Ybf2/Sas3, Sas2, and Tip60 (23). Additional members have more recently been identified, including yeast Esa1, Drosophila MOF, and human HBO1 and MORF. These proteins are grouped together on the basis of their close sequence similarities (Fig. 4) and their possession of a particular acetyltransferase homology region (part of motif A of the GNAT superfamily) (174), as shown in Fig. 1A. Although containing regions similar in sequence, the members of the MYST family are involved in a wide range of regulatory functions in various organisms.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Alignment of the MYST family of HATs and putative HAT proteins. The MYST homology region is indicated, with the acetyl-CoA-binding site, corresponding to GNAT family motif A, shown as a black box. Z, zinc finger motifs: an atypical C2HC motif in the MYST region (Esa1 diverges from this motif), a typical C2HC in the N-terminal region of HBO1, and two adjacent C4HC3 (or PHD) fingers in MOZ and MORF. C, chromo-like domain found in Esa1, MOF, and Tip60.

Sas2 and Sas3. One of the diverse functions mentioned above is transcriptional silencing, which in S. cerevisiae involves at least two MYST proteins, Sas2 and Sas3 (also known as Ybf2). The SAS2 (something about silencing) gene was originally discovered in a screen for defects in epigenetic silencing in a sir1 genetic background (194). sir1 null mutation leads to loss of mating in most cells due to defects in silencing at the HM mating type loci, but a subpopulation of cells remain able to mate. Additional mutation of SAS2, however, led to absence of mating, even though a sas2 single mutant was phenotypically normal. Interestingly, Sas2 seems to have opposite regulatory effects depending on the silenced locus, promoting silencing at HML while inhibiting it at HMR (64). Other tests demonstrated that Sas2 was required for telomeric silencing (194). Sas3 is a second silencing-related yeast MYST protein, identified by its close homology to Sas2. A sas3 single mutant was also phenotypically normal, and subsequent mutant studies showed that Sas3 has overall weaker effects than Sas2: it is involved in silencing at mating loci, since a sas3 mutation (like sas2) restored silencing to a partially defective HMR locus but did not affect silencing at telomeres (194).

Esa1. A third yeast MYST family protein, Esa1, has recently been identified and characterized as an essential HAT required for cell cycle progression. Esa1 was originally identified through its homology with Sas2, Sas3, and other MYST proteins, and a null mutant of its gene was inviable, hence its name (essential Sas family acetyltransferase 1) (216). Esa1 is a HAT, as recombinant protein was able to acetylate free histones H2A, H3, and H4 in vitro, with its strongest activity on histone H4, particularly at lysine-5. It was unable, however, to acetylate nucleosomes in vitro. In vivo, loss of Esa1 led to specific defects in histone acetylation and growth (47). When esa1 temperature-sensitive mutants were grown at the restrictive temperature, the lysine-5-acetylated form of histone H4 was partially lost (extracts were probed with antibody specific to this isoform). Furthermore, flow cytometric and microscopic analyses of these mutants revealed that cells that lose Esa1 exhibit G₂/M arrest, blocked in the cell cycle subsequent to DNA replication but prior to mitosis and cell division (47). Taken together, these findings demonstrate the importance of the Esa1 protein in yeast cellular function, and its direct connection to transcription has recently been shown by studies with a native Esa1-containing complex, NuA4 (described below).

MOF. In Drosophila melanogaster, the MOF protein is a MYST family member with an important role in another transcriptional regulatory process, dosage compensation. Since male fruit flies have only one copy of the X chromosome compared to females' two, dosage compensation occurs in males to cause a twofold increase in the expression of X-linked genes (reviewed in reference 121). Association of a dosage compensation complex (123) with the chromosome is correlated with increased acetylation of histone H4 at a specific residue (lysine-16) (22, 245). The mechanism of this process was elucidated with the characterization of the mof (males absent on the first) mutation, which made male flies inviable. The gene product MOF was found to have MYST homology, and its direct link to histone acetylation was demonstrated by the fact that dying mof mutant males lack the lysine-16-acetylated isoform of histone H4 normally associated with the X chromosome (103). Interestingly, the mutation (mof¹) leading to nonfunctional MOF was a single glutamate substitution at a GNAT motif A invariant glycine residue implicated in acetyl-CoA substrate binding.

