Role of histone acetylation in the control of gene expression1

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Role of histone acetylation in the control of gene expression1 Loredana Verdone, Micaela Caserta, and Ernesto Di Mauro

Abstract: Histone proteins play structural and functional roles in all nuclear processes. They undergo different types of covalent modifications, defined in their ensemble as epigenetic because changes in DNA sequences are not involved. Histone acetylation emerges as a central switch that allows interconversion between permissive and repressive chromatin domains in terms of transcriptional competence. The mechanisms underlying the histone acetylationdependent control of gene expression include a direct effect on the stability of nucleosomal arrays and the creation of docking sites for the binding of regulatory proteins. Histone acetyltransferases and deacetylases are, respectively, the enzymes devoted to the addition and removal of acetyl groups from lysine residues on the histone N-terminal tails. The enzymes exert fundamental roles in developmental processes and their deregulation has been linked to the progression of diverse human disorders, including cancer. Key words: gene expression, transcription, HATs, HDACs, nucleosome. Résumé : Les protéines histoniques jouent des rôles structurales et fonctionnels dans tous les processus nucléaires. Elles sont soumises à types différents de modifications covalentes, sont définies dans leur ensemble comme épigénétiques à cause de la non-implication des séquences de l’ADN. L’acétylation des histones constitue un interrupteur central permettant la interconversion entre les domaines permissifs et répressifs en termes de compétence transcriptionelle. Les mécanismes à la base du contrôle de l’expression des gènes dépendante de l’acétylation des histones incluent un effet direct sur la stabilité des ensembles nucleosomiques et la création de sites d’amarrage pour la liaison des protéines régulatrices. Les acétyltransferases et les déacétylases sont enzymes dédiés respectivement à l’addition et au déplacement de groups acétyliques des résidus de lysine des queues N-terminales des histones. Ces enzymes-ci jouent des rôles fondamentaux dans les processus du développement et, encore plus important, leur dérégulation a été liée à la progression de nombreux désordres humains, cancer inclus. Mots clés : expression génique, transcription, HATs, HDACs, nucleosomes. Verdone et al.

Introduction The genetic information of eukaryotic organisms is tightly packed in chromosomes, which represent the structural and functional supports of all nuclear processes. Eukaryotic Received 11 November 2004. Revision received 18 February 2005. Accepted 22 February 2005. Published on the NRC Research Press Web site at http://bcb.nrc.ca on 11 June 2005. L. Verdone. Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, Rome, Italy. M. Caserta and E. Di Mauro.2,3 Istituto di Biologia e Patologia Molecolari, CNR, Università La Sapienza, Rome, Italy. 1

This paper is one of a selection of papers published in this Special Issue, entitled Epigenetics in Chromatin, and has undergone the Journal’s usual peer review process. 2 Corresponding author (e-mail: [email protected]). 3 Present address: Fondazione Istituto Pasteur-Fondazione Cenci Bolognetti, Rome, Italy. Biochem. Cell Biol. 83: 344–353 (2005)

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chromosomes are highly organized; they consist of topologically distinct domains, physically attached to a protein scaffold, and are made of chromatin, the actual template of all genetic processes. Basically, chromatin consists of DNA, histones, and a plethora of different protein complexes that assist the dynamic changes that occurr during DNA replication, cell-cycle progression, regulated transcriptional and post-transcriptional events, and DNA repair and recombination. Two main types of proteins have been detected in these complexes: ATP-dependent molecular motors and enzymatic activities able to covalently modify histones and other nuclear proteins. All changes in chromatin that do not involve a change in DNA sequence are defined as epigenetic. As a rule, these modifications of chromatin affect genome functions at many different levels. One interesting aspect of epigenetics is that the crucial components of these protein complexes are well conserved from lower to higher eukaryotes, suggesting that once this powerful mechanism to control chromatin dynamics emerged, it was kept as a tool for the further evolution. Epigenetic modifications,

doi: 10.1139/O05-041

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therefore, increase the coding potential of each genome, especially considering the combinatorial property of the proposed histone code (Strahl and Allis 2000). The interplay among different patterns of modifications has been the topic of several recent reviews (Berger 2002; Fischle et al. 2003a, 2003b; Peterson and Laniel 2004; Schübeler et al. 2004).

