Gcn5p is involved in the acetylation of histone H3 in nucleosomes

Ruiz-García, Ana Belén; Sendra, Ramón; Pamblanco, Mercè; Tordera, Vicente

doi:10.1016/s0014-5793(97)00049-5

Cited by 54 publications

(60 citation statements)

References 27 publications

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“…Our results indicate that Gcn5p action and chromatin decondensation may be directly linked. Although it has been proposed that Gcn5p must interact with other protein subunits to acetylate nucleosomal arrays (10,(12)(13)(14)30), our studies have demonstrated unequivocally that rGcn5p per se is capable of acetylating both nucleosomes and nucleosomal arrays under appropriate ionic conditions, including those that approximate the ionic environment present in the nucleus. Furthermore, rGcn5p exhibits a marked preference for extensively folded nucleosomal arrays as 3 H]acetate incorporation was ascertained by scintillation counting (see "Experimental Procedures").…”

Section: Discussioncontrasting

confidence: 53%

Gcn5p, a Transcription-related Histone Acetyltransferase, Acetylates Nucleosomes and Folded Nucleosomal Arrays in the Absence of Other Protein Subunits

Tse

Georgieva

Ruiz-García

et al. 1998

Journal of Biological Chemistry

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Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl 2 , purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays ϳ40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.Histone acetylation is a reversible dynamic process that occurs at specific lysine residues in the N termini of all the core histone proteins and has been correlated with several key biological processes, including nucleosome assembly and modulation of gene expression (1-4). The recent discoveries of the specific histone acetyltransferases (HATs), 1 Hat1p (5, 6) and Gcn5p (7), have directly linked histone acetylation with nucleosome assembly and transcriptional activation, respectively. Hat1p and Gcn5p are specific examples of the two general families of HATs, termed HAT-A and HAT-B. The HAT-A enzymes reside in the nucleus and acetylate the core histone N termini following their assembly into nucleosomes and chromatin. HAT-B type enzymes are primarily cytoplasmic and acetylate only free histones prior to nucleosome assembly (6), although Hat1p has recently been localized in the nucleus as well (8).Hat1p is the catalytic subunit of both the major cytoplasmic HAT-B complex and the nuclear HAT-A3 complex (8). Consistent with their roles in nucleosome assembly, recombinant Hat1p, the HAT-B complex, and the HAT A-3 complex all acetylate free histone H4 in vitro but do not acetylate H4 after its incorporation into nucleosomes (5, 6, 8, 9). However, seemingly at odds with its role as a HAT-A enzyme, Gcn5p has been reported to acetylate free histones H3 and H4 but not nucleosomal H3 or H4 (10 -13). This result has been reconciled by a model in which Gcn5p acetylates nucleosomal substrates only when a component of specific multiprotein complexes and is supported by the recent identification of several Gcn5-Ada complexes that are capable of acetylating histone H3 and to a lesser extent H4 in nucleosomes (12, 14 -16). Nevertheless, because all previous studies of rGcn5p activity were performed using a very...

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Section: Discussioncontrasting

confidence: 53%

Gcn5p, a Transcription-related Histone Acetyltransferase, Acetylates Nucleosomes and Folded Nucleosomal Arrays in the Absence of Other Protein Subunits

Tse

Georgieva

Ruiz-García

et al. 1998

Journal of Biological Chemistry

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“…This suggests that the Ada2/Ada3/Gcn5 subcomplex recapitulates SAGA's HAT function of acetylating nucleosomes. However, in contrast to SAGA which is apparently directed to the promoter through its Tra1 subunit, the Ada2/Ada3/Gcn5 complex (which may be identical to the HAT-A2 complex [10,11]) may act as a global, untargeted nucleosome acetyltransferase in the cell [12]. We have similarly identified the Piccolo NuA4 complex of Epl1, Yng2 and Esa1 as the catalytic core of the megadalton NuA4 complex [13,14].…”

