histone acetyltransferase complexes: one size doesn't fit all

When Conrad H. Waddington coined the term ‘epige-netic landscape’ in the 1940s, he could not have envi-sioned that more than a half century later his metaphor would become the basis for a whole field of research1,2 (TIMELINE). Originally used to explain how gene regulation determines development, the definition of epigenetics has evolved to mean the heritable changes in gene function that occur in the absence of changes in DNA sequence. Now, more than 50 years after introducing the term epigenetics, it is abundantly clear that we are more than the sum of our genes.

In the nucleus of eukaryotic cells, DNA is highly compacted and organized into chromatin by both histone and non-histone proteins. The basic unit of chromatin is the nucleosome, which consists of 147 base pairs of DNA wrapped 1.6 times around an octamer of core his-tone proteins H2A, H2B, H3 and H4 (REF. 3). These four canonical histone proteins are composed of a structured central (globular) domain that is in close contact with the DNA and a less well-structured amino-terminal tail domain. Histones are also distinguished by the fact that they undergo abundant post-translational modifications to both the globular and tail domains. The combination of such histone modifications is indicative of whether a gene is transcriptionally active or inactive4,5. Several histone modifications have been identified, including acetylation, phosphorylation, methylation, ubiquitylation and sumoylation. In this review we focus on the enzymes that acetylate histones and how they are regulated.

A histone acetyltransferase (HAT) can be defined as an enzyme that acetylates core histones, which results in important regulatory effects on chromatin structure

and assembly, and gene transcription. The current understanding of HATs can be traced back over 40 years. In 1964, Allfrey and colleagues showed that histones can be modified by the addition of acetyl and methyl groups6. Over the course of the next 15 years, a number of studies correlated the acetylation of histones with gene activity (for a review, see REF. 7). Then, in 1979, Cano and Pestana isolated protein fractions with HAT activity from the larvae of brine shrimp8. Another 15 or so years passed before the first HATs, Hat1 and Gcn5, were isolated and cloned from Saccharomyces cerevisiae9,10. Two years later, the first multisubunit nuclear HAT complex, SAGA (Spt–Ada–Gcn5–acetyltransferase), was isolated11.

Over the past 10 years, the study of HATs has advanced significantly as they have become much more amenable to molecular and biochemical analysis. A number of HAT enzymes have been isolated from various organisms12. The identification of new HATs has resulted in other important findings. It has been dem-onstrated that HATs are evolutionarily conserved from yeast to humans (TABLE 1), that HATs generally contain multiple subunits13, and that the functions of the catalytic subunit depend largely on the context of the other sub-units in those complexes14 (TABLE 1). Furthermore, recent work on HAT complexes has resulted in their categoriza-tion on the basis of their catalytic domains. The picture is complicated by the observation that some HAT enzymes can modify different histone substrates and that some HAT enzymes also acetylate an ever growing number of non-histone substrates15. In addition, histone acetylation is a dynamic reversible process. The balance of histone acetylation is important for proper cellular function and

Stowers Institute, 1000 East 50th Street, Kansas City, Missouri 64110, USA. Correspondence to J.L.W.e-mail: [email protected]:10.1038/nrm2145

Histone acetyltransferase complexes: one size doesn’t fit allKenneth K. Lee and Jerry L. Workman

Abstract | Over the past 10 years, the study of histone acetyltransferases (HATs) has advanced significantly, and a number of HATs have been isolated from various organisms. It emerged that HATs are highly diverse and generally contain multiple subunits. The functions of the catalytic subunit depend largely on the context of the other subunits in the complex. We are just beginning to understand the specialized roles of HAT complexes in chromosome decondensation, DNA-damage repair and the modification of non-histone substrates, as well as their role in the broader epigenetic landscape, including the role of protein domains within HAT complexes and the dynamic interplay between HAT complexes and existing histone modifications.

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© 2007 Nature Publishing Group

BromodomainAn evolutionarily conserved domain that has been shown to bind to acetylated residues.

ChromodomainA conserved structural motif that is common to some chromosomal proteins. It interacts with chromatin by binding to methylated lysine residues in histone proteins.

WD40 repeatA poorly conserved repeat sequence of 40–60 amino acids, which usually ends with tryptophan and aspartic acid (WD). Several consecutive repeats fold into a circular structure, a so-called β-propeller, in which each blade is a four-stranded β-sheet. This domain is found in proteins of various functions.

Tudor domainA domain first identified in the Drosophila melanogaster Tudor protein. Originally identified as an RNA-binding motif, it has also been shown to bind methyl arginine residues and methylated histones.

PHD finger(Plant homeodomain). A ~50-amino-acid motif found mainly in proteins that function in eukaryotic transcription. The characteristic sequence feature is a conserved Cys4-His-Cys3 zinc-binding motif.

the cell has evolved enzymes that catalyse the removal of acetyl groups, termed histone deacetylases (HDACs) (reviewed in REF. 16).

Although the discovery of the various HATs has been insightful, we are just beginning to understand the com-plexity of their role in epigenetics. Recent reviews have catalogued the known HAT complexes, their subunits and their substrates13 (TABLE 1) and the role of histone acetylation in transcription17. Here, we first discuss the complexity of HATs and their specialized roles in chromo some decondensation, DNA-damage repair and the modification of non-histone substrates. Next, we address the role of HAT complexes in the broader epi-genetic landscape, including the role of protein domains within HAT complexes and the dynamic interplay between HAT complexes and existing histone modi-fications. We conclude by offering possible directions for future research in this area.

The diversity of HATsHistone acetyltransferases are a diverse set of enzymes that can be grouped on the basis of their catalytic domains (TABLE 1). Gcn5 is the founding member of the Gcn5 N-acetyltransferases (GNATs), and this family includes Gcn5, PCAF, Elp3, Hat1, Hpa2 and Nut1 (reviewed in REF. 13). The MYST HATs are named for the founding members of this family: Morf, Ybf2 (Sas3), Sas2 and Tip60 (reviewed in REF. 13). Although these two families of HATs are the predominant ones, other proteins including p300/CBP (CREB-binding protein), Taf1 and a number of nuclear receptor co-activators, have also been shown to possess intrinsic HAT activity. However, they do not contain true consensus HAT domains and therefore represent an ‘orphan class’ of HAT enzymes (reviewed in REF. 13). Most recently, the circadian rhythm protein CLOCK has also been

described as having a MYST-like HAT domain, which is important for its function18 (BOX 1).