345 Fig. 1. Schematic representation of lysine residues on the histone N-terminal tails.

Histone acetylation and the enzymes involved One of the first-studied types of histone modification is acetylation. It was initially linked to transcriptional activation (Brownell et al. 1996; Struhl 1998), and subsequently linked to such diverse processes as gene silencing, DNA repair, and cell-cycle progression (Carrozza et al. 2003). This particular modification is carried out by enzymes dubbed histone acetyltransferases (HATs), which catalyze the transfer of an acetyl group from acetyl-CoA to the lysine ⑀-amino groups on the N-terminal tails of histones (Yang 2004) (Fig. 1). The enzymes that counteract this activity are known as histone deacetylases. Two main superfamilies of HATs have been identified: the GNAT and the MYST families. The GNAT superfamily (Gnc5-related N-acetyltransferase) is the best understood set of acetyltransferases. In mammals, the Gcn5 subclass of acetyltransferases is represented by 2 closely related proteins: GCN5 and p300/CREB-binding protein-associated factor (PCAF). Homologs of Gcn5 have been cloned and sequenced from numerous divergent organisms, such as human (Candau et al. 1996), mouse (Xu et al. 1998a), Saccharomyces cerevisiae (Georgakopoulos and Thireos 1992; Kleff et al. 1995), Drosophila melanogaster (Smith et al. 1998), Arabidopsis thaliana, and Toxoplasma gondii (Hettmann and Soldati 1999), suggesting that its function is highly conserved throughout the eukaryotes. The function of human GCN5 has been investigated in vitro and in vivo, and it has been found to perform transcriptional adaptor roles analogous to those of yeast Gcn5 (Candau et al. 1996). Studies conducted using the recombinant short-form (lacking the N-terminal region) of human GCN5 have shown that it can acetylate histone H3 (and to a lesser extent H4) only as free histones (Wang et al. 1997; Yang et al. 1996). The full-length form is able to acetylate nucleosomal histones, which implicates the N-terminal region in chromatin substrate recognition (Xu et al. 1998a). The C-terminal bromodomain is another domain for GCN5 function; it interacts with the DNA-dependent kinase holoenzyme, which inhibits GCN5 HAT activity through phosphorylation (Barlev et al. 1998). The gene coding for PCAF was identified because of its homology to GCN5, and in vivo and in vitro studies have found that PCAF interacts with p300 and CBP (Yang et al. 1996). Studies investigating the role of PCAF in transcription have shown its recruitment as a HAT and as a coactivator in several processes, such as myogenesis (Puri et al. 1997), nuclear-receptor-mediated activation (Blanco et al. 1998; Korzus et al. 1998), and growth-factor-signaled activation (Xu et al. 1998b). In addition to its histone acetyltransferase activity, PCAF is able to acetylate nonhistone transcription-

