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The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Eukaryotic chromosomal DNA is packaged into nucleosomes to form a dynamic structure known as chromatin. The compaction of DNA within chromatin poses a unique hindrance with regards to the accessibility of the DNA to enzymes involved in replication, transcriptional regulation, and repair. The physical structure and physiological activity of chromatin are regulated through a diverse set of posttranslational modifications, histone exchange, and structural remodeling. Of the covalent chromatin modifications, the acetylation of lysine residues within histone proteins by acetyltransferase enzymes, such as GCN5, is one of the most prevalent and important steps in the regulation of chromatin function. Alteration of histone acetyltransferase activity can easily result in the dysregulation of gene transcription and ultimately the onset of a disease state. Many transcription factors contain polyglutamine regions within their primary sequence. Mutations resulting in the elongation of these polyglutamine tracts are associated with a disease family known as the polyglutamine expansion disorders. Spinocerebellar ataxia type 7 (SCA7) is one of the nine diseases that are grouped in this family and is caused by polyglutamine expansion of the ataxin-7 protein, which is a component of the GCN5-containing human SAGA histone acetyltransferase complex. Mutation of ataxin-7 in this manner has been shown to disrupt the structural integrity of the SAGA complex and result in aberrant chromatin acetylation patterns at the promoters of genes involved in the normal function of tissues that are affected by the disease. The specific aspects of molecular pathology are not currently understood; however, studies carried out in laboratory systems ranging from the budding yeast Saccharomyces cerevisiae to transgenic mouse models and cultured human cells are poised to allow for the elucidation of disease mechanisms and subsequent therapeutic approaches., (Copyright 2010 Elsevier Inc. All rights reserved.)

Histone acetyltransferases have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous multisubunit histone acetyltransferase complexes that were originally identified in the yeast Saccharomyces cerevisiae. Here, we identify the protein Sgf73/Sca7 as a component of SAGA and SLIK, and a homologue of the human SCA7-encoded protein ataxin-7, which, in its polyglutamine expanded pathological form, is responsible for the neurodegenerative disease spinocerebellar ataxia 7 (SCA7). Our findings indicate that yeast Sca7 is necessary for the integrity and function of both SAGA and SLIK, and that the human ataxin-7 is able to compliment the loss of Sca7 in yeast. A polyglutamine-expanded version of ataxin-7 assembles a SAGA complex that is depleted of critical proteins that regulate the ability of SAGA to acetylate nucleosomes. These observations have significant implications for the function of the human Sca7 protein in disease pathogenesis.

The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication. Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes. For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B. Here we identify the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1) as a component of SAGA and SLIK. Our findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity. Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo. Our study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties.

In our efforts to understand how transcription may be regulated in Entamoeba histolytica, we have examined if this parasite has conserved enzymatic mechanisms for targeted acetylation and deacetylation of histones. Western blotting indicated that basic nuclear proteins in the size range of 16-23 kDa were acetylated in amebic trophozoites, suggesting histone acetylation. Single representatives of the GNAT and MYST family of histone acetyltransferases (HATs) were identified in the E. histolytica genome and their expression in amebic trophozoites was detected by reverse transcription of RNA followed by the polymerase chain reaction (RT-PCR). Full-length recombinant EhMYST protein demonstrated HAT activity with calf thymus histones and showed a preference for histone H4, similar to the yeast MYST protein, Esa1. However, ehMYST did not complement a yeast esa1 mutation. Histone deacetylase (HDAC) activity was detected in nuclear extracts from E. histolytica, and characteristically, was inhibited by trichostatin A (TSA). Consistent with the observation of HDAC activity, RT-PCR analysis demonstrated that an amebic hdac1 homolog (ehHDAC) is expressed and appropriately spliced in E. histolytica trophozoites. Our results suggest that mechanisms for histone acetylation and deacetylation are operational in E. histolytica.