Just as HATs are a diverse set of enzymes, the multi-protein complexes in which they reside also vary. Different HAT complexes are composed of various unique sub-units. The combinations of these subunits contribute to the unique features of each HAT complex. For example, some subunits have domains that cooperate to recruit the HAT to the appropriate location in the genome; these include bromodomains, chromodomains, WD40 repeats, Tudor domains and PHD fingers (TABLE 2). Next, we discuss the various histone substrates of HAT complexes and the various HAT subunits, and how protein domains read and interpret the ‘histone code’ — that is, the combination of post-translational marks on the histones.

HATs as HATs. Interestingly, HAT complexes can perform specific tasks even if they do not have unique substrates. The substrates for several HAT complexes overlap, but the result of acetylation by HATs can vary (TABLE 1). An example in yeast that highlights the dichotomy between the overlapping substrates and the specialized functions of HATs involves the SAGA, NuA3 and Elongator com-plexes. The SAGA complex preferentially modifies histone H3 on Lys9 (H3K9) and to a lesser extent Lys14 (H3K14), whereas the NuA3 complex preferentially modifies H3K14 (REF. 19). Meanwhile, the Elongator complex has overlap-ping substrate specificity with the SAGA complex, but is thought to function in gene-coding regions, rather than at promoters (as is the case with SAGA) to acetylate nucleo-somes during transcription20. Adding to this complexity is the existence of three Gcn5-containing complexes in yeast, bringing the total number of HAT complexes that acetylate H3 to at least five. Therefore, something besides their catalytic activity must help these HAT complexes to identify the correct region of the genome to be modified.

Timeline | Histone acetylation in an epigenetics context

The structure of DNA is solved by Watson and Crick119.

The concept of the epigenotype is introduced by Waddington1.

The term ‘epigenetic landscape’ was coined by Waddington in reference to factors that influence the developing organism2.

Allfrey and colleagues isolated acetylated and methylated histones, demonstrating that these proteins undergo modification6.

Cano and Pestano isolated the first protein fraction that contained HAT activity from brine shrimp8.

Luger, Richmond and colleagues solved the first nucleosome structure, opening up a new way to look at epigenetic modifications125.

The Marmorstein and Allis laboratories solved the crystal structure for Gcn5 and subsequently solved co-crystal structures, demonstrating the manner in which the enzyme recognizes its substrate126,127.

The genetic code was revealed by Nirenberg and Crick120,121.

Kornberg defined the nucleosome as the basic unit of chromatin122.

The Gottschling and Allis laboratories identified and cloned the A-type HAT, Hat1, and the B-type HAT, Gcn5, respectively123,124.

The Workman, Berger, Allis and Winston laboratories purified the first HAT complex, SAGA11. The Winston and

Shultz laboratories solved the first electron microscopy structure of the SAGA complex, providing the first insight into the layout of the complex43.

The Cole and Roeder laboratories crystallized the first HAT inhibitor in the context of the corresponding enzyme128.

The Tora and Shultz laboratories solved the first electron microscopy structure of the TFTC complex from humans, leading to the first insight into how this macromolecular machine can recognize and bind DNA79.

1942 1953 1956 1961 1964 1974 1979 1995 1997 1999 2000 2001 2004

Building on a concept formulated by Turner, Jenuwein and Allis proposed the histone code whereby modifications to the tails of the histone proteins create a code84,129.

HAT, histone acetyltransferase; SAGA, Spt–Ada–Gcn5–acetyltransferase; TFTC, TBP-free TAFII complex.

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Domains: readers and interpreters of the histone code. Bromodomains, chromodomains, WD40 repeats, Tudor domains and PHD fingers are all chromatin-binding domains that recognize modified histone tails (TABLE 2). Bromodomains are known to recognize and bind acetylated lysine residues21–23. Interestingly, Gcn5 contains a bromodomain that is important for the bind-ing, recognition and retention of SAGA on acetylated promoter nucleosomes24 (TABLE 2).

Chromodomains have been shown to specifically bind methylated lysine tails (TABLE 2). In Drosophila melanogaster, the chromodomain of heterochromatin protein 1 (HP1) binds methylated H3K9, whereas the chromodomain of Polycomb binds methylated H3K27 (REFS 25–29). More recently, the chromodomain of Eaf3 — a protein subunit shared by the HAT complex NuA4 and the HDAC complex Rpd3S — was shown to be important for the recognition of and binding to methyl-ated H3K36 (REFS 30–32). Loss of this binding leads to increased acetylation in transcribed regions and the

formation of spurious transcripts that are initiated within open reading frames32. Importantly, unlike acetylation, methylation occurs in three states, mono-, di- and tri-, and the protein domains that bind them have been shown to differentially associate with the varying states of methylation (TABLE 2).

As additional chromatin-binding domains are char-acterized, it is likely to be shown that other domains interact with modified histones. The WD40-domain-containing protein WDR5 has been reported to pref-erentially bind dimethylated H3K4 in vitro, in addition to being associated with a complex that also contains HAT activity 33. The crystal structure, which demonstrates the recognition by WDR5 of dimethyl H3K4, shows how the WD repeats distinguish between dimethyl and tri methyl H3K4 (REFS 34,35). Although recent reports dispute the specificity of WD repeats for modified his-tones35,36, this does not take into account conformational changes that might occur to the WD repeat when it resides in its native chromatin-modifying complex.