related factors (HMGN2 and HMGA1; p53; MyoD; HIV Tat and TFIIE and TFIIF). P300/CBP is another family of histone acetyltransferases (Bannister and Kouzarides 1996; Shikama et al. 1997). p300 and CBP are ubiquitously expressed global transcriptional coactivators that play critical roles in a large variety of cellular phenomena, including cell-cycle control, differentiation, and apoptosis. The p300 and CBP enzymes are often referred to as a single entity, because they are considered structural and functional homologs, although functional differences between them have been acknowledged (Glass and Rosenfeld 2000). In addition, it has been demonstrated that these two cofactors associate at different times during the formation of the ER transcription complex at the promoters of estrogen-responsive genes (Shang et al. 2000; Reid et al. 2002). At the molecular level, p300/CBP stimulates transcription of specific genes by interacting 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. p300/CBP also binds the HAT PCAF, an interaction that competes with the adenoviral oncoprotein E1A (Yang et al. 1996). The HAT activity of p300/CBP was discovered in an E1A pulldown from HeLa nuclear extracts and in direct CBP immunoprecipitations from Cos cell extracts. The amino-terminal tails of all 4 core histones were acetylated by recombinant p300 and CBP (Bannister and Kouzarides 1996; Shikama et al. 1997). Like PCAF, p300/CBP is known to acetylate and regulate transcription-related proteins other than histones (Sterner and Berger 2000). The known FAT (factor acetyltransferase) substrates of p300/CBP include HMGA1, activators p53, GATA-1, erythroid Kruppel-like factor (EKLF), HIV Tat, nuclear receptor factor SRC-1, ACTR and TIF2, and general factors TFIIE and TFIIF. Other identified acetyltransferases are Hat1, Hat2, and Elp3. Yeast Hat1 and Hat2 were found to be part of nuclear HAT activity on free (but non-nucleosomal) histones, indicating its potential involvement in chromatin assembly in a more direct manner, perhaps at the replication forks or silenced telomeres (Ruiz-Garcia et al. 1998). Homologs of Hat1 and Hat2 were found in a HAT complex characterized from human S-phase nuclei, which showed in vitro specificity similar to that of the yeast enzyme, suggesting conservation of its function © 2005 NRC Canada

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throughout eukaryotes (Verreault et al. 1998). Elp3, a yeast A-type HAT, is part of the RNA polymerase II holoenzyme and is involved in transcriptional elongation (Otero et al. 1999). Elp3 has a GNAT homology, which is the reason recombinant Elp3 was produced from insect cells and tested for HAT activity. As a result, the ability of Elp3 to acetylate all 4 histones was shown. The importance of Elp3, even though its gene is not essential, is demonstrated by its evolutionary conservation in numerous eukaryotes, including mammals (Wittschieben et al. 1999). In S. cerevisiae, the double-deletion mutant elp3 gcn5 shows a widespread and severe histone H3 hypoacetylation in chromatin, suggesting that the 2 enzymes play similar, although not identical, roles (Kristjuhan et al. 2002). The MYST family, named after its founding members (MOZ, Ybf2/Sas3, Sas2, and Tip60), is another group of evolutionary-related proteins (Borrow et al. 1996). Other members have since been identified, including yeast Esa1, Drosophila MOF, human HBO1, and MORF. Although containing regions similar in sequence, the members of the MYST family are involved in a wide range of regulatory functions in various organisms. Tip60 (Tat-interactive protein, 60 kDa) was the first human MYST protein to be discovered. The Tip60 histone acetyltransferase shares many properties with the tumor suppressor p53. Both proteins are involved in the cellular response to DNA damage. Overexpression of a dominant negative HATdefective Tip60 mutant decreases both DNA repair and apoptosis upon induction of DNA double-breaks (Momand et al. 1992). Tip60 seems to participate in transcriptional activation of specific genes through local histone acetylation, particularly H4 acetylation (Yamamoto and Horikoshi 1997). MOZ (monocytic leukemia zinc finger protein) is a MYST protein involved in a specific human disease process: the 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 MOZ and CBP. The chimeric protein contained threequarters of the N-terminal of MOZ fused to the C-terminal of CBP (Borrow et al. 1996). MOZ–CBP may be the cause of aberrant chromatin acetylation that results from the mistargeting of specific HAT activities. MOZ fusion with another transcription-related protein, TIF2, has also been observed in other cases of leukemia (Carapeti et al. 1998; Liang et al. 1998; Rozman et al. 2004). MORF (monocytic leukemia zinc finger protein-related factor, or MYST4) is a member of the MYST family of histone acetyltransferases and has been found to be rearranged in some types of acute myeloid leukemia (Champagne et al. 1999). Recombinant full-length MORF expressed in insect cells and a bacterially produced MYST domain fragment were both able to acetylate free histones in vitro, with a preference for H3 and H4. Furthermore, the insect-derived protein was capable of nucleosome acetylation, strongly preferring histone H4. HBO1 is a protein that interacts with the human origin recognition complex (ORC) (Iizuka and Stillman 1999). Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1, supporting the model in which HBO1-associated HAT activity may play a direct role in the process of DNA replication (Burke et al. 2001). Hyper-