Tip60. The first human MYST protein to be discovered, Tip60, also demonstrated a potential direct relationship between activation and histone acetylation. Tip60 (Tat-interactive protein, 60 kDa) was identified in a yeast two-hybrid/human library screen seeking proteins that interact with the activation domain of the HIV-1 transactivator protein Tat; specific physical interaction was further demonstrated by binding of expressed Tip60 to purified Tat in vitro (120). A recombinant construct of Tip60 lacking the N-terminal 40% but containing the MYST domain homology region was subsequently shown to have in vitro HAT activity, acetylating free histones H2A, H3, and H4 on specific lysines but acetylating nucleosomes poorly (125, 277). The findings of HAT activity and Tat interaction have recently provided insights into the cellular function of Tip60, as the Tat-repressed gene for Mn-dependent superoxide dismutase (262) was tested and found to be positively regulated by Tip60 in vivo. Furthermore, Tat was found to prevent this activation by specifically inhibiting the HAT activity of Tip60, leading to the hypothesis that Tip60 normally activates a set of genes by histone acetylation but that their expression can be opposed by Tat-mediated HAT inhibition (50). More information on the physiological functions of Tip60 may be provided by the very recent identification of a native, nucleosome-acetylating Tip60 complex, described later in this review (Y. Nakatani, unpublished results).

MOZ and MORF. While Tip60 is apparently associated with the action of HIV, MOZ is a MYST protein involved in another specific human disease process, oncogenic transformation leading to leukemia. When a particular chromosomal translocation in acute myeloid leukemia was characterized, it was found to have resulted in the fusion of two apparent HATs, the novel protein MOZ (monocytic leukemia zinc finger protein) (23) and CBP (described below). This created a chimeric protein consisting of the N-terminal three-quarters of MOZ (including its MYST and zinc finger domains) fused to the C-terminal 90% of CBP, containing its HAT domain and activator interaction regions. Although acetyltransferase activity of MOZ has not been directly demonstrated, it is hypothesized that MOZ-CBP may cause aberrant chromatin acetylation due to mistargeting of specific HAT activities, ultimately leading to leukemogenesis.

HBO1. A fourth human MYST protein is HBO1 (histone acetyltransferase bound to ORC), which was discovered in a two-hybrid screen on the basis of its interaction with the ORC1 subunit of the origin recognition complex (ORC) (112). ORC is conserved throughout the eukaryotes and is primarily known to bind DNA replication origins and to be critical for the initiation of replication (13, 60). ORC also has a transcriptional function, however, since it has been demonstrated to be involved in silencing at yeast mating type loci (12, 69, 71) and Drosophila heterochromatin regions (184). In the case of S. cerevisiae, a relationship with the MYST proteins Sas2 and Sas3 is suggested by the fact that ORC binds Sir1 (70, 242) and that Sas2 displays genetic interactions with ORC (SAS2 knockout results in partial suppression of orc2 and orc5 mutant phenotypes) and antagonizes ORC-mediated silencing at the HMR locus (64).

After the discovery of histone acetylation by Gcn5 and PCAF, the critical role of acetyltransferases in transcriptional regulation was also demonstrated by the fact that a pair of previously well-characterized coactivators of multicellular eukaryotes, p300 and its close homolog CBP (CREB-binding protein), are themselves HATs (8, 178) and FATs (as described below). The interactions of p300/CBP (p300 and CBP are often referred to as a single entity, since the two proteins are considered structural and functional homologs) with PCAF and GCN5, described above, and with nuclear receptor coactivators, described below, are examples of transcriptional regulatory complexes with multiple acetyltransferase activities.

p300/CBP is a ubiquitously expressed, global transcriptional coactivator that has critical roles in a wide variety of cellular processes, including cell cycle control, differentiation, and apoptosis (81, 211), and mutations in p300 and CBP are associated with certain cancers and other human disease processes (80). On the molecular level, p300/CBP stimulates transcription of specific genes by interacting, either directly or through cofactors, with numerous promoter-binding transcription factors such as CREB, nuclear hormone receptors, and oncoprotein-related activators such as c-Fos, c-Jun, and c-Myb. As described above, p300/CBP also binds the HAT PCAF, an interaction with which adenoviral oncoprotein E1A competes (279). p300/CBP is a large protein of about 300 kDa and more than 2,400 residues, and at least four interaction domains with different sets of factors have been characterized throughout its sequence, as shown in Fig. 5. Furthermore, its central region contains a bromodomain motif (98, 117), which is also found in the HATs Gcn5, PCAF, and TAF_II250.