Table 1a | Classes and substrates of histone acetyltransferases*

HAT complexes of the GNAT family

SAGA(Sc)

SLIK(Sc)

ADA(Sc)

HAT-A2 (Sc)

SAGA (Dm)

ATAC (Dm)

PCAF(Hs)

STAGA (Hs)

TFTC(Hs)

HATB (Sc)

Elongator (Sc)

Hpa2 (Sc)

Catalytic subunit

Gcn5 Gcn5 Gcn5 Gcn5 GCN5 GCN5 PCAF GCN5L GCN5L Hat1 Elp3 Hpa2

Histones modified

H2B/ H3/H4

H2B/ H3/H4

H3 H3 H3 H3/H4 H3/H4 H3/H4 H3/H4 H2A/H4 H3 H3/H4

Associated complex subunits

Tra1 Tra1 TRA1 PAF400 TRRAP TRRAP Hat2 Elp1 Hpa2

Spt7 Spt7‡ SPT7 STAF65γ Hif1 Elp2

Spt8 Elp4

Spt3 Spt3 SPT3 SPT3 SPT3 SPT3 Elp5

Spt20 Spt20 Elp6

Ada1 Ada1 ADA1 STAF42

Ada2 Ada2 Ada2 Ada2 ADA2B ADA2A ADA2

Ada3 Ada3 Ada3 Ada3 ADA3 ADA3 ADA3 STAF54 ADA3

Sgf29 Sgf29 Sgf29 Sgf29 SGF29

Sgf73 Sgf73 SCA7 SCA7

Ubp8 Ubp8 TAF5L TAF5L TAF5L

Sgf11 Sgf11 TAF6L TAF6L TAF6L

Taf5 Taf5 TAF5 TAF9 TAF9 TAF9



Taf10 Taf10 TAF10B TAF2

Taf12 Taf12 TAF12 STAF36 TAF4

Rtg2 STAF46 TAF5

Chd1 Chd1 TAF6

Ahc1 WDA ATAC1

Ahc2 HCF1

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The Tudor domain of several chromatin-related proteins has been shown to interact with various meth-ylated lysine and arginine residues37,38. Both WD40-domain- and Tudor-domain-containing proteins are present in various HAT complexes, including SAGA (TABLE 2). Most recently, the plant homeodomain, or PHD finger, which is present in a number of proteins in various HAT complexes, was shown biochemically and structurally to bind trimethylated H3K4 (REFS 39–41).

An emerging theme for these various chromatin-binding domains is that they are often associated with enzymes that modify or alter chromatin, such as Eaf3 and WDR5 (REFS 33,42), or even carry the enzymatic activity themselves, as is the case with the Gcn5 HAT21. The SAGA complex itself, which contains 19 known subunits, contains 15 putative chromatin-interacting domains (TABLE 2). In addition, the context in which these domains exist is equally important for their function. For example, although SAGA contains two proteins with bromodomains, Gcn5 and Spt7, only the

bromodomain of Gcn5 functions in binding acetylated nucleosomes in vitro24,43. To fully understand the func-tions of these domains and how the domain-containing subunits help the HATs carry out their various func-tions, the domains need to be studied within their resident protein complexes.

HATs serve a multiplicity of functionsA number of different HAT complexes exist in yeast and other organisms (TABLE 1), and these HAT com-plexes are composed of assorted proteins with various chromatin-binding domains that influence HAT recruitment. What is the reason for such complexity? In large part, the answer lies in the fact that HATs serve a multiplicity of functions.

First, HAT complexes modify histones in relation to the state and/or function of the DNA that the histones are compacting. As discussed above, HAT enzymes take advantage of their associated proteins as well as cellular signals for their recruitment to distinct regions

Table 1b | Classes and substrates of histone acetyltransferases*

HAT complexes of the MYST family

NuA4 (Sc)

Pic. NuA4 (Sc)

NuA3 (Sc)

SAS (Sc)

TIP60 (Dm/Hs)

HBO1 (Hs)

MOZ/MORF(Hs)

MSL (Dm)

Catalytic subunit

Esa1 Esa1 Sas3 Sas2 TIP60 HBO1 MOZ/ MORF MOF

Histones modified

H2A/H4 H2A/H4 H3 H4 H2A/H4 H3/H4 H3 H4

Associated complex subunits

Tra1 Yng1 Sas4 TRRAP ING5 ING5 MSL1

Yng2 Yng2 Taf14 Sas5 ING3 ING4 BRPF1 MSL2

Yaf9 Nto1 p400 JADE1 MSL3

Eaf1 BRD8 MLE

Eaf2 EPC1 roX RNA

Eaf3 EPC2

Eaf5 DMAP1

Eaf6 RUVBL1 EAF6 EAF6

Eaf7 MRG15

Epl1 Epl1 BAF53a

Act1 Actin

Arp4 GAS41

MRGX

MRGBP

FLJ11730

YL1

TIP49a

TIP49b

TRCp120 *The histone acetyltransferases (HATs) are grouped according to their substrate recognition motifs (reviewed in REF. 13). ‡C-terminal truncation of Spt is present in SLIK. Complexes for GNAT (Gcn5 N-acetyltransferase)- and MYST (Morf–Ybf2–Sas2–Tip60)-family HATs have been purified in organisms from yeast to humans. Substrates, associated complexes and known components are listed for each of the complexes. Much of the information is reviewed in REF. 13, with the exception of the Drosophila melanogaster Gcn5 complexes117,118. Dm, Drosophila melanogaster; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae.

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CRYCLOCK

AcAc

AcAc

AcAc

Transcriptional activation Transcriptional repression

BMAL1

CLOCK

BMAL1

CLOCK

BMAL1CRYKinase?

P

P

E-box

E-box

E-box

E-box

of the genome. In turn, these associations allow them to carry out specialized functions, which the enzyme would not be capable of on its own44,45. Such specialized functions of HATs include roles in processes such as genome stability and DNA repair (see below). Second, HAT enzymes do not solely modify histones, but func-tion to acetylate an increasing number of non-histone substrates15. For example, many HATs also function as transcription factor acetyltransferases (FATs), which means that these same enzymes can acetylate non-histone substrates to regulate their activities in the cell15,46 (see below). Recently, much research has gone into deciphering specific functions of HATs and the mechanisms by which they carry out these functions. We will highlight two elegant, but different, methods by which HATs attain their uniqueness.

As is the case with the function of many proteins, there are a number of factors that can influence the manner in which they function. The same is true for HATs. As we mentioned above, HATs carry out a broad range of functions and the regulation of these functions is in many cases vital for maintaining the integrity of the organism. In the examples that follow, we illustrate two ways in which HATs are specialized to carry out

specific functions to establish and maintain genomic stability. In the case of H4K16 acetylation, the ability for a specific histone modification to function throughout the genome as well as in specialized regions underlies the importance of the specific recruitment and regulation of HAT complexes. Although the same residue is being modified in yeast, flies and humans, the function and regulation of this modification is different among these three species, with each using specific HATs to regulate this single modification. In the case of DNA repair, we focus on how different HAT complexes function to repair different types of DNA damage. GNAT HATs function at sites of nucleotide excision repair (NER), whereas MYST HATs function at sites of double-strand break (DSB) repair. In keeping with the theme of this article, the protein subunits associated with these dif-ferent HAT complexes contribute to the function and specificity of the various HATs.