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acetylation of histone H3 was also observed at the Kaposi’s sarcoma–associated Herpesvirus latent replication origin (Stedman et al. 2004). Most HATs exist as multisubunit complexes in vivo (Yang 2004). The complexes are typically more active than their respective catalytic subunits and the specificities of their substrates are disctinct (Grant et al. 1997; Ogryzko et al. 1998; Boudreault et al. 2003), suggesting that associated subunits regulate the activities of the respective catalytic subunits. Noncatalytic subunits are also involved in recruiting substrates for targeted action to ensure the specificity (Yang 2004). Human histone acetyltransferase complexes are analogous in composition to known yeast HAT complexes. The PCAF complex contains more than 20 different polypeptides (Ogryzko et al. 1998; Vassilev et al. 1998). Some of the PCAF subunits were identified by protein sequencing and appear to be homologs of components of yeast SAGA, suggesting strong evolutionary conservation of this type of complex (Sterner and Berger 2000). Besides the GCN5 homolog PCAF, the complex contains the human adaptor homologs hADA2 and hADA3, hSPT3 (Yu et al. 1998), the transcriptional cofactor TRRAP (McMahon et al. 1998), and different TAFII-related proteins. Two other human complexes, purified on the basis of TAF subunits, are very similar to the human GCN5 complex (Sterner and Berger 2000). One is the TFTC complex (TBPfree TAFII-containing complex) (Wieczorek et al. 1998); its subunits were identified immunochemically. TFTC contains the PCAF/GCN5 complex subunits (except hADA2) and several TAFIIs, such as TAFII150, TAFII135, and TAFII100. TFTC can acetylate free and nucleosomal histones (with a preference for H3) in addition to linker histone H1. The other TAFII-derived complex is STAGA (SPT3-TAFII31GCN5-L acetyltransferase), which is purified using an antiboby against the histone-H3-like TAFII31 (Martinez et al. 1998). Cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes have been shown with direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machinery (Martinez et al. 2001). The oncoprotein Myc, implicated in many types of cancer, induces TRRAP recruitment and histone hyperacetylation at specific Myc-activated genes in vivo. The HAT complex recruited is the human STAGA (Liu et al. 2003). The human Tip60 complex consists of at least 14 distinct subunits, 3 of which are homologs of known proteins involved in DNA remodeling. In fact, the purified Tip60 complex possesses ATPase, DNA helicase, and structural DNA-binding activities. Mutated Tip60 lacking HAT activity causes defects in the ability of the cell to repair DNA and to trigger DNAdamage-induced apoptosis (Ikura et al. 2000). It has been suggested that Tip60, like p53, induces DNA repair or an apoptotic response, depending on the level of damage. Tip60 might interact with cell-cycle checkpoint proteins to activate an apoptotic pathway. Whereas recombinant Tip60 is able to acetylate free but not nucleosomal histones, the Tip60 complex can acetylate both substrates. Interestingly, the Tip60 complex and its yeast homolog Esa1 acetylate in vivo histone H4 and H2A most efficiently (Yamamoto and Horikoshi 1997). The Tip60 complex is considered the © 2005 NRC Canada