Post-translational modifications of the histone protein components of eukaryotic chromatin play an important role in the regulation of chromatin structure and gene expression (1). Given the requirement of Rad6/Bre1-dependent ubiquitination of histone H2B for H3 dimethylation (at lysines 4 and 79) and gene silencing (2-7), removal of ubiquitin from H2B may have a significant regulatory effect on transcription. Here we show that a putative deubiquitinating enzyme, Ubp8, is a structurally nonessential component of both the Spt-Ada-Gcn5-acetyltransferase (SAGA) and SAGA-like (SLIK) histone acetyltransferase (HAT) complexes in yeast. Disruption of this gene dramatically increases the cellular level of ubiquitinated-H2B, and SAGA and SLIK are shown to have H2B deubiquitinase activity. These findings demonstrate, for the first time, how the ubiquitin moiety can be removed from histone H2B in a regulated fashion. Ubp8 is required for full expression of the SAGA- and SLIK-dependent gene GAL10 and is recruited to the upstream activation sequence (UAS) of this gene under activating conditions, while Rad6 dissociates. Furthermore, trimethylation of H3 at lysine 4 within the UAS increases significantly under activating conditions, and remarkably, Ubp8 is shown to have a role in regulating the methylation status of this residue. Collectively, these data suggest that the SAGA and SLIK HAT complexes can regulate an integrated set of multiple histone modifications, counteracting repressive effects that alter chromatin and regulate gene expression.

The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF(II)s), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.

Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Here we show that distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, our data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly.

Previous studies have shown that the transcriptional coactivator protein Gcn5 functions as a catalytic histone acetyltransferase (HAT). In this work, we examine the roles of the Ada2 and Ada3 coactivator proteins that are functionally linked to Gcn5. We show that yeast Ada2, Ada3, and Gcn5 form a catalytic core of the ADA and Spt-Ada-Gcn5-acetyltransferase HAT complexes, which is necessary and sufficient in vitro for nucleosomal HAT activity and lysine specificity of the intact HAT complexes. We also demonstrate that Ada3 is necessary for Gcn5-dependent nucleosomal HAT activity in yeast extracts. Our results suggest that Ada2 potentiates the Gcn5 catalytic activity and that Ada3 facilitates nucleosomal acetylation and an expanded lysine specificity.

We have purified and characterized a Gcn5-independent nucleosomal histone H3 HAT complex, NuA3 (Nucleosomal Acetyltransferase of histone H3). Peptide sequencing of proteins from the purified NuA3 complex identified Sas3 as the catalytic HAT subunit of the complex. Sas3 is the yeast homolog of the human MOZ oncogene. Sas3 is required for both the HAT activity and the integrity of the NuA3 complex. In addition, NuA3 contains the TBP- associated factor, yTAF(II)30, which is also a component of the TFIID, TFIIF, and SWI/SNF complexes. Sas3 mediates interaction of the NuA3 complex with Spt16 both in vivo and in vitro. Spt16 functions as a component of the yeast CP (Cdc68/Pob3) and mammalian FACT (facilitates chromatin transcription) complexes, which are involved in transcription elongation and DNA replication. This interaction suggests that the NuA3 complex might function in concert with FACT-CP to stimulate transcription or replication elongation through nucleosomes by providing a coupled acetyltransferase activity.

Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast. The N-terminal transactivation domain of GR, tau1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo. Using in vitro techniques, we demonstrate that the GR-tau1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex. Mutations in tau1 that reduce tau1 transactivation activity in vivo lead to a reduced binding of tau1 to the SAGA complex and conversely, mutations increasing the transactivation activity of tau1 lead to an increased binding of tau1 to SAGA. In addition, the Ada-independent NuA4 HAT complex also interacts with tau1. GAL4-tau1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner. Low-activity tau1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity tau1 mutants increase transcriptional activation, specifically from chromatin templates. Our results demonstrate that the targeting of native HAT complexes by the GR-tau1 activation domain mediates transcriptional stimulation from chromatin templates.

Modification of histone amino terminal tails by acetylation has long been linked to the transcriptional capacity of genes in chromatin and to various aspects of chromatin dynamics. Over the last few years a flurry of reports have described the purification and identification of a large number of histone acetyltransferases. Many of these enzymes had previously been described as transcriptional regulators and have frequently been isolated as part of larger multisubunit protein complexes. This review describes the association of acetyltransferases with partner proteins and the additional functional attributes of such complexes beyond catalytic function., (Copyright 1999 Academic Press.)

