Histone Acetyltransferase GCN5 Research Articles

TRRAP and GCN5 are used by c-Myc to activate RNA polymerase III transcription

Activation of RNA polymerase (pol) II transcription by c-Myc generally involves recruitment of histone acetyltransferases and acetylation of histones H3 and H4. Here, we describe the mechanism used by c-Myc to activate pol III transcription of tRNA and 5S rRNA genes. Within 2 h of its induction, c-Myc appears at these genes along with the histone acetyltransferase GCN5 and the cofactor TRRAP. At the same time, occupancy of the pol III-specific factor TFIIIB increases and histone H3 becomes hyperacetylated, but increased histone H4 acetylation is not detected at these genes. The rapid acetylation of histone H3 and promoter assembly of TFIIIB, c-Myc, GCN5, and TRRAP are followed by recruitment of pol III and transcriptional induction. The selective acetylation of histone H3 distinguishes pol III activation by c-Myc from mechanisms observed in other systems.

The Chromatin Remodeling Protein, SRCAP, Is Critical for Deposition of the Histone Variant H2A.Z at Promoters

Studies in Saccharomyces cerevisiae indicate the histone variant H2A.Z is deposited at promoters by the chromatin remodeling protein Swr1 and plays a critical role in the regulation of transcription. In higher eukaryotes, however, little is known about the distribution, method of deposition, and function of H2A.Z at promoters. Using biochemical studies, we demonstrated previously that SRCAP (SNF-2-related CREB-binding protein activator protein), the human ortholog of Swr1, could catalyze deposition of H2A.Z into nucleosomes. To address whether SRCAP directs H2A.Z deposition in vivo, promoters targeted by SRCAP were identified by a chromatin immunoprecipitation (ChIP)-on-chip assay. ChIP assays on a subset of these promoters confirmed the presence of SRCAP on inactive and active promoters. The highest levels of SRCAP were observed on the active SP-1, G3BP, and FAD synthetase promoters. Detailed analyses of these promoters indicate sites of SRCAP binding overlap or occur adjacent to the sites of H2A.Z deposition. Knockdown of SRCAP levels using siRNA resulted in loss of SRCAP at these promoters, decreased deposition of H2A.Z and acetylated H2A.Z, and a decrease in levels of SP-1, G3BP, and FAD synthetase mRNA. Thus, these studies provide the first evidence that SRCAP is recruited to promoters and is critical for the deposition of H2A.Z.

Crystal structure of a binary complex between human GCN5 histone acetyltransferase domain and acetyl coenzyme A

Becauseof this, enzymes catalyzing histone acetylation, the his-tone acetyltransferases (HATs, EC 2.3.1.48), have a majorrole in the cell fate, proliferation, and differentiation.The histone acetyltransferase GCN5 acts as a supervi-sor in the normal cell cycle progression having compre-hensive control over expressions of these cell cycle-related genes, as well as apoptosis-related genes, viaalterations in the chromatin structure, mimicked bychanging acetylation status of core histones, which sur-round these genes.

Domains of Arabidopsis ADA2a and ADA2b necessary for proper biological functioning

The way DNA is packaged into chromatin influences gene function by regulating transcription, DNA replication, and recombination. Modifications of histones and chromatin remodeling mechanisms are critical for an organism's proper development. This research focuses on histone acetylation, a form of chromatin remodeling accomplished when protein complexes add acetyl groups to histone proteins, specifically the lysine residues on the N-terminal histone “tails”. The histone acetyltransferase GCN5 works with the transcriptional coactivator ADA2. Previous studies have shown that mutations in ADA2 or GCN5 effect normal growth, development, and gene expression patterns. In our work, we use Arabidopsis thaliana as an experimental model allowing us to study of the effects of chromatin modifying proteins on development. In Arabidopsis there are two genes, ADA2a and ADA2b, which resemble the Ada2 coactivator in yeast. Disruptions in ADA2b result in dwarfed plants but disruptions in ADA2a have no phenotypic effect. In this experiment recombinant DNA technology is used to create chimeric transgenic constructs in order to determine the domains of ADA2b necessary for normal phenotype and plant development.

Actively Transcribed GAL Genes Can Be Physically Linked to the Nuclear Pore by the SAGA Chromatin Modifying Complex

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy (Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. (2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.