Specialized roles of H4K16-specific HATsThe significance of H4K16 acetylation has become increasingly evident. First, a global loss of H4K16 acetylation has been linked to tumorigenesis47 (FIG. 1). Second, H4K16 acetylation has been shown to destabi-lize nucleosomes and to correlate with regions of chrom-atin decondensation48. This, in turn, could regulate the access of transcription factors and chromatin-remodelling enzymes to specific regions of the genome and counter chromatin condensation by the Sir proteins at yeast telomeres48.

Although all characterized H4 acetyltransferases can modify H4K16, there are two HAT complexes that modify this residue for very specific functions — the SAS (something about silencing) complex in yeast and the MSL (male-specific lethal) complex in flies and humans49–53.

In yeast, the SAS complex was originally identi-fied as a complex that is involved in gene silencing (hence the name ‘something about silencing’)52,53. It was subsequently shown that the SAS complex works antagonistically with the NAD-dependent HDAC Sir2 to create a silencing boundary at telomeres through the regulation of H4K16 acetylation45,54 (FIG. 1). In this scen-ario, Sir2 spreads from the telomere and encounters the SAS complex and H4K16 acetylation, which pre-vents further spreading of Sir2, setting up a boundary of heterochromatin45,54. Esa1, the catalytic subunit of the NuA4 HAT complex, can also acetylate H4K16 and other lysine residues55, and is required for H4K16 acetylation at the INO1 gene promoter45. However, the loss of Esa1 does not have the same effect at telomeres as the loss of Sas2, which is the catalytic subunit of the SAS complex45,54. Additionally, genome-wide chro-matin immunoprecipitation (ChIP-on-chip) analysis clearly demonstrates that Sas2 is the telomere-specific H4K16 acetyltransferase, because deletion of Sas2 results in a specific decrease in H4K16 acetylation at the telomere56.

The MSL complex in flies and humans carries out a specialized role through the acetylation of H4K16. In D. melanogaster, transcriptional upregulation

Box 1 | Identification of CLOCK as a histone acetyltransferase

It was widely recognized from early work on histone acetylation that this type of modification had a crucial role in transcriptional activation105,106. It is therefore not surprising that several histone acetyltransferases (HATs), such as PCAF and Gcn5, were originally identified as transcriptional co-activators7,107. However, since this discovery, many other previously characterized proteins have been identified as having intrinsic HAT activity, including Taf1 and activator or thyroid and retinoid receptors (ACTR; also known as NCOA3)108,109. By extension, other transcriptional regulators could also have a dual role as HATs.

The circadian regulator CLOCK was recently identified as a HAT that is related to the MYST (Morf–Ybf2–Sas2–Tip60) family of HATs18. About 10% of all mammalian transcripts undergo circadian oscillations and it is clear that these transcripts are subjected to tight regulation, most likely through changes in histone modification and chromatin remodelling18. In an interesting parallel with other HATs, the HAT activity of CLOCK requires the presence of another protein, BMAL1 (also known as ARNTL)18, and both contain bHLH–PAS domains that bind E-boxes18 (see figure). As CLOCK is ubiquitously expressed, its function as a HAT must be regulated. A specific subset of the blue-light receptors, known as the cryptochromes (CRY), have long been known to regulate circadian rhythms in mammalian systems110. Recent work indicates that the association of CLOCK and BMAL1 with cryptochrome causes transcriptional repression, and it is therefore possible that changes in associated subunits rather than the regulation of CLOCK itself controls the activity111–113.

bHLH–PAS domainA structural motif that is characterized by two helices connected by a loop. Transcription factors that contain this domain are typically dimeric, each with one helix that contains basic amino-acid residues that facilitate DNA binding. Basic helix–loop–helix (bHLH) proteins typically bind to a consensus sequence, CANNTG, which is known as an E-box. The PAS (PER–ARNT–SIM) domain mediates interactions between transcription factors, and most PAS-domain-containing proteins also contain a bHLH domain.

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correlates with the specific acetylation of H4K16 on the male X chromosome by the MOF (males absent on the first) HAT49,57,58. Again, although other D. melanogaster HATs can acetylate H4 (on residue K16), including the

TIP60 complex, it is the MSL complex with its catalytic subunit MOF that carries out this specialized function on this unique chromosome (FIG. 1). Another level of complexity is added when the human MSL complex is considered. Although both the fly and the human MSL complexes specifically acetylate H4K16, the human MSL complex is responsible for the majority of genome-wide H4K16 acetylation, whereas the D. melanogaster MSL complex is much more restricted in its function to the male X chromosome. This is due to the fact that another MSL component, MSL2, is only translated in males, therefore leading to the specific targeting of MSL to the male X chromosome51,59. This marked difference between flies and humans provides an example of how a conserved HAT is used for dif-ferent functions. Because the mechanism of dosage compensation in humans does not involve the select upregulation of transcription from a specific set of genes on one chromosome, MOF in humans does not need to perform the same function as it does in flies. Therefore, it seems that human MOF has taken on the role of regulating global H4K16 acetylation (FIG. 1). In addition, the other subunits within the human and fly MOF direct its function. For example, the MSL proteins and the associated rox RNA components of MOF target it to the male X chromosome in D. melanogaster57,60. In humans, the MOF protein is associated with other complexes, including the MLL (mixed lineage leukae-mia) complex, which confers a more global function for MOF61.

HATs and DNA repairAll organisms are subjected to environmental stresses that affect the genome in countless ways. Although the functions of various DNA-repair pathways and checkpoints have been elucidated in much detail62, the role of specific HATs in these processes, by catalysing transient or constitutive acetylation, has only recently come to light (reviewed in REF. 63). We focus our attention on the two major classes of HAT enzymes, the MYST and GNAT families, and discuss their varied roles in the repair of different types of DNA damage (FIG. 2). It is clear that these mechanisms, as well as other DNA repair mechanisms, are conserved from yeast to humans64.