The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

The SAGA histone acetyltransferase/transcriptional adaptor complex is composed of multiple transcriptional regulators including Ada, Spt, and TAFII proteins. Here we identify an additional novel subunit of the complex, Tra1, an ATM/PI-3-kinase-related homolog of the human TRRAP cofactor, which is essential for c-Myc and E2F-mediated oncogenic transformation. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable association of this protein within SAGA. In addition, the Tra1 protein is a component of at least two other histone acetyltransferase protein complexes. These results indicate a role for Tra1 in the regulation of transcriptional activation through the recruitment of HAT activity to an activator-bound promoter.

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.

Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core histones to transcriptional activity of cellular chromatin. New insights into this field were gained on the identification of the first nuclear, type A histone acetyltransferase (HAT). The yeast transcriptional adaptor protein Gcn5 was identified as a nuclear HAT and thus provided a direct link between pathways of transcriptional activation and histone acetylation. However, while recombinant Gcn5 can efficiently acetylate free histone H3 and, to a lesser extent, H4 it is unable to acetylate nucleosomal histones. It is therefore very likely that additional proteins are required for Gcn5-mediated acetylation of chromosomal histones. We have recently shown that Gcn5 is the catalytic subunit of two high-molecular-weight histone acetyltransferase complexes in yeast. In addition to the Gcn5-containing ADA and SAGA HAT complexes we have identified two other HAT complexes in yeast. These are called NuA3 and NuA4 for their predominant specificity to acetylate histones H3 and H4, respectively. Here we describe the identification and characterization of four native nuclear high-molecular-weight HAT complexes in Saccharomyces cerevisiae. These purified HATs can be used in a variety of functional assays to further address questions of how acetylation has an impact on transcriptional regulation.

Transcriptional co-activators were originally identified as proteins that act as intermediaries between upstream activators and the basal transcription machinery. The discovery that co-activators such as Tetrahymena and yeast Gcn5, as well as human p300/CBP, pCAF, Src-1, ACTR and TAFII250, can acetylate histones suggests that activators may be involved in targeting acetylation activity to promoters. Several histone deacetylases have been linked to transcriptional co-repressor proteins, suggesting that the action of both acetylases and deacetylases is important in the regulation of many genes. Here we demonstrate the binding of two native yeast histone acetyltransferase (HAT) complexes to the herpesvirus VP16 activation domain and the yeast transcriptional activator Gcn4, and show that it is their interaction with the VP16 activation domain that targets Gal4-VP16-bound nucleosomes for acetylation. We find that Gal4-VP16-driven transcription from chromatin templates is stimulated by both HAT complexes in an acetyl CoA-dependent reaction. Our results demonstrate the targeting of native HAT complexes by a transcription-activation domain to nucleosomes in order to activate transcription.

A number of transcriptional coactivator proteins have been identified as histone acetyltransferase (HAT) proteins, providing a direct molecular basis for the coupling of histone acetylation and transcriptional activation. The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex requires the coactivator protein Gcn5 for HAT activity. Identification of protein subunits by mass spectrometry and immunoblotting revealed that the TATA binding protein-associated factors (TAF(II)s) TAF(II)90, -68/61, -60, -25/23, and -20/17 are integral components of this complex. In addition, TAF(II)68 was required for both SAGA-dependent nucleosomal HAT activity and transcriptional activation from chromatin templates in vitro. These results illustrate a role for certain TAF(II) proteins in the regulation of gene expression at the level of chromatin modification that is distinct from the TFIID complex and TAF(II)145.

Several previously characterized transcriptional adaptors and coactivators are now known to be histone acetyltransferases (HATs). Recent studies in Saccharomyces cerevisiae indicate that the Gcn5p HAT exists in large complexes containing several phenotypic classes of transcription factors. Genetic and biochemical studies of these transcription factors and their functions within HAT complexes suggest that acetylation of histones is one function of an integrated system of modular activities. These activities include interaction with activators, histone acetylation and interaction with basal factors. Coordination of these functions may well be an important component of gene activation in vivo.

The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).