Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes.
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 Escherichia 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.
Barrios
Adam
A
Center for Gene Regulation, Department of Biochemistry and Molecular Biology, 108 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802-1014, USA.
Selleck
William
W
Hnatkovich
Brian
B
Kramer
Ryan
R
Sermwittayawong
Decha
D
Tan
Song
S
D000107:Acetylation; D042002:Chromatin Assembly and Disassembly / Q000502:physiology; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D051548:Histone Acetyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics; D000099224:p300-CBP-Associated Factor
D051548:Histone Acetyltransferases; D006657:Histones; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C090575:ADA2 protein, S cerevisiae; C083323:NGG1 protein, S cerevisiae; C501392:GCN5 protein, S cerevisiae; D000099224:p300-CBP-Associated Factor; C504740:NuA4 protein, S cerevisiae