Site-specific acetylation of ISWI by GCN5

BackgroundThe tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition.ResultsWe found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF.ConclusionAcetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(Kac)xPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

The Histone Chaperone Anti-silencing Function 1 Stimulates the Acetylation of Newly Synthesized Histone H3 in S-phase

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. We have found that budding yeast lacking Asf1 has greatly reduced levels of histone H3 acetylated at lysine 9. Lysine 9 is acetylated on newly synthesized budding yeast histone H3 prior to its assembly onto newly replicated DNA. Accordingly, we found that the vast majority of H3 Lys-9 acetylation peaked in S-phase, and this S-phase peak of H3 lysine 9 acetylation was absent in yeast lacking Asf1. By contrast, deletion of ASF1 has no effect on the S-phase specific peak of H4 lysine 12 acetylation; another modification carried by newly synthesized histones prior to chromatin assembly. We show that Gcn5 is the histone acetyltransferase responsible for the S-phase-specific peak of H3 lysine 9 acetylation. Strikingly, overexpression of Asf1 leads to greatly increased levels of H3 on acetylation on lysine 56 and Gcn5-dependent acetylation on lysine 9. Analysis of a panel of Asf1 mutations that modulate the ability of Asf1 to bind to histones H3/H4 demonstrates that the histone binding activity of Asf1 is required for the acetylation of Lys-9 and Lys-56 on newly synthesized H3. These results demonstrate that Asf1 does not affect the stability of the newly synthesized histones per se, but instead histone binding by Asf1 promotes the efficient acetylation of specific residues of newly synthesized histone H3.

Gcn5 Promotes Acetylation, Eviction, and Methylation of Nucleosomes in Transcribed Coding Regions

We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequences during transcriptional induction, dependent on PIC assembly and Ser5 phosphorylation of the Pol II CTD. Induction of GAL1 increases H3 acetylation per nucleosome in the ORF, dependent on SAGA integrity but not the alternative Gcn5p-HAT complex ADA. Unexpectedly, H3 acetylation in ARG1 coding sequences does not increase during induction due to the opposing activities of multiple HDAs associated with the ORF. Remarkably, inactivation of Gcn5p decreases nucleosome eviction from both GAL1 and a long ( approximately 8 kb) ORF transcribed from the GAL1 promoter. This is associated with reduced Pol II occupancy at the 3' end and decreased mRNA production, selectively, for the long ORF. Gcn5p also enhances H3-K4 trimethylation in the ARG1 ORF and bulk histones. Thus, Gcn5p, most likely in SAGA, stimulates modification and eviction of nucleosomes in transcribed coding sequences and promotes Pol II elongation.

Catalytic Mechanism of a MYST Family Histone Acetyltransferase

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 1.6 s(-1)) and of CoA formation (0.0021 s(-1)) were inconsistent with a kinetically competent acetyl-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat approximately 200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.

Quinoline Derivative MC1626, a Putative GCN5 Histone Acetyltransferase (HAT) Inhibitor, Exhibits HAT-Independent Activity against Toxoplasma gondii

We report that quinoline derivative MC1626, first described as an inhibitor of the histone acetyltransferase (HAT) GCN5, is active against the protozoan parasite Toxoplasma gondii in vitro. However, MC1626 does not inhibit Toxoplasma GCN5 HATs or reduce HAT-mediated activity; rather, this quinoline may target the plastid organelle called the apicoplast.

Arabidopsis GCN5, HD1, and TAF1/HAF2 Interact to Regulate Histone Acetylation Required for Light-Responsive Gene Expression

We previously showed that Arabidopsis thaliana histone acetyltransferase TAF1/HAF2 is required for the light regulation of growth and gene expression, and we show here that histone acetyltransferase GCN5 and histone deacetylase HD1/HDA19 are also involved in such regulation. Mutation of GCN5 resulted in a long-hypocotyl phenotype and reduced light-inducible gene expression, whereas mutation of HD1 induced opposite effects. The double mutant gcn5 hd1 restored a normal photomorphogenic phenotype. By contrast, the double mutant gcn5 taf1 resulted in further loss of light-regulated gene expression. gcn5 reduced acetylation of histones H3 and H4, mostly on the core promoter regions, whereas hd1 increased acetylation on both core and more upstream promoter regions. GCN5 and TAF1 were both required for H3K9, H3K27, and H4K12 acetylation on the target promoters, but H3K14 acetylation was dependent only on GCN5. Interestingly, gcn5 taf1 had a cumulative effect mainly on H3K9 acetylation. On the other hand, hd1 induced increased acetylation on H3K9, H3K27, H4K5, and H4K8. GCN5 was also shown to be directly associated with the light-responsive promoters. These results suggest that acetylation of specific histone Lys residues, regulated by GCN5, TAF1, and HD1, is required for light-regulated gene expression.