MYST-family HATs. The MYST-family HAT complex TIP60 and its orthologue in yeast, NuA4, are well known for their role in DNA repair in conjunction with other post-translational modifications of histones that serve as signals for these complexes (FIG. 2). The original observation that the TIP60 complex in humans con-tains a homologue of the bacterial RuvB protein, which is involved in branch migration during homologous recombination, sparked interest in the exact role this complex might have in DNA repair65,66. It was first demonstrated in mammalian cells that the loss of TIP60 HAT activity resulted in cells that were defective in DSB repair and failed to undergo apoptosis66. Subsequently, it was shown in D. melanogaster that the TIP60 complex, in addition to having HAT activity, also possessed the

Table 2 | Chromatin-binding domains in known HAT complexes in yeast*

Domain Structure Proposed role Complexes Proteins

Bromo Binds acetylated lysine

SAGA Gcn5, Spt7

NuA3 Yta7

Chromo Binds methylated lysine

SAGA Chd1

NuA4 Esa1, Eaf3

Tudor Binds methylated lysine and arginine

SAGA Sgf29

SANT Predicted to bind to histone tails

SAGA Ada2

NuA4 Eaf1, Eaf2

SWIRM Predicted to regulate transcription through protein–protein interactions

SAGA Ada2

WD40 Binds methylated lysine

SAGA Spt8

HatB Hat2

Elongator Elp1, Elp2

PHD Binds methylated lysine

NuA4 Yng2

NuA3 Yng1, Nto1

YEATS Unknown Predicted to regulate transcription through protein–protein interactions

NuA4 Yaf9

NuA3 Taf14

SAS Sas5

EPC-N Unknown Predicted to bind methylated lysine

NuA3 Nto1

NuA4 Eaf6*Using yeast as an example, a number of chromatin-binding domains have been found associated with various histone acetyltransferase (HAT) complexes. Here we illustrate the domains with their associated crystal structures if known (Protein Data Bank accession numbers: bromo, 1F68; chromo, 1KNA; Tudor, 1G5V; SANT, 2CU7; SWIRM, 2FQ3; WD, 2G9A; PHD, 2G6Q), as well as which residues this domain recognizes. In addition, we list the subunits of various HAT complexes that contain proteins with these domains.

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Tumorigenesis Normal transcription

Male-specificlethal

Dosage compensationfor X chromosome

Boundary function at the telomere;facilitates the incorporationof Htz1 into nucleosomes

Heterochromatin boundaryis disrupted, leading toSir2 spreading ineuchromatic regions

Condensedchromatin

Decondensedchromatin

H4K16 acetylation

H4K16 deacetylation

Human Fly Yeast

Ac

Ac

Ac

ability to swap the histone variant H2Av (known as H2Az in yeast) in and out of nucleosomes, a process that is important in the DNA-damage response44. Following DNA damage, H2Av is phosphorylated, and this phos-phorylation is recognized by the TIP60 complex. The TIP60 complex then acetylates the phosphorylated form of H2Av and signals the ATPase component of the TIP60 complex to remove the phosphorylated H2Av and replace it with unmodified H2Av, which allows the cells to recover from the damage44,67.

An analogous situation exists in yeast. Originally, Esa1, the catalytic subunit of NuA4, was shown to be required for DSB repair68. The original report showed that the NuA4 complex could be directly bound to a DSB site in vivo68. Subsequently, other subunits of NuA4 were also found to be important for DSB response and repair, including the PHD-finger-containing protein Yng2 (REF. 69). Unlike its mammalian counterpart, the NuA4 complex does not contain an ATPase component.

However, recent work has demonstrated two alternative modes by which NuA4 functions in DSB repair in vivo, providing an elegant mechanism for the ordered recruitment and dissociation of a HAT com-plex through combinations of histone modifications. One involves the recruitment of NuA4 following DNA-damage-mediated phosphorylation of histone H2A, and a second involves the phosphorylation of Ser1 of histone H4 (REFS 70,71). Phosphorylation of Ser129 of histone H2A results in the early recruitment of NuA4 to the DSB site and subsequent acetylation, whereas phosphorylation of Ser1 of histone H4 results in the inhibition of NuA4 binding and subsequent local deacetylation70,71. This recruitment and subsequent modifications help to set up local changes to ensure that DNA repair occurs at the proper site. Although the

NuA4 and TIP60 complexes have evolved to contain different components, this might be a result of neces-sity that is based on how the damage is presented in lower versus higher eukaryotes. This is a theme that holds true for the GNAT HATs discussed below.

GNAT-family HATs. The role of GNAT-family HATs is less well understood. Originally, these HATs were divided into classes on the basis of their subcellular localization; A-type HATS are characterized by nuclear localization, whereas B-type HATs are found in the cytoplasm. In yeast and humans, roles are emerging for Gcn5 in NER72,73. Interestingly, the early work that identified a role for Gcn5 in repair occurred in higher eukaryotes and has only recently become evident in yeast72,74. In these studies, the SAP130 subunit of the human TFTC (TBP-free TAFII complex) was shown to be homologous to a component of the UV-damaged DNA-binding factor DDB1 (REF. 72) (TABLE 1). The TFTC complex was shown to preferentially bind and acetylate nucleosomes on UV-damaged DNA. In support of this, UV damage results in a global increase in H3 acetyla-tion, which is also seen in yeast72,73. The presence of SAP130 in TFTC and STAGA (SPT3–TAFII31–GCN5L acetylase) suggests a direct recruitment of these com-plexes to a specific type of DNA damage and a prefer-ence of Gcn5 complexes for UV-damaged DNA, which facilitates NER.

