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.
While we possess a good understanding of how transcription factors bind to their DNA targets, we lack equivalent biochemical or structural insight into how chromatin factors bind to their nucleosome substrate. To address this deficiency, we are studying how transcription factors and chromatin enzymes interact with nucleosomes. Our studies of histone acetyltransferase chromatin modification enzymes shows that although the yeast histone Gcn5 and Esa1 acetyltransferase subunits can modify histone substrates, neither acetylates the physiological nucleosome substrate efficiently. In contrast, the parent megadalton, multicomponent SAGA and NuA4 complexes possess strong nucleosome acetyltransferase activity. We have identified minimal catalytic SAGA and NuA4 subcomplexes sufficient and necessary to acetylate nucleosomes. Deletion analysis of the catalytic NuA4 subcomplex, Piccolo NuA4 (comprised of the Epl1, Yng2 and Esa1 subunits) identifies the Epl1 EPcA homology region and the Esa1 chromodomain as critical for Piccolo's ability to specifically act on nucleosomes.
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