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mammalian homolog of the yeast NuA4 complex (Allard et al. 1999). The largest subunit of the Tip60 complex is TRRAP, a transcriptional regulatory protein also found in human GCN5 and PCAF complexes. Tra1 is the yeast homolog of TRRAP, and is a component of the SAGA and NuA4 complexes. At least two other complexes have been identified in human cells as having HAT subunits and activity. One of these is the TFIIIC multisubunit complex. Purified TFIIIC was able to alleviate chromatin-mediated transcriptional repression in vitro (Kundu et al. 1999). The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery, and contains a histone-specific acetyltransferase activity (Hsieh et al. 1999). The other complex contains the MYST protein HBO1 (Iizuka and Stillman 1999). Recombinant HBO1 alone has not been found to acetylate free histones; however, an HBO1-containing complex, isolated from nuclear extracts, was able to acetylate free H3 and H4 histones. Full activity of the HBO1 protein may therefore require other factors or in vivo modifications. Although the precise mechanism of deacetylation is still being debated, some evidence suggests that reversible acetylation also plays an important role in many cellular processes (Kouzarides 2000). The enzymes able to remove acetyl groups from chromatin substrates are called histone deacetylases (HDACs). In mammals, they have been ordered into 3 major classes: class I contains the RPD3-like enzymes; class II, the HDA1-like enzymes; and class III is represented by the sirtuin family, named after the Sir2 protein (Marks et al. 2003). As with histone acetyltransferases, which are recruited to specific promoters by activators and coactivators, deacetylases also interact with repressors and corepressors. Little is known about HDAC substrates. However, different types of deacetylases recognize specific acetylation patterns on nucleosomes (Kölle et al. 1999; Clemente et al. 2001), even though site specificity seems to depend mainly on the cofactors forming complexes with HDACs (Clemente et al. 2001). In yeast, the function of all known histone deacetylases has been determined (Kurdistani and Grunstein 2003). Histone acetylation and deacetylation are indeed dynamic events, and transcriptional activation can be considered a cyclical process that requires both activating and repressive epigenetic events. In the case of the human pS2 gene, which is controlled by the estrogen receptor-α, the concept of a transcriptional clock has been introduced to define the sequential and combinatorial assembly of protein complexes that contain different chromatin modifiers (Métivier et al. 2003, 2004). In yeast, the osmotic-stress-activated MAPK Hog1 has been shown to recruit the Rpd3–Sin3 histone deacetylase complex (de Nadal et al. 2004) and, at the same time, to convert a protein acting as a repressor into an activator capable of recruiting SAGA and SWI/SNF at the same promoter (GRE2) (Edmunds and Mahadevan 2004).

Genome-wide analysis Genome-wide approaches to epigenomic profiling based on chromatin immunoprecipitation (ChIP) experiments (Orlando 2000) have been extensively applied in many different eukaryotic organisms. Simple organisms, such as S. cerevisiae, have turned out to be extremely useful in the application of this type of analysis. The first studies used

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various deletion mutants, including HDACs, to develop expression profiles (Sun and Hampsey 1999; Hughes et al. 2000). The analysis was improved by combining the expression profiles derived from deletion mutants with those obtained by treating the cells with histone deacetylases inhibitors (Bernstein et al. 2000). Finally, the introduction of ChIP-on-chip experiments has been particularly helpful in S. cerevisiae. The relatively small size and low complexity of its genome has allowed the construction of true wholegenome arrays, including all transcription units and all intergenic regions. In particular, Kurdistani and coworkers (2002) set up a genome-wide approach to map, in vivo, the distribution of the histone deacetylase and repressor Rpd3. It was found that Rpd3 associates preferentially with the upstream regions of genes that direct high transcriptional activity. The most likely explanation for this finding is that more deacetylase activity may be needed to counteract the increased acetyltransferase activity at heavily transcribed genes. This fact is consistent with the observation that the acetyl groups in the 4 core histones have a short turnover time (Waterborg 2001). The most interesting aspect of this work is based on the complementarity of the Rpd3-binding map with acetylation and expression arrays. The triple approach establishes where a histone-modifying enzyme (in this case, deacetylase Rpd3) binds, whether it is active at that particular site, and what the effect is on the expression of the nearby gene. Histone-acetylation microarrays have been used to identify the intergenic regions at which acetylation is increased when all the known yeast HDACs are deleted, thus uncovering the division of labor for yeast histone deacetylases (Robyr et al. 2002). A more recent analysis has revealed that both hyper- and hypoacetylation of individual lysines are associated with transcription, creating distinct acetylation patterns that specify groups of biologically related genes (Kurdistani et al. 2004).