More recent work on the role of yeast Gcn5 in NER has involved the MFA2 promoter in yeast, which func-tions in the production of a-factor. It has been shown that UV irradiation triggers genome-wide hyper-acetylation of histones H3 and H4. However, in nucleo-somes at the repressed MFA2 promoter only histone H3 is hyperacetylated following UV irradiation. This Gcn5-mediated histone H3 hyperacetylation enables efficient NER at MFA2 (REFS 73,75,76). Because yeast is more amenable to genetic techniques, it was shown that the deletion of GCN5 impairs the ability to repair DNA damage at the MFA2 promoter; however, deletion of GCN5 does not impair NER in the genome overall76. Because Gcn5 is also required for the transcriptional activation of the MFA2 gene, it is intriguing to consider that specific Gcn5 complexes, such as SAGA, might be involved in MFA2 transcription, whereas other Gcn5 complexes, such as the SLIK (also known as SALSA) or ADA complex, might be involved in NER. Although this remains to be shown, such a scenario would illus-trate how Gcn5 carries out two different functions at a specific gene depending on its associated subunits. In addition, it will be important to decipher which protein in the Gcn5 complexes in yeast mediates the recruitment of Gcn5 to the sites of damage (that is, the orthologue of SAP130) in order to understand how evolutionarily related these two processes are.

The importance of proper DNA-damage repair is illustrated by the efforts made by the cell to take advantage of specific HATs to participate in and repair distinct types of damage. Furthermore, the fact that these conserved proteins carry out a conserved function in evolution emphasizes their importance. However, the

Figure 1 | Consequences of histone H4 lysine 16 acetylation. The acetylation of histone H4 on lysine 16 (H4K16) serves different functions in yeast, flies and humans. Accurate regulation of the levels of H4K16 acetylation is important for the integrity of the organism. In humans a decrease in H4K16 acetylation leads to global increases in transcription and tumorigenesis, whereas in flies H4K16 acetylation on the male X chromosome is controlled by the MOF (males absent on the first) catalytic subunit of the MSL (male-specific lethal) histone acetyltransferase (HAT) complex and is essential for dosage compensation. Aberrant regulation of H4K16 acetylation in male flies leads to lethality. In yeast, the SAS (something about silencing) HAT complex mediates H4K16 acetylation at telomeres to set up heterochromatic boundaries through its antagonistic actions with the NAD-dependent histone deacetylase Sir2, as well as by the incorporation of the histone variant Htz1. Loss of H4K16 acetylation leads to heterochromatin spreading and the subsequent silencing of genes.

E-boxA DNA location with the consensus sequence CANNTG. E-boxes have a regulatory role in the control of transcription. They bind to basic helix–loop–helix (bHLH)-type transcription factors. Binding specificity is determined by the specific bHLH heterodimer or homodimer combination and by the specific nucleotides at the third and fourth position of the E-box sequence.

Nucleotide excision repairA DNA-repair process, in which a small region of the DNA strand that surrounds the UV-induced DNA damage is recognized, removed and replaced.

Double-strand break (DSB) repairA group of DNA-repair processes that includes homologous recombination and non-homologous end-joining; both processes recognize and repair the DNA double-strand break.

NAD-dependent histone deacetylaseAn enzyme that catalyses the NAD-dependent removal of an acetyl group from lysine residues in histones.

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DSB P

P P

H2ASer129

H4Ser1-P

Tel1Mec1

Acetylation andremodelling

Acetylation andremodelling

P

PAc AcP

P

P P

P

P

P P

P

Esa1

NuA4

Rsc2

RSC

Gcn5

SAGA

Snf2

Swi/Snf

HATB

Rad52

Hat1

NuA4

DNA repair,assembly of nucleosomesat repair site

HeterochromatinA highly condensed and transcriptionally less active form of chromatin that occurs at defined sites such as centromeres, silencer DNA elements or telomeres.

role of HATs in cellular processes goes far beyond DNA repair and genome stability and extends to processes that require additional factors and additional substrates to function.

HATs and non-histone substratesThe discovery that the tumour suppressor p53 is acetylated by p300/CBP, a member of the orphan class of HATs, showed that some HATs can also acetylate non-histone substrates77. Since then, many other non-histone substrates have been identified for a number of HATs15. The mechanism by which the same enzymes that acetylate histones are also able to acetylate other proteins became clear when it was found that amino acids surrounding the substrate lysine residue contribute

to the affinity for the Gcn5 complex78 (FIG. 3). Although Gcn5 recognizes H3, H4 and p53, its affinity for H3 was 10 times as strong owing to the 10 N-terminal amino acids of the H3 peptide78. This insight, along with the knowledge that other protein subunits associated with the catalytic Gcn5 subunit in the respective complexes could direct their catalytic activity to specific areas of the genome, helps to clarify the different specificities of these HATs (FIG. 3). Because the structures shown focus on the enzyme and substrate in isolation (that is, in the absence of other subunits of the HAT complex), it is interesting how small changes in the substrate can result in drastic conformational changes. When observing the contrast between the non-phosphorylated H3 peptide bound to the Gcn5 HAT domain versus the phospho-rylated peptide bound to the Gcn5 HAT domain, it is clear that this phosphorylation leads to a conformational change as well as new contacts between the enzyme and its substrate78. The fact that a HAT enzyme can accom-modate both a modified and unmodified peptide will be discussed below in terms of histone crosstalk and regulation of HAT function.

In addition, at first glance the structure of the HAT domain of Gcn5 bound to the H4 peptide and to the p53 peptide appears almost indistinguishable, and indeed most of the contacts are conserved between the enzyme and these two peptides. Interestingly, many of the same protein residue interactions are conserved, despite the divergence in sequence of the three resi-dues that are C-terminal to the reactive lysine residue78. Therefore, it could be that the role of one or more of the subunits within these Gcn5 HAT complexes is to facilitate more optimal contacts with substrates that have inherently poor affinity for the isolated catalytic subunit, such as in the case of histone H4 and p53 (REF. 78). This becomes more evident when one observes the three-dimensional structures of mammalian TFTC and yeast SAGA HAT complexes43,79. Particularly in the case of the yeast SAGA complex, one can observe the arrangement of the different protein subunits, which would allow recognition and binding of Gcn5 to its varied substrates43.

Revisiting the most well-characterized non-histone substrate, the oncoprotein p53, we realize that the nature by which acetylation regulates p53 is much more complicated than simply acetylating specific residues. Since it was first shown to be acetylated by p300/CBP, the mechanism by which p53 is regulated by acetylation has become increasingly clear. It is now known that binding of p53 to p300/CBP through its PXXP proline-rich domain mediates the DNA-dependent acetylation of p53, thereby stabilizing the acetylated p53–p300/CBP complex80. Moreover, the binding of interferon regulatory factor 1 (IRF1) to p300/CBP stimulates the acetylation of p53 (REF. 81). Again, an underlying theme is that the context of HATs is important for recognition and subsequent substrate modification. So, as HATs can function in both transcriptional activation and repres-sion it is necessary to analyse further how these single enzymes use their associated proteins and the existing status of the epigenome to allow this switch to occur.

