CHARACTERIZATION OF GCN5 HISTONE ACETYLTRANSFERASE ACTIVITY AND BROMODOMAIN ACETYL-LYSINE BINDING FUNCTION
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Date
2015-02-12
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Johns Hopkins University
Abstract
In eukaryotes, DNA is packaged into chromatin, which consists of the DNA wrapped around an octameric core of histone proteins (H2A, H2B, H3, and H4) which collectively comprise the nucleosome. Gene activation and silencing determined by the access of transcriptional machinery to DNA can be dictated in part by dynamic post-translational modifications (PTMs) on histone proteins. These PTMs can be added, removed, and interpreted by chromatin effector complexes that interact with chromatin. In yeast, the highly 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 ≈ 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 wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. Consistent with our in vitro results, when the Gcn5 bromodomain was impaired in vivo we observed a global decrease in H3K18 acetylation.
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. Through our development of in vitro assays that contain selectively modified histones and intact epigenetic complexes and modules, we can gain biologically relevant insight into the activity and chromatin interactions of effector protein complexes. 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."
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Epigenetics, histone, acetylation, Gcn5, bromodomain, histone acetyltransferase