Why is histone acetylation relevant in gene regulation? The correlation between histone acetylation and transcriptional activity was proposed 40 years ago by Allfrey et al. (1964). Since then it has been solidly established (reviewed in Csordas 1990; Turner 1991; Grunstein 1997; Struhl 1998). Although transcriptional activation is generally correlated with histone acetylation, and repression with histone deacetylation (Struhl 1998; Kadosh and Struhl 1998; Shang et al. 2000), several studies have proven that this correlation is not exclusive (Deckert and Struhl 2001; Mulholland et al. 2003). Nevertheless, histone acetylation emerges as a central switch that allows interconversion between permissive and repressive chromatin structures and domains (Eberharter and Becker 2002) (Fig. 2). One major point still to be clarified concerns the role of histone acetylation in controlling gene expression. What is the molecular mechanism underlying the effects exerted by this particular modification? There are two possible explanations: histone acetylation changes the structure of the nucleosomes; or histone acetylation provides a signal for protein binding. According to the first possible explanation, changes in the structure of the nucleosomes, the addition of an acetyl group © 2005 NRC Canada

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Fig. 2. Switch from repressed to transcriptionally competent chromatin. The removal of acetyl groups from the lysine residues induces compaction of chromatin; their addition allows transcription. HAT, histone acetyltransferases; HDAC, histone deacetylases.

to the lysine ⑀-amino groups of histone N-terminal tails neutralizes the positive charge of the lysine side chain. This modification may affect the interaction between the lysine residue and DNA, leading to weaker contact with the negatively charged DNA backbone. As a consequence, nucleosome fluidity would increase (Kingston and Narlikar 1999); this could be the reason why, during the activation of several promoters, 1 or 2 nucleosomes undergo sliding, which facilitates the interaction of the transcription machinery with RNA initiation sites. In vivo nucleosome repositioning during transcription activation has been reported in the ADH2 gene in yeast (Di Mauro et al. 2002); one of the Adr1-dependent promoters requires the activity of Gcn5 for transcription initiation (Agricola et al. 2004). In higher eukaryotes, nucleosome sliding of the interferon (IFN)-β promoter has been correlated with the ability of TBP to bend DNA (Lomvardas and Thanos 2001). Evidence of a direct effect of histone acetylation on nucleosome structure and (or) on the stability of nucleosomal arrays has been provided in vitro by several groups (Bauer et al. 1994; Garcia-Ramirez et al. 1995; Wang et al. 2001; Hansen et al. 2002), and in vivo has been provided in the case of the S. cerevisiae ADH2 TATA-box-containing nucleosome (Verdone et al. 2002). According to the second possible mechanism, in which histone acetylation provides a signal for protein binding, the addition of an acetyl group to lysine residue creates a new surface for protein association, thus exerting its effects through gain-of-function mechanisms. Lysine acetylation creates docking sites for a protein module known as bromodomain,