Figure 2 | Histone acetyltransferases and DNA repair. Here we outline the complex and finely orchestrated roles of various histone acetyltransferase (HAT) complexes in DNA repair. After the introduction of a DNA double-strand break (DSB), the Mec1 and Tel1 kinases are recruited and phosphorylate histone H2A on serine 129 (reviewed in REF. 114). This phosphorylation event leads to the recruitment of the NuA4 HAT complex and the RSC chromatin remodelling complex, which are proposed to carry out events that allow repair proteins to access the break site (reviewed in REF. 114). Once NuA4 and RSC are recruited, the Swi/Snf chromatin remodelling complex, the SAGA (Spt–Ada–Gcn5–acetyltransferase) and HATB HAT complexes and the Rad52 repair protein are recruited to the DSB site115. This leads to the subsequent repair of the break with the DNA wrapped in a nucleosome that contains histone H4 acetylated on lysines 5 and 12 in a Hat1-dependent manner (Hat1 is the catalytic subunit of HATB)115. The mechanistic basis for these steps is not yet clear. H4Ser1 phosphorylation follows, leading to the inhibition of NuA4 binding and subsequent local deacetylation and finally to the restoration of the undamaged DNA state.

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a Gcn5 with H3 peptide b Gcn5 with phosphorylated H3 peptide

c Gcn5 with H4 peptide d Gcn5 with p53 peptide

Dosage compensationA process by which the expression of sex-linked genes is equalized in species in which males and females differ in the number of sex chromosomes. In Drosophila melanogaster dosage compensation is achieved by hypertranscription of the single male X chromosome.

Dynamic interplay of histone modificationsAs discussed above, HAT-associated proteins have vari-ous domains that function by targeting the HAT complex to specific regions in the genome. This specified bind-ing helps to determine the way the HAT will function. Without these associated proteins and their domains, the various HATs could acetylate almost any protein based on the enzyme’s ability to recognize various substrates78. However, this is clearly not what happens. In addition, the cell has evolved mechanisms to regulate the function of HATs through other histone modifications. Over the past few years, many examples have come to light about how histone modifications might function together; for example, how phosphorylation in combination with acetylation can lead to increased transcription82. Such studies are leading to a greater understanding of how the arrangement of lysine and serine residues in histone

proteins might regulate transcription29. In addition, several studies have demonstrated that other histone modifications, such as methylation and sumoylation, can have varied effects on the ability to recruit HATs and on subsequent histone acetylation (see below).

Histone acetylation in context. Many HAT complexes are composed of proteins with domains that can rec-ognize and bind histone modifications, leading to subsequent acetylation. What, then, is the functional significance of histone acetylation in the context of other modifications? The ‘charge hypothesis’ asserts that the neutralization of positively charged lysine residues by acetylation and the negative charge brought about by phosphorylated serine residues functionally influences the interaction surface of histones with DNA83. This allows for the loosening of the protein–DNA contacts, which facilitates transcription.

Along similar lines, particular histone modifications might directly alter chromatin structure. As mentioned above, acetylation of H4K16 leads to decreased con-densation of nucleosome arrays into chromatin fibres48. However, different modifications might constitute a histone code, such that the modifications would act sequentially and in combination to create distinct out-puts that could not necessarily be predicted from single modifications84–87.

Finally, it has been proposed that signalling through histone modifications resembles what is seen in recep-tor tyrosine kinase signal transduction88. In this model, the cascade of signals is initiated by the HAT complex that is recruited to acetylate a histone tail, and this acetylation recruits other complexes to remodel the chromatin, thereby setting up a ‘docking site’ that is analogous to signal transduction at the cell membrane88. These models need not be mutually exclusive, and there are a growing number of examples of interdependent histone modifications, the effects of which vary greatly depending on the type of modification.

Methylation-associated recruitment of HATs. At the same time that histones were identified as being acetylated, they were also shown to be methylated6. Since then, much progress has been made in understanding the role of histone methylation in heterochromatin formation, transcriptional activation and transcriptional repression (reviewed in REF. 89). Recently, it has become apparent that there is a link between histone methylation and histone acetylation, and that in many cases HATs use methylated histones as docking sites to carry out subsequent histone acetylation events. For example, the HAT complexes NuA3 and SAGA both bind methylated histones, and this ability to bind such histones enhances their activity90,91.

Recently, four new SAGA components have been identified90,92–95, including the ATP-dependent chromatin-remodelling factor Chd1, which contains several poten-tial chromatin-binding domains96 (TABLE 2). Recent work demonstrates that the second chromodomain of Chd1 binds dimethylated and trimethylated Lys4 of histone H3 (REF. 90). Purified SLIK also showed enhanced acetylation

Figure 3 | Structural basis for histone acetyltransferase substrate recognition. a | Crystal structure of the histone acetyltransferase (HAT) domain of Tetrahymena thermophila Gcn5 with bound coenzyme A and a 19-residue histone H3 peptide (1PU9)116. b | Crystal structure of the HAT domain of T. thermophila Gcn5 with bound coenzyme A and a phosphorylated, 19-residue histone H3 peptide (1PUA)116. c | Crystal structure of the HAT domain of T. thermophila Gcn5 with bound coenzyme A and a 19-residue histone H4 peptide (1Q2C)78. d | Crystal structure of the HAT domain of T. thermophila Gcn5 with bound coenzyme A and a 19-residue p53 peptide (1Q2D)78. These four structures demonstrate the ability of the catalytic core of Gcn5 to accommodate a variety of potential substrates. In all cases, the substrate is depicted in green and acetyl-CoA is indicated in red. Although Gcn5 can recognize all four of these peptides, it does so at different affinities with the affinity for phosphorylated H3 being the strongest, followed by unphosphorylated H3, and the affinity for H4 and p53 being weakest116. The difference in affinity is due to the different surface contacts that occur between the different peptides and Gcn5. For example, the phosphorylation of the H3 peptide causes it to undergo structural changes such that it comes into contact with more residues within the catalytic site of Gcn5, which leads to an (up to 10 times) increase in enzyme–substrate affinity116.

