The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3
The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3
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DOI：
10.1074/mcp.m114.038174
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发表时间：
2014-11-01
影响因子：
7
通讯作者：
Taverna, Sean D.
中科院分区：
文献类型：
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作者：
Cieniewicz, Anne M.;Moreland, Linley;Taverna, Sean D.
在酵母中，保守的组蛋白乙酰转移酶（HAT）Gcn 5与Ada 2和Ada 3结合形成ADA和佐贺转录共激活因子复合物的催化模块。Gcn 5还含有乙酰基-赖氨酸结合布罗莫结构域，其已经涉及在体外调节核小体乙酰化，以及在细胞中的基因启动子。然而，Gcn 5布罗莫结构域在调节HAT活性的位点特异性中的贡献仍不清楚。在这里，我们使用酸-尿素凝胶和定量质谱相结合的方法来比较野生型和Gcn 5溴结构域突变ADA亚复合物（Gcn 5-Ada 2-Ada 3）的HAT活性。野生型ADA亚复合物乙酰化H3赖氨酸具有以下特异性：H3 K14> H3 K23> H3 K9，近似于H3 K18> H3 K27> H3 K36。然而，当Gcn 5布罗莫结构域在乙酰基-赖氨酸结合中有缺陷时，ADA亚复合物在游离和核小体H3上表现出改变的位点特异性乙酰化，其中H3 K18 ac减少得最严重。在野生型HAT反应中，H3 K18 ac在H3 K14 R而非H3 K23 R底物上也严重减少，进一步表明Gcn 5催化的H3 K14乙酰化和溴结构域结合H3 K14 ac是H3 K18 ac之前的重要步骤。总之，这项工作详细说明了Gcn 5布罗莫结构域阅读器功能和酶促HAT活性之间的先前未表征的串扰，其可能最终影响基因表达。溴结构域或其他组蛋白翻译后修饰阅读器中的突变如何影响染色质模板化酶活性的未来研究将对潜在的组蛋白/表观遗传密码产生前所未有的洞察力。MS数据可通过ProteomeXchange获得，标识符为PXD 001167。
In yeast, the 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 approximate to 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. 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. 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. MS data are available via ProteomeXchange with identifier PXD001167.