which has been found to be present in many transcription and chromatin regulators (Yang 2004). In S. cerevisiae, there are 10 bromodomain proteins; in Drosophila, about 16; and in humans, more than 30. Several studies have led to the identification of at least 5 functions for bromodomains (Yang 2004). However, whether all bromodomains are able to recognize acetyllysine is currently unknown. In addition, evidence has been reported of a bromodomain-containing protein, p300, that binds to histones in an acetylation-independent manner (Manning et al. 2001). One of the functions of bromodomains in gene regulation is chromatin acetylation by HATs. In S. cerevisiae, the bromodomain of the acetyltransferase Gcn5 is required for the SAGA complex to associate with acetylated chromatin (Hassan et al. 2002), and for the coordination of nucleosome remodeling (Syntichaki et al. 2000). In particular, Gcn5 bromodomain recognizes a Lys16-acetylated H4 peptide, in spite of a rather weak binding (Hudson et al. 2000; Owen et al. 2000). The histone H4 tail is the target of another important yeast protein, Bdf1 (Matangkasombut et al. 2000). This protein has been shown to be required for anchoring TFIID to the S. cerevisiae PHO5 promoter when nucleosome positioning of the TATA box is altered in an H4-acetylation-leveldependent manner (Martinez-Campa et al. 2004). Another function of bromodomains is to link the activity of ATP-dependent chromatin remodelers to the acetylation of specific lysines. In the case of the human IFN-β gene, the SWI/SNF complex is recruited to the promoter by the Gcn5dependent acetylation of H4 Lys8, inducing chromatin © 2005 NRC Canada

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Fig. 3. Influence of acetylation level on transcriptional state. Gcn5 and Esa1 contribute to nucleosome-1 destabilization of the Adr1dependent genes allowing transcription.

remodeling, which, in turn, allows the binding of TFIID to acetylated Lys9 and Lys14 of H3 (Agalioti et al. 2002). Selective recognition of acetylated histones by bromodomaincontaining proteins has been recently established in the intact nuclei of living cells by FRET analysis (Kanno et al. 2004). For example, the transcriptional regulator Brd2 has been shown to interact principally with acetylated lysine 12 of histone H4 and, to a lesser degree, with histone H2B, but not with H3 and H2A. On the other hand, TAFII250, a component of the transcription factor TFIID, was found to recognize H3 and H4 (in the latter case with no preference for specific lysines), and to a lesser extent histone H2B, but not H2A. As a third bromodomain-containing protein PCAF was analyzed and found to produce FRET signals with both H3 and H4, but not with H2A or H2B (Kanno et al. 2004).

A model system in yeast Among the genes influenced by the activity of both Hda1 and Rpd3, the best characterized is ADH2, which codes for the glucose-regulated alcohol dehydrogenase II enzyme. It has been shown that, in a strain in which both RPD3 and HDA1 are deleted, the structure of the TATA-box-containing nucleosome is destabilized, the recruitment of the transcriptional factor Adr1 is facilitated, and the kinetics of ADH2 mRNA accumulation is faster (Verdone et al. 2002). This study represents the first example describing a direct in vivo effect of a change in histone acetylation level on the structure of a specific nucleosome. When genes for acetyltransferases GCN5 and ESA1 were disrupted, ADH2 promoter structure and function were shown to be affected. In particular, the chromatin remodeling occurring in derepressing conditions in the GCN5 mutant is less pronounced, and the kinetics of mRNA accumulation is slower; in the presence of an ESA1 temperature-sensitive mutation, the mRNA amount is lower even in permissive conditions. Therefore, histone deacetylation/ acetylation must be directly involved in modulating the accessibility of chromatin at the ADH2 gene. The requirement for the Esa1 and Gcn5 acetyltransferases in transcriptional activation has been recently described in the case of another glucose-repressed gene, SUC2, in which chromatin remodeling of the TATA-box-containing nucleosome was also observed (Geng and Laurent 2004). The similar