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on dimethylated and trimethylated H3K4 substrates, and this activity is dependent on the second chromodomain of Chd1 (REF. 90). As chromo domain proteins exist in other HAT complexes, including NuA4 and TIP60, it is interesting to speculate that these proteins might also recruit their respective complexes to methylated histone substrates in order to mediate transcriptional activa-tion and other related processes. Interestingly, SAGA and SLIK contain additional sub units with potential methyl-binding activities, such as WD40-repeat- and Tudor-domain-containing proteins, which might also contribute to the affinity of SAGA for methylated histones (TABLE 2).

The binding and recognition of methylated his-tones has also been linked to another major histone H3-specific HAT in yeast, NuA3, and this binding has been shown to mediate NuA3 association with chro-matin91. In contrast to SAGA, the binding of NuA3 to chrom atin is mediated through the methylation of both Lys4 and Lys36 of histone H3. Loss of both modi-fications through the deletion of the histone methyl-transferases Set1 (Lys4) and Set2 (Lys36) results in an increase in the non-chromatin-bound fraction of NuA3 and, subsequently, a decrease in Gcn5-independent H3 acetylation91. How does NuA3 recognize methylated Lys4 and Lys36 of histone H3? NuA3 is a 400-kDa com-plex that contains the catalytic subunit Sas3 (REF. 19), but also contains subunits that have the potential to mediate binding to methylated histones. The Yng1 protein con-tains a PHD domain that has recently been show to bind methylated histones39,40,97, and the Nto1 subunit con-tains an Epc-N domain, a proposed chromatin-binding domain98. Indeed, recent work has demonstrated that the Yng1 protein of the NuA3 complex is important for binding and recognition of Lys4-methylated histone H3 (REF. 99). However, the protein or proteins that mediate binding and recognition of methylated Lys36 by NuA3 remain unknown. The more we understand about these domains and their ability to use modified histones as substrates, the closer we will come to an understand-ing of the intricate functions of histone modification in transcriptional regulation.

Inhibition of acetylation by sumoylation. In contrast to methylation, which facilitates the interactions of HATs with chromatin, sumoylation has recently been shown to inhibit acetylation by HATs at specific H3 residues, thereby resulting in transcriptional repression100. Histone sumoylation was first reported to correlate with transcriptional repression in mammals101. Recent work in this area, however, demonstrates that all four histones in yeast are subjected to sumoylation, and specific sumoylation sites have been identified in the N-terminal domains of histone H2B and H4 (REF. 100). Furthermore, several pieces of evidence have strength-ened our understanding of the relationship between histone sumoylation and acetylation in yeast. First, sumoylation sites map at or near known sites of histone acetylation; second, a decrease in histone sumoyla-tion results in an increase in acetylation; and third, in genomic regions with low levels of histone acetylation,

such as the subtelomeric regions, histone sumoylation levels are increased100.

Such evidence reinforces the idea that sumoylation can inhibit other positive modifications. Previous work demonstrated a similar relationship between sumoyla-tion and ubiquitylation in the regulation of the IκB (inhibitor of NF-κB; also known as NFKBIA) gene102. Accordingly, because HATs are recruited to chromatin through methylated histones, which results in transcrip-tional activation, it is possible that sumoylation not only inhibits HAT recruitment, but it might also facilitate the recruitment of other negatively acting complexes, such as HDACs, which are known to interact with sumoylated substrates, resulting in transcriptional repression100,103.

Conclusions and future directionsIn the past 15 years, the field of epigenetics has vastly expanded through the identification and characteriza-tion of the biochemical processes associated with distinct chromatin states. It is now clear that the complexity of HAT complexes is much greater than originally thought. We have examined the diversity of HATs, the multipro-tein complexes in which they reside, HAT functionality and how HAT function is regulated. In so doing, we have highlighted the specialized functions of HATs in genome stability and DNA repair, as well as the interplay between HATs and sumoylation and methylation. Building on a historical perspective, we also highlight recent findings in the literature to demonstrate the diversity of this single modification. It is important to note that HATs function in several other processes that are becoming clear, including DNA replication and recombination. The study of HATs is a broad and complex field that is still expanding, and although our knowledge of these important proteins and complexes has increased sub-stantially in recent years there is still much to learn. In time, acetylation and subsequent deacetylation of proteins might be looked at in a similar fashion as phosphorylation and dephosphorylation in regulating cellular processes.

Since the coining of the term epigenetic landscape, epigenetics has become synonymous with chromatin regulation. In the coming years, it will become increas-ingly important to revisit Waddington’s original use of the term by putting chromatin modifications in the context of development2. As we continue our research on HATs and acetylation, we must revisit and build on the findings by Marzluff reported in 1970 (REF. 104); it is possible that HATs have developmental-stage-specific and tissue-specific functions in complex organisms. This will undoubtedly lead to further complexity, as HAT-associated proteins will probably vary depending on tissue type and developmental stage, where it is already known that epigenetic marks vary.

In addition, as HDAC inhibitors garner much of the attention in anticancer and ageing diseases, work on understanding and developing effective reagents that target HATs in similar processes is equally important, as it is clear that keeping the proper balance of protein acetylation underlies the efficacy of these reagents.

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AcknowledgementsWe are grateful to L. Krom, K. Smith, V. Weake, B. Li, P. Prochasson and M. Carey for helpful discussions and criti-cal reading of the manuscript. We thank W.-J. Shia for the concept in Figure 1 and B. Li and B. Geisbrecht for assistance with Figure 2. We apologize to our colleagues for not being able to quote all references owing to space limitations. K.K.L. was supported by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation. Research in the Workman laboratory is supported by the US National Institutes of Health.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:RCSB Protein Data Bank: http://www.rcsb.org/pdb/home/home.do1PU9 | 1PUA | 1Q2C | 1Q2D Saccharomyces Genome Database: http://genome-www.stanford.edu/SaccharomycesElp3 | Gcn5 | Hat1 | Hpa2 | Nut1 | Sas2 | Sas3Access to this links box is available online.

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histone acetyltransferase complexes: one size doesn't fit all

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