Bromodomain and Histone Acetyltransferase Domain Specificities Control Mixed Lineage Leukemia Phenotype

A critical unanswered question about mixed lineage leukemia (MLL) is how specific MLL fusion partners control leukemia phenotype. The MLL-cyclic AMP-responsive element binding protein-binding protein (CBP) fusion requires both the CBP bromodomain and histone acetyltransferase (HAT) domain for transformation and causes acute myelogenous leukemia (AML), often preceded by a myelodysplastic phase. We did domain-swapping experiments to define whether unique specificities of these CBP domains drive this specific MLL phenotype. Within MLL-CBP, we replaced the CBP bromodomain or HAT domain with P300/CBP-associated factor (P/CAF) or TAF(II)250 bromodomains or the P/CAF or GCN5 HAT domains. HAT, but not bromodomain, substitutions conferred enhanced proliferative capacity in vitro but lacked expression of myeloid cell surface markers normally seen with MLL-CBP. Mice reconstituted with domain-swapped hematopoietic progenitors developed different disease from those with MLL-CBP. This included development of lymphoid disease and lower frequency of the myelodysplastic phase in those mice developing AML. We conclude that both the CBP bromodomain and HAT domain play different but critical roles in determining the phenotype of MLL-CBP leukemia. Our results support an important role for MLL partner genes in determining the leukemia phenotype besides their necessity in leukemogenesis. Here, we find that subtleties in MLL fusion protein domain specificity direct cells toward a specific disease phenotype.

Stress-Specific Role of Fission Yeast Gcn5 Histone Acetyltransferase in Programming a Subset of Stress Response Genes

Gcn5 is a coactivator protein that contributes to gene activation by acetylating specific lysine residues within the N termini of histone proteins. Gcn5 has been intensively studied in the budding yeast, Saccharomyces cerevisiae, but the features of genes that determine whether they require Gcn5 during activation have not been conclusively clarified. To allow comparison with S. cerevisiae, we have studied the genome-wide role of Gcn5 in the distantly related fission yeast, Schizosaccharomyces pombe. We show that Gcn5 is specifically required for adaptation to KCl- and CaCl(2)-mediated stress in S. pombe. We have characterized the genome-wide gene expression responses to KCl stress and show that Gcn5 is involved in the regulation of a subset of stress response genes. Gcn5 is most clearly associated with KCl-induced genes, but there is no correlation between Gcn5 dependence and the extent of their induction. Instead, Gcn5-dependent KCl-induced genes are specifically enriched in four different DNA motifs. The Gcn5-dependent KCl-induced genes are also associated with biological process gene ontology terms such as carbohydrate metabolism, glycolysis, and nicotinamide metabolism that together constitute a subset of the ontology parameters associated with KCl-induced genes.

SWI/SNF Binding to the HO Promoter Requires Histone Acetylation and Stimulates TATA-Binding Protein Recruitment

We use chromatin immunoprecipitation assays to show that the Gcn5 histone acetyltransferase in SAGA is required for SWI/SNF association with the HO promoter and that binding of SWI/SNF and SAGA are interdependent. Previous results showed that SWI/SNF binding to HO was Gcn5 independent, but that work used a strain with a mutation in the Ash1 daughter-specific repressor of HO expression. Here, we show that Ash1 functions as a repressor that inhibits SWI/SNF binding and that Gcn5 is required to overcome Ash1 repression in mother cells to allow HO transcription. Thus, Gcn5 facilitates SWI/SNF binding by antagonizing Ash1. Similarly, a mutation in SIN3, like an ash1 mutation, allows both HO expression and SWI/SNF binding in the absence of Gcn5. Although Ash1 has recently been identified in a Sin3-Rpd3 complex, our genetic analysis shows that Ash1 and Sin3 have distinct functions in regulating HO. Analysis of mutant strains shows that SWI/SNF binding and HO expression are correlated and regulated by histone acetylation. The defect in HO expression caused by a mutant SWI/SNF with a Swi2(E834K) substitution can be partially suppressed by ash1 or spt3 mutation or by a gain-of-function V71E substitution in the TATA-binding protein (TBP). Spt3 inhibits TBP binding at HO, and genetic analysis suggests that Spt3 and TBP(V71E) act in the same pathway, distinct from that of Ash1. We have detected SWI/SNF binding at the HO TATA region, and our results suggest that SWI/SNF, either directly or indirectly, facilitates TBP binding at HO.

CAT in the HAT: Catabolic inhibition by the histone acetyltransferase GCN5

The nuclear hormone receptor coactivator PGC-1alpha is a key regulator of gluconeogenic genes during fasting. In this issue of Cell Metabolism, Puigserver and colleagues (Lerin et al., 2006) report that the histone acetyltransferase GCN5 inhibits gluconeogenesis by acetylating and sequestering PGC-1alpha in nuclear foci.

Myc influences global chromatin structure.