View larger version (8K): [in this window] [in a new window]   FIG. 5.   Domains and interaction regions of the global coactivator HATs p300/CBP. Labeled below the polypeptide diagram are several domains and sequence motifs, including a bromodomain, the HAT domain, and ZZ and TAZ putative zinc fingers (190). Above are indicated some of the proteins demonstrated to interact with p300/CBP at certain regions. PCAF has been shown to interact with two regions of p300/CBP (130).

The HAT activity of p300/CBP was first discovered in an E1A pulldown from HeLa (human) nuclear extract (178) and in direct CBP immunoprecipitations from Cos (primate) cell extracts (8). In vitro studies with recombinant p300 and CBP proteins confirmed that these proteins were indeed HATs, strongly acetylating the amino-terminal tails of all four core histones with little apparent specificity. Unlike other HATs, recombinant p300/CBP was able to acetylate all four histones within nucleosomes as well as in free-histone form. Deletion mutant analysis mapped the HAT domain of p300/CBP to an interior region between the bromodomain and the PCAF/E1A/MyoD/c-Fos interaction region (8, 178). p300/CBP represents a unique class of acetyltransferase, although it may be distantly related to other HATs. Careful sequence analysis identified regions with limited homology to GNAT motifs A, B, and D, in addition to another short motif shared with PCAF and Gcn5 (158). Site-directed mutagenesis demonstrated that all four of these motifs contribute to CBP's HAT function. Furthermore, the connection between p300/CBP's HAT function and transcription in vivo was demonstrated by the fact that a promoter-tethered CBP HAT domain resulted in activation, and HAT-impaired mutant versions showed a direct correlation of acetylation competence with this transcriptional activity (158). p300/CBP's HAT function was also shown to be required for certain types of nuclear receptor-mediated activation in vivo (130).

In addition, the HAT activity of p300/CBP is apparently regulated by other factors. As observed for PCAF, the viral protein E1A and the regulatory protein Twist were shown to bind to p300 and inhibit its HAT activity (40, 96, 187). However, another report indicates that E1A has a HAT-stimulatory effect on CBP (2), suggesting a possible functional difference between p300 and CBP (this study also found that cell cycle-dependent phosphorylation of CBP by Cdk2 increases its HAT activity). Since another study reported no effect of E1A binding on CBP's HAT activity (8), it is possible that these HAT effects are due to experimental discrepancies that need to be resolved.

Overall, p300/CBP is one of the most potent and versatile of the acetyltransferases, consistent with its role as a global coactivator in higher eukaryotes. Like PCAF, p300/CBP is known to acetylate and regulate various transcription-related proteins other than histones. The known FAT substrates of p300/CBP, described later in this review, include HMG I(Y), activators p53, GATA-1, erythroid Krüppel-like factor (EKLF), Drosophila T-cell factor (dTCF), and HIV Tat, nuclear receptor coactivators SRC-1, ACTR, and TIF2, and general factors TFIIE and TFIIF. Another phenomenon relevant to the regulatory activities of p300/CBP is that human chromosomal translocations fusing CBP to either the putative HAT MOZ (23) or the MLL gene (232) can result in leukemogenesis; the mechanisms of these processes, however, and whether they involve HAT or FAT activity remain to be elucidated.

SRC-1. Steroid receptor coactivator-1 (SRC-1), also known as p160 (119) and NCoA-1 in mice (240), is a human nuclear receptor cofactor originally discovered by way of its interaction with the human progesterone receptor (PR) in a yeast two-hybrid screen. In vivo experiments in mammalian cells established the coactivator function of SRC-1, as it was able to stimulate ligand-dependent activation by numerous nuclear receptors, including PR, glucocorticoid receptor (GR), estrogen receptor (ER), thyroid hormone receptor (TR), and retinoid X receptor (RXR) (180). Because of this coactivator function, recombinant SRC-1 was assayed in vitro and found to have HAT activity, acetylating H3 and H4 either as free histones or in mononucleosomes (220). Truncation analysis revealed that the HAT domain is located in the C-terminal region of SRC-1, as diagrammed in Fig. 6. SRC-1 was known to interact with p300/CBP (119, 214, 280), and interestingly, it also interacted with PCAF in vitro and in vivo (220), indicating that multiple HATs are employed to regulate hormone-signaled transcription. In addition, p300/CBP was recently shown to acetylate SRC-1, an event that is likely relevant to its nuclear receptor coactivator function (43).

