Abstract
The Spt-Ada-Gcn5 acetyltransferase (SAGA) family of transcriptional coactivators are prototypical nucleosome acetyltransferase complexes that regulate multiple steps in gene transcription. The size and complexity of both the SAGA enzyme and the chromatin substrate provide numerous opportunities for regulating the acetylation process. To better probe this regulation, here we developed a bead-based nucleosome acetylation assay to characterize the binding interactions and kinetics of acetylation with different nucleosomal substrates and the full SAGA complex purified from budding yeast (Saccharomyces cerevisiae). We found that SAGA-mediated nucleosome acetylation is stimulated up to 9-fold by DNA flanking the nucleosome, both by facilitating the binding of SAGA and by accelerating acetylation turnover. This stimulation required that flanking DNA is present on both sides of the nucleosome and that one side is >15 bp long. The Gal4-VP16 transcriptional activator fusion protein could also augment nucleosome acetylation up to 5-fold. However, contrary to our expectations, this stimulation did not appear to occur by stabilizing the binding of SAGA toward nucleosomes containing an activator-binding site. Instead, increased acetylation turnover by SAGA stimulated nucleosome acetylation. These results suggest that the Gal4-VP16 transcriptional activator directly stimulates acetylation via a dual interaction with both flanking DNA and SAGA. Altogether, these findings uncover several critical mechanisms of SAGA regulation by chromatin substrates.
Highlights
The Spt-Ada-Gcn5 acetyltransferase (SAGA) family of transcriptional coactivators are prototypical nucleosome acetyltransferase complexes that regulate multiple steps in gene transcription
To better understand the requirements and mechanism of activator-mediated stimulation of SAGA acetylation, we previously developed a nucleosome acetylation assay that utilized chromatin model systems assembled from individual nucleosomes and purified endogenous SAGA complex from budding yeast [36]
As well as to characterize the effect of activator proteins, here we develop several new assays for characterizing nucleosome acetylation and binding, and we apply them to studying the role of flanking DNA and activator on SAGA-mediated nucleosome acetylation
Summary
To characterize how different factors affect the mechanism of SAGA-mediated nucleosome acetylation, we developed a beadbased, initial rate, steady-state nucleosome acetylation assay that extends our previously described acetylation sequencing assay [36]. To perform the initial rate, steady-state assays, tritium-labeled acetyl-CoA, and purified native yeast SAGA complex were added to the bead-bound GBY nucleosomes, and aliquots of the reaction were removed at specific time points (Fig. 1A). These aliquots were washed to remove unincorporated acetyl-CoA and counted by liquid scintillation. To characterize the mechanism by which flanking DNA stimulates nucleosome acetylation, we performed initial rate, steady-state kinetic analysis on the 147-bp nucleosome and compared the results with that of the GBY nucleosome (Fig. 2B). Utilizing recombinant Gal4 –VP16 and the GBY DNA template used to make GBY nucleosomes, we showed that increasing amounts of activator
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