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

Barrios, Adam F.; Selleck, William; Hnatkovich, Brian; Kramer, Ryan M.; Sermwittayawong, Decha; Tan, Song

doi:10.1016/j.ymeth.2006.08.007

Cited by 32 publications

(37 citation statements)

References 20 publications

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“…Cloning, Recombinant Expression, and Purification of Full-length ADA (Gcn5/Ada2/Ada3) Subcomplex-The pST44-yAda3t2HISx3-yAda2 ϫ 3-yGcn5 ϫ 5 polycistronic vector containing yeast GCN5, ADA2, and ADA3, obtained courtesy of Dr. Song Tan (Penn State, PA), originally encoded a truncated form of Ada3 (missing N-terminal residues 1-184) (33). Full-length Ada3 was generated using nested primers to extend the truncated ADA3 fragment, which was cloned into the pST44 vector (supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3

Cieniewicz

Moreland

Ringel

et al. 2014

Molecular & Cellular Proteomics

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In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 Ϸ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wildtype HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167. Molecular &

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Section: Methodsmentioning

confidence: 99%

The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3

Cieniewicz

Moreland

Ringel

et al. 2014

Molecular & Cellular Proteomics

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“…Wild-type Esa1, Yng2(2-18), and hexahistidine-tagged Epl1(51-380) subunits of Piccolo NuA4 and appropriate mutational variants were expressed using the pST44 polycistronic expression vector in BL21(DE3)pLysS cells as described previously (17,23). The coexpressed complexes were purified by Talon (Clontech) cobalt affinity and SourceQ anion-exchange chromatography (24), and the engineered mutations did not adversely affect Piccolo NuA4 complex stability.…”

Section: Methodsmentioning

confidence: 99%

Piccolo NuA4-Catalyzed Acetylation of Nucleosomal Histones: Critical Roles of an Esa1 Tudor/Chromo Barrel Loop and an Epl1 Enhancer of Polycomb A (EPcA) Basic Region

Huang

Tan

2013

Molecular and Cellular Biology

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Piccolo NuA4 is an essential yeast histone acetyltransferase (HAT) complex that targets histones H4 and H2A in nucleosome substrates. While Piccolo NuA4's catalytic subunit Esa1 alone is unable to acetylate nucleosomal histones, its accessory subunits, Yng2 and Epl1, enable Esa1 to bind to and to act on nucleosomes. We previously determined that the Tudor domain of Esa1 and the EPcA homology domain of Epl1 play critical roles in Piccolo NuA4's ability to act on the nucleosome. In this work, we pinpoint a loop within the Esa1 Tudor domain and a short basic region at the N terminus of the Epl1 EPcA domain as necessary for this nucleosomal HAT activity. We also show that this Esa1 Tudor domain loop region is positioned close to nucleosomal DNA and that the Epl1 EPcA basic region is in proximity to the N-terminal histone H2A tail, the globular region of histone H4, and also to nucleosomal DNA when Piccolo NuA4 interacts with the nucleosome. Since neither region identified is required for Piccolo NuA4 to bind to nucleosomes and yet both are needed to acetylate nucleosomes, these regions may function after the enzyme binds nucleosomes to disengage substrate histone tails from nucleosomal DNA.

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“…The Ada2-Ada3-Gcn5 and Ada2-Ada3-Gcn5-Y413A subcomplexes were expressed and purified as described (22). Protein concentrations were determined by comparison with known amounts of recombinant Gcn5 on 15% SDS-polyacrylamide gel, stained with Coomassie Blue.…”

Section: Methodsmentioning

confidence: 99%

The Gcn5 Bromodomain of the SAGA Complex Facilitates Cooperative and Cross-tail Acetylation of Nucleosomes

Shogren-Knaak

2009

Journal of Biological Chemistry

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Bromodomains are acetyl lysine binding modules found in many complexes that regulate gene transcription. In budding yeast, the coactivator complex SAGA (Spt-Ada-Gcn5-acetyltransferase) predominantly facilitates transcription of stressactivated genes and requires the bromodomain of the Gcn5 subunit for full activation of a number of these genes. This bromodomain has previously been shown to promote retention of the complex to H3 and H4 acetylated nucleosomes. Because the SAGA complex mediates histone H3 acetylation, we sought to determine to what extent the Gcn5 bromodomain directly modulates histone acetylation activity. Kinetic analysis of SAGAmediated acetylation of nucleosomal substrates reveals that this bromodomain: 1) is required for the cooperative acetylation of nucleosomes, 2) enhances acetylation of an H3 histone tail when the other H3 tail within a nucleosome is already acetylated, and 3) augments the acetylation turnover of nucleosomes previously acetylated at lysine 16 of the histone H4 tails. These results indicate that the Gcn5 bromodomain promotes the establishment of nucleosome acetylation through multiple mechanisms and more generally show how chromatin recognition domains can modulate the enzymatic activity of chromatin modifying complexes.

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

Cited by 32 publications

References 20 publications

The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3

The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3

Piccolo NuA4-Catalyzed Acetylation of Nucleosomal Histones: Critical Roles of an Esa1 Tudor/Chromo Barrel Loop and an Epl1 Enhancer of Polycomb A (EPcA) Basic Region

The Gcn5 Bromodomain of the SAGA Complex Facilitates Cooperative and Cross-tail Acetylation of Nucleosomes

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