View larger version (16K): [in this window] [in a new window]   FIG. 6.   Alignment of the p160 family of mammalian nuclear receptor coactivators. Indicated are the PAS/basic helix-loop-helix homology (bHLH) domain, nuclear receptor interaction regions, and the general area of interaction for coactivators p300/CBP and PCAF. ACTR and SRC-1 each have a HAT domain near their C terminus (42, 220), although the boundaries of these domains have only been approximately defined, and TIF2's domain is inferred by homology.

ACTR. To identify additional human proteins that interact with nuclear hormone receptors, a yeast one-hybrid screen was employed which used reporter genes with retinoic response elements and a human retinoic acid receptor (RAR) as bait. Screening with a cDNA library resulted in several known receptor interactors (including SRC-1) and one novel cofactor, termed ACTR (42), also known as RAC3 (142), AIB1 (6), and TRAM-1 (231) in humans and p/CIP in mice (240). Like SRC-1, ACTR was shown to interact with multiple nuclear hormone receptors and stimulate transactivation. Further, it was tested in vitro and also found to be a HAT capable of acetylating free or nucleosomal histones H3 and H4, and its HAT domain similarly mapped to the C-terminal end of the protein (42). In fact, ACTR shows significant sequence similarity to SRC-1 in several regions (Fig. 6): an N-terminal, basic helix-loop-helix/PAS region (236), receptor and coactivator interaction domains, and the C-terminal HAT region, defining, along with TIF2, the p160 (or SRC) family of nuclear receptor coactivators (42, 139, 252).

TIF2. A third potential HAT in the human nuclear receptor coactivator family is TIF2 (transcriptional intermediary factor 2) (252), also known as GRIP1 (106) and NCoA-2 (240) in mice. Like SRC-1 and ACTR, TIF2 binds to a number of nuclear hormone receptors, stimulates transcriptional activation (252), and interacts with (251) and is acetylated by (43) CBP. Although its HAT activity has not yet been demonstrated, TIF2 has all of the homology regions shared by SRC-1 and ACTR, including the putative HAT domain (42). Because of the sequence and functional similarities of this protein to the other two coactivators, it stands as a likely HAT candidate whose activity remains to be characterized. Another potentially interesting aspect of TIF2 is its fusion to MOZ in leukemia-associated translocations, as noted above (38, 144). Future studies will be required to determine the mechanism of this oncogenic effect and whether it involves either putative HAT activity.

Another direct connection between acetylation and activated transcription was demonstrated with the discovery that one of the TAF_II (TATA-binding protein [TBP]-associated factor) subunits of the general transcription factor TFIID is itself a HAT. Specifically, homologs of this proteinTAF_II250 in humans, TAF_II230 in Drosophila, and Taf_II145/130 in S. cerevisiaewere shown to have HAT activity in vitro (169).

TFIID is one of the general factors required for the assembly of the RNA polymerase II transcription preinitiation complex, along with TFIIA, TFIIB, TFIIE, and TFIIF (32, 97). TFIID is in fact the first factor needed in the stepwise assembly: through its TBP subunit, TFIID binds to specific promoter DNA sequences and allows subsequent formation of the transcription complex. Although TBP without TAF_IIs is able to bind promoters and allow basal transcription in vitro, the TAF_II subunits promote activated transcription. Furthermore, TAF_IIs have been shown to interact with certain activators and initiation-related factors (250).

The potential involvement of acetylation in TAF_II function was realized with the discovery that a 250-kDa band from human nuclear extract (in an in-gel assay) and immunoprecipitated human TFIID had HAT activity (169). Further characterization of the TAF_II HAT activity was performed with recombinant Drosophila TAF_II230, which was found to acetylate H3 (preferentially on lysine-14, like Gcn5) and H4 in a free histone mixture (and H2A as an individual histone). It should be noted that TAF_II250 and its homologs, like the p160 nuclear receptor coactivators, have some of the weaker in vitro HAT activities observedp300/CBP and PCAF, for example, have more potent activities (130, 177; unpublished results). The in vivo significance of these apparent differences in catalytic strength, however, is not yet known.