Section: Introductionmentioning

confidence: 91%

Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes

Barrios

Selleck

Hnatkovich

et al. 2007

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Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates, we have identified the catalytic SAGA and NuA4 subcomplexes sufficient to act on nucleosomes. We describe here expression and purification procedures to prepare recombinant yeast Ada2/Ada3/Gcn5 subcomplex of SAGA which acetylates histones H3 and H2B on nucleosomes, and the Piccolo NuA4 complex which acetylates histones H4 and H2A on nucleosomes. We demonstrate an unexpected benefit of using the BL21-CodonPlus strain to enhance the purity of metal affinity purified Ada2/Ada3/Gcn5 complex. We also identify E. coli EF-Tu as a contaminant that copurifies with both complexes over multiple chromatographic steps and use of hydrophobic interaction chromatography to remove the contaminant from the Piccolo NuA4 complex. The methods described here will be useful for studies into the molecular mechanism of these enzymes and for preparing the enzymes as reagents to study the interplay of nucleosome acetylation with other chromatin modification and remodeling enzymes.

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“…Whole cell extracts were obtained by the salt dissociation/ultracentrifugation method previously described (11). After dialysis against buffer B (15 mM Tris-HCl, pH 7.9, 0.25 mM EDTA, 5 mM 2-mercaptoethanol, 0.05% (v/v) Tween 20, 10% (v/v) glycerol, 10 mM NaCl), cell extracts were made to 80 mM NaCl by addition of solid NaCl, centrifuged at 27,000 ϫ g for 10 min and used for chromatographic fractionation.…”

Section: Subcellular Fractionation and Preparation Of Extracts-yeastmentioning

confidence: 99%

“…GCN5 is a nuclear HAT-A enzyme that acetylates H3 and, to a lesser extent, H4 in their free forms but is unable to modify any nucleosome-bound histone (9,10). However, we have recently demonstrated that GCN5 is a member of a multisubunit complex possessing HAT activity that modifies H3 in nucleosomes (11). Moreover, it has also been reported that GCN5 functions in two multisubunit complexes that are able to acetylate nucleosomal histones.…”

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confidence: 93%

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HAT1 and HAT2 Proteins Are Components of a Yeast Nuclear Histone Acetyltransferase Enzyme Specific for Free Histone H4

Ruiz-García¹,

Sendra²,

Galiana³

et al. 1998

Journal of Biological Chemistry

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We have analyzed the histone acetyltransferase enzymes obtained from a series of yeast hat1, hat2, and gcn5 single mutants and hat1,hat2 and hat1,gcn5 double mutants. Extracts prepared from both hat1 and hat2 mutant strains specifically lack the following two histone acetyltransferase activities: the well known cytoplasmic type B enzyme and a free histone H4-specific histone acetyltransferase located in the nucleus. The catalytic subunits of both cytoplasmic and nuclear enzymes have identical molecular masses (42 kDa), the same as that of HAT1. However, the cytoplasmic complex has a molecular mass (150 kDa) greater than that of the nuclear complex (110 kDa). The possible functions of HAT1 and HAT2 in the yeast nucleus are discussed. In addition, we have detected a yeast histone acetyltransferase not previously described, designated HAT-A4. This enzyme is located in the nucleus and is able to acetylate free and nucleosome-bound histones H3 and H4. Finally, we show that the hat1,gcn5 double mutant is viable and does not exhibit a new phenotype, thus suggesting the existence of several histone acetyltransferases with overlapping functions.

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Gcn5p is involved in the acetylation of histone H3 in nucleosomes

Cited by 54 publications

References 27 publications

Gcn5p, a Transcription-related Histone Acetyltransferase, Acetylates Nucleosomes and Folded Nucleosomal Arrays in the Absence of Other Protein Subunits

Gcn5p, a Transcription-related Histone Acetyltransferase, Acetylates Nucleosomes and Folded Nucleosomal Arrays in the Absence of Other Protein Subunits

Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes

HAT1 and HAT2 Proteins Are Components of a Yeast Nuclear Histone Acetyltransferase Enzyme Specific for Free Histone H4

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