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

Michael A. Shogren-Knaak,Shanshan Li

doi:10.1074/jbc.m809617200

Abstract

Bromodomains are acetyl lysine binding modules found in many complexes that regulate gene transcription. In budding yeast, the coactivator complex SAGA (Spt-Ada-Gcn5-acetyl-transferase) predominantly facilitates transcription of stress-activated 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 SAGA-mediated 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.

Highlights

Kinetic constants are the average of three independent trials, except for some of the experiments with SAGA Gcn5-Y413A, which were the average of two trials
Initial velocity analysis of SAGA-mediated acetylation of nucleosomal arrays containing either unacetylated or H4 Lys-16 acetylated histones revealed that overall preacetylation reduced the acetylation half-saturation substrate concentration 1.7-fold and increased the apparent turnover rate constant by 1.86-fold (Fig. 4A). These results suggests that the presence of H4 Lys-16 acetylation augments nucleosome acetylation but do not indicate how because H4 Lys-16 acetylation could either facilitate Gcn[5] bromodomain binding (14) or disrupt higher-order chromatin structure (19)
Our results show that in the SAGA complex, the bromodomain of Gcn[5] promotes the establishment of nucleosomal suggests that SAGA subunits outside of the core subcomplex may play a role in promoting enzyme cooperativity

Summary

EXPERIMENTAL PROCEDURES

DNA, Octamer, and Nucleosome Preparation—Recombinant Xenopus laevis histones were expressed, purified, and characterized according to standard methods (18). Assays were performed at 30 °C in HAT buffer (50 mM Tris, pH 7.5, 5% glycerol, 0.125 mM EDTA, 50 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium butyrate) with various concentrations of nucleosomal arrays or mononucleosomes, 4.0 ␮M acetyl-CoA (1.25 ␮Ci/␮M [3H]acetyl-CoA), and either 1.0 nM SAGA or 2.5 nM Ada2-Ada3-Gcn[5] subcomplex. In addition to the gel-based quantification of the enzymes described above, equivalence of activity for WT and mutated enzymes was confirmed by acetylation assay In these assays, for equal amounts of enzyme, the extent of acetylation after 3 min for 300 nM nucleosome was determined. Characterization of Acetylation Products—The histone specificity of SAGA-mediated acetylation was determined in assays with 0.12 ␮M nucleosomes (1 ␮g of total histone), 0.83 ␮M acetyl-CoA (1.20 ␮Ci/␮M [3H]acetyl-CoA), and 2.0 nM SAGA complex in HAT buffer at 30 °C for 40 min.

RESULTS

DISCUSSION

Methods