Truncation studies with yeast and Drosophila TAF mapped the HAT domain to the conserved central region of the protein. This region has little apparent similarity to other known proteins, so TAF_II250 may define a unique HAT class. However, a potential acetyl-CoA binding site has been identified within this region; it shares a Gly-X-Gly pattern with Gcn5 and other acetyltransferases, and mutation of these glycines led to reduced HAT activity (58). Like Gcn5, PCAF, and p300/CBP, TAF_II250 also has a bromodomain (and Drosophila TAF_II230 has two), but truncation studies demonstrated that it is not required for HAT activity (169); this and the fact that the yeast homolog contains no bromodomain argue against a major role for it in TAF_II250's HAT function.

The HAT activity of TAF_II250 and its homologs suggests a model for the initiation of transcription complex formation at chromatin-packaged promoters. Nucleosomes are known to inhibit binding of TBP to the TATA box (164, 273), and this inhibition is apparently mediated by histone tails (82, 115). As part of TFIID, TAF_II250 may well facilitate TBP binding directly by acetylating histones at the TATA box, allowing formation of the preinitiation complex. Also potentially relevant to TAF_II250 function is that TFIID is proposed to contain a histone octamer-like structure (104, 274), which may displace nucleosomal histones in concert with TAF_II250's HAT activity. Although the widespread involvement of TFIID in initiation (including at TATA-less promoters) is expected to bring TAF_II250 to very many genes, recent mutant studies suggest that its HAT activity is required for transcription at only a subset of promoters (e.g., certain cell cycle regulators) (58, 176). The mechanism of this specificity, however, is not yet known.

Although all of the A-type HATs discussed so far in this review are proposed to be involved with transcription by RNA polymerase II (primarily of mRNA), chromatin structure is expected to affect any kind of transcription, such as the synthesis of rRNA by RNA polymerase I or tRNA precursors by RNA polymerase III. Evidence that histone acetylation is a generally employed mechanism in transcription is the fact that subunits of TFIIIC, a general transcription factor in the RNA polymerase III basal machinery, were also recently identified as HATs (109, 133). The known function of TFIIIC is to initiate transcription complex formation by binding to promoter DNA and recruiting TBP-containing TFIIIB and RNA polymerase III (137). Recent in vitro studies with purified human TFIIIC showed that it harbored HAT activity, acetylating H3, H4, and H2A as free histones and also in nucleosomes. Interestingly, an in-gel assay of TFIIIC revealed that three of its nine subunits have apparent HAT activity. The HAT functions of two of these subunits, TFIIIC110 and TFIIIC90, have been confirmed and further investigated. A bacterially expressed C-terminal fragment of TFIIIC110 had HAT activity in an in-gel assay (133), while recombinant TFIIIC90 was competent for the acetylation of either nucleosomal or free histone H3, with an apparent preference for lysine-14 (like Gcn5 and TAF_II230) (109). Future studies should better clarify the function of these HAT activities in this type of transcription, but a logical hypothesis is that it fulfills a role similar to that of TAF_II250 in the RNA polymerase II transcription complex. In both cases, a HAT enzyme is intimately associated with the first step in DNA binding of the transcription complex and likely acts to destabilize promoters' nucleosomes to facilitate this process. Furthermore, it is reasonable to predict that RNA polymerase I transcription is also associated with HAT activity, although this has not yet been demonstrated.

Most known HATs are able to acetylate free histones in vitro when assayed as a single polypeptide. Many, however, such as Gcn5, are unable to acetylate their probable physiological substrate, nucleosomal histones, under standard conditions in vitro, apparently due to the requirement for other factors to allow this level of substrate specificity. Because of this, a study was performed which sought to identify native yeast complexes capable of acetylating nucleosomal substrates (84). Through fractionation of S. cerevisiae extracts and assays of nucleosomal HAT activity, four distinct complexes were discovered and have been further characterized: SAGA, ADA, NuA4, and NuA3.