acetylation requirement for ADH2 and SUC2 activation could be due to the fact that both genes are glucose-repressed and both undergo derepression by lowering the amount of glucose in the medium. On the other hand, glucose starvation can be considered a cellular stress. Interestingly, genes that are commonly upregulated during general environmental stress are dependent on SAGA, the protein complex containing the histone acetyltransferase Gcn5 (Huisinga and Pugh 2004). Among them, the PHO5 promoter should be mentioned; it shows a SAGA-dependent increase in the histone H3 acetylation level during activation, which actually precedes nucleosome loss (Reinke and Hörz 2003). Moreover, Esa1-dependent histone H4 acetylation at this promoter correlates with specific recruitment of the NuA4 complex in repressed conditions (Nourani et al. 2004). This recruitment is required for subsequent chromatin remodeling and transcription. Therefore, PHO5-promoter induction is dependent on at least 2 different complexes: NuA4, recruited by Pho2 (Nourani et al. 2004); and SAGA (Huisinga and Pugh 2004). A group of coregulated genes, all repressed by glucose and all derepressed in the presence of a common transcription activator, the Adr1 protein, was recently found to be dependent on both Esa1 and Gcn5 acetyltransferases (Agricola et al. 2004) (Fig. 3). This family of coregulated genes offers the unique opportunity to test whether the common requirement, in terms of activator (Adr1) and coactivator (acetyltransferase) functions, is reflected by a common chromatin architecture at the promoter level. Indeed, in repressing conditions, a nucleosome particle always obstructs the TATA box immediately adjacent to an upstream-located nucleosome-free region that contains a cluster of Adr1 binding sites; upon derepression, the TATA-box-containing nucleosome is destabilized by a mechanism shared by all the genes studied. The transcription factor Adr1 is always required for the chromatin remodeling observed. An increase in the histone H3 acetylation level during gene activation is required to reach the normal mRNA amount. The unexpected finding is that the acetylation increase occurs specifically at some but not all the histone H3 acetylatable lysines in a Gcn5-dependent manner. In addition to Gcn5, the activity of the acetyltransferase Esa1 is required for the transcription of all Adr1-dependent genes (Agricola et al. 2004). Unlike histone H3, histone H4 is constitutively acetylated on all acetylatable sites; this could be © 2005 NRC Canada

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necessary and sufficient for the binding of Gcn5, which, in turn, acetylates specific lysine residues on histone H3 only during transcriptional activation. This suggests the existence of unique patterns of acetylation for a defined set of coregulated genes, as was recently proposed (Kurdistani et al. 2004).

Concluding remarks A final issue needs to be discussed when trying to clarify the role of histone acetylation in gene expression. It has to do with the interplay with other histone modifications. For instance, acetylation and methylation of histone H3 at Lys9 have completely opposite functional consequences; acetylation is associated with active chromatin and methylation with heterochromatin or inactive chromatin (Strahl and Allis 2000; Turner 2002). On the other hand, there is evidence that one type of histone modification affects other histone modifications (Ng et al. 2002; Sun and Allis 2002). It has been proposed that the variety of histone modifications and their recognition by other proteins act sequentially or in combination to form a histone code (Strahl and Allis 2000). However, it has been pointed out that the total number of modifications does not necessarily contain more information than the sum of individual modifications (Turner 2002; Kurdistani and Grunstein 2003). In Drosophila, evidence of a binary pattern of histone modifications among euchromatic genes has been reported (Schübeler et al. 2004). This issue is quite controversial, and more work is required to resolve this debate. Nevertheless, what really matters is that disruption of the balance of epigenetic networks can cause major pathologies, including cancer, syndromes involving chromosomal instabilities, and mental retardation. Tumors that are late-arising were found to involve epigenetic rather than genetic alterations, whereas early-arising tumors had classic genetic changes (Feinberg and Tycko 2004). The functional importance of HATs and HDACs is highlighted by the fundamental regulatory roles that they play in developmental processes, and by the evidence that their deregulation has been linked to the progression of cancers and diverse human disorders, such as the Rubinstein–Taybi and fragile X syndromes (Timmermann et al. 2001). Germ-line mutations in CBP cause Rubinstein–Taybi syndrome, a developmental disorder, and these patients suffer an increased cancer risk (Petrij et al. 1995; Murata et al. 2001). Finally, geneticablation studies of CBP and p300 in mouse models have confirmed that both proteins function as tumor suppressors (Yao et al. 1998; Kung et al. 2000; Kang-Decker et al. 2004).

Acknowledgements This work was supported by CNR Genomica Funzionale and Center of Excellence BEMM La Sapienza.

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