The family of myc proto-oncogenes encodes transcription factors (c-, N-, and L-Myc) that regulate cell growth and proliferation and are involved in the etiology of diverse cancers. Myc proteins are thought to function by binding and regulating specific target genes. Here we report that Myc proteins are required for the widespread maintenance of active chromatin. Disruption of N-myc in neuronal progenitors and other cell types leads to nuclear condensation accompanied by large-scale changes in histone modifications associated with chromatin inactivation, including hypoacetylation and altered methylation. These effects are largely reversed by exogenous Myc as well as by differentiation and are mimicked by the Myc antagonist Mad1. The first chromatin changes are evident within 6 h of Myc loss and lead to changes in chromatin structure. Myc widely influences chromatin in part through upregulation of the histone acetyltransferase GCN5. This study provides the first evidence for regulation of global chromatin structure by an oncoprotein and may explain the broad effects of Myc on cell behavior and tumorigenesis.

Identification of small molecules inhibitors of GCN5 histone acetyltransferase activity

Starting from a yeast phenotypic screening performed on 21 chemically different substances we described the discovery of two small molecules as GCN5 inhibitors. The 2-methyl-3carbethoxyquinoline 9 and its 2-desmethyl analogue 18 were able to significantly reduce the yeast cell growth, thus miming the effect of GCN5 deletion mutant. Tested to evaluate their effect on GCN5-dependent transcription of the HIS3 gene, 9 and 18 showed high inhibitory activity of gene transcription, more evident in activated conditions. Compound 9 was also able to reduce the acetylation levels of H3 and, to a lesser extent, H4 in yeast at 0.6 mM. In human leukemia U937 cells, at 1 mM concentration 9 showed 27% apoptosis induction, while 18 had just a little effect in the same conditions. Further studies on 9 and 18 will be performed to deepen their effects on GCN5-related phenomena.

Regulation of c‐Myc by p300 via Control of Myc Protein Turnover and Myc‐Induced Transcription

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N-terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1-110 and acetylates Myc and Max in a TAD-dependent manner in vivo at several lysine residues. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. While p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions also as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation. Preliminary results also indicate that p300-mediated acetylation of Max residues may control Myc transactivation by regulating Max:Max/Myc:Max nuclear localization and DNA binding. Supported by NIH grant CA100464 and a UCR GI Core Instrumentation Facility grant.

A Role for Gcn5-Mediated Global Histone Acetylation in Transcriptional Regulation

Transcriptional activators often require histone acetyltransferases (HATs) for full activity. The common explanation is that activators directly recruit HATs to gene promoters to locally hyperacetylate histones and thereby facilitate transcription complex formation. However, in addition to being targeted to specific loci, HATs such as Gcn5 also modify histones genome-wide. Here we provide evidence for a role of this global HAT activity in regulated transcription. We show that activation by direct recruitment of the transcriptional machinery neither recruits Gcn5 nor induces changes in histone acetylation yet can strongly depend on Gcn5 at promoters showing a high basal state of Gcn5-mediated histone acetylation. We also show that Gcn5 dependency varies among core promoters and is influenced by the strength of interaction used to recruit the machinery and by the affinity of the latter for the core promoter. These data support a role for global Gcn5 HAT activity in modulating transcription independently of its known coactivator function.

Glutamine-Expanded Ataxin-7 Alters TFTC/STAGA Recruitment and Chromatin Structure Leading to Photoreceptor Dysfunction

Spinocerebellar ataxia type 7 (SCA7) is one of several inherited neurodegenerative disorders caused by a polyglutamine (polyQ) expansion, but it is the only one in which the retina is affected. Increasing evidence suggests that transcriptional alterations contribute to polyQ pathogenesis, although the mechanism is unclear. We previously demonstrated that theSCA7 gene product, ataxin-7 (ATXN7), is a subunit of the GCN5 histone acetyltransferase–containing coactivator complexes TFTC/STAGA. We show here that TFTC/STAGA complexes purified from SCA7 mice have normal TRRAP, GCN5, TAF12, and SPT3 levels and that their histone or nucleosomal acetylation activities are unaffected. However, rod photoreceptors from SCA7 mouse models showed severe chromatin decondensation. In agreement, polyQ-expanded ataxin-7 induced histone H3 hyperacetylation, resulting from an increased recruitment of TFTC/STAGA to specific promoters. Surprisingly, hyperacetylated genes were transcriptionally down-regulated, and expression analysis revealed that nearly all rod-specific genes were affected, leading to visual impairment in SCA7 mice. In conclusion, we describe here a set of events accounting for SCA7 pathogenesis in the retina, in which polyQ-expanded ATXN7 deregulated TFTC/STAGA recruitment to a subset of genes specifically expressed in rod photoreceptors, leading to chromatin alterations and consequent progressive loss of rod photoreceptor function.