SAGA. After their discovery, the four separable nucleosomal HAT activities were initially analyzed by Western blot and null mutation studies, and it was found that the two nucleosomal histone H3/H2B-specific complexes contained Gcn5 as their HAT catalytic subunit, along with two other transcriptional adaptor proteins, Ada2 and Ada3 (84). Interestingly, one of these complexes also contained several Spt proteins, which were originally identified via another transcription-related genetic screen (suppression of Ty and  insertions at promoters) (reviewed in reference 267). This complex was therefore named SAGA (Spt-Ada-Gcn5 acetyltransferase) (reviewed in reference 88); the other complex, containing Ada proteins but not Spts, was called ADA (described below).

View larger version (42K): [in this window] [in a new window]   FIG. 7.   Schematic diagram of known subunits and functions of the yeast SAGA and human PCAF or GCN5 HAT complexes. The yeast SAGA complex (top) contains Gcn5 as its HAT catalytic subunit. SAGA has been shown to interact with acidic activation domains, and this function may be mediated by its adaptor components (Ada2, Ada3, and Gcn5) or possibly through Tra1 or other subunits. Another subset of SAGA proteins (Ada1, Spt7, and Spt20/Ada5) are required for its structural integrity and overall function. The Spt3 and Spt8 subunits have been implicated in interaction with TBP, and the TafII group, which also participate in TFIID, may provide TBP-binding function to SAGA as well. The human SAGA-analogous complexes (bottom) contain either GCN5 or PCAF. The PCAF complex has been more thoroughly studied; it has nucleosome acetylation function, and known subunits include TRRAP, hADA2, hADA3, hSPT3, and TAFIIs/PAFs (PCAF-associated factors)all homologs of proteins found in SAGA. Activator and TBP interaction functions are hypothesized for these human complexes but have not yet been demonstrated.

ADA. The other known Gcn5-containing complex is ADA, which has a size of about 800 kDa. Like SAGA, the ADA complex acetylates nucleosomes primarily on histones H3 and H2B in vitro, and it contains Ada2 and Ada3 but none of the other known subunits of SAGA (84). Recently, peptide analysis revealed a novel subunit unique to ADA, demonstrating that it is a distinct complex and not a subcomplex or artifactual fragment of SAGA (63). This subunit, Ahc1 (ADA HAT complex component 1), is required for the structural integrity of ADA, as a knockout mutation disrupted the complex.

NuA4. Another yeast HAT complex identified by Grant et al. (complex 2) was immediately distinguishable from the others in that its nucleosomal substrate was primarily histone H4 (as well as H2A, to a lesser degree) and it did not significantly acetylate histone H3 (84). Further purification and characterization of this 1.3-MDa complex, called NuA4 (nucleosomal acetyltransferase of histone H4), has revealed that its HAT catalytic subunit is the MYST protein Esa1 (3). It also contained Tra1, identified previously as a component of SAGA. Also like SAGA, NuA4 interacted with acidic activation domains in vitro and stimulated transcription in an acetylation-dependent manner in various in vitro assays with chromatin templates (3, 113, 246, 254). Interestingly, extensive acetylation of nucleosomal templates with NuA4 led to transcriptional activation even with other types of activators that do not interact with NuA4, an effect not seen with SAGA (113). This general activation by histone H4/H2Aas opposed to H3/H2Bacetylation shows the potential impact of nucleosomal histone specificity on transcription. NuA4's composition (it contains at least seven additional unknown subunits) and in vivo function remain to be fully characterized, but its possession of two essential transcription-related subunits, including a HAT needed for cell cycle progression (47), suggests that it plays a critical role in the cell.

NuA3. A fourth yeast HAT complex that has been identified and further investigated is NuA3 (also referred to as complex 3), a 500-kDa complex that exclusively acetylates histone H3 in nucleosomes (84). This is perhaps the least well characterized complex in terms of composition, but its catalytic subunit was recently determined to be Sas3, a MYST protein involved in silencing (S. John and J. L. Workman, unpublished results). Some in vitro studies have been performed with NuA3, and like ADA, it failed to interact with activation domains or to activate transcription in a specific way (246, 254). The function of this complex in vivoi.e., its role, if any, in transcription or its relationship to silencingremains to be determined by future studies.