Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity.
Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity.
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DOI：
10.1093/nar/gks1247
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发表时间：
2013-02-01
影响因子：
14.9
通讯作者：
Cheung P
中科院分区：
文献类型：
--
作者：
Lau PN;Cheung P
组蛋白翻译后修饰（PTM）通常形成复杂的组合模式，并协同指定下游生物过程。为了系统地分析组合PTM和组蛋白PTM之间的串扰，我们开发了一种新的核小体纯化方法，称为生物素化辅助分离CO修饰的核小体（BICON）。该技术基于组蛋白修饰酶的酶活性与生物素连接酶BirA的体内生物素化的物理偶联，并使用链霉亲和素纯化共修饰的核小体。通过BICON分离的核小体的分析允许鉴定在修饰的核小体上富集并在核小体环境中一起起作用的PTM组合。我们使用这种新的方法来研究MSK1介导的H3磷酸化，发现MSK1不仅直接磷酸化H3，而且还诱导核小体内组蛋白H3和H4的超乙酰化。此外，我们确定了一个新的串扰之间的H3磷酸化和H4乙酰化K12的途径。通过染色质免疫沉淀试验进一步证实了这些乙酰标记参与MSK 1介导的转录，从而验证了BICON结果的生物学相关性。这些研究作为这种新技术方法的原理证明，并证明BICON可以进一步适用于研究与其他组蛋白修饰酶相关的PTM和串扰。
Histone post-translational modifications (PTMs) often form complex patterns of combinations and cooperate to specify downstream biological processes. In order to systemically analyse combinatorial PTMs and crosstalks among histone PTMs, we have developed a novel nucleosome purification method called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique is based on physical coupling of the enzymatic activity of a histone-modifying enzyme with in vivo biotinylation by the biotin ligase BirA, and using streptavidin to purify the co-modified nucleosomes. Analysing the nucleosomes isolated by BICON allows the identification of PTM combinations that are enriched on the modified nucleosomes and function together within the nucleosome context. We used this new approach to study MSK1-mediated H3 phosphorylation and found that MSK1 not only directly phosphorylated H3, but also induced hyperacetylation of both histone H3 and H4 within the nucleosome. Moreover, we identified a novel crosstalk pathway between H3 phosphorylation and H4 acetylation on K12. Involvement of these acetyl marks in MSK1-mediated transcription was further confirmed by chromatin immunoprecipitation assays, thus validating the biological relevance of the BICON results. These studies serve as proof-of-principle for this new technical approach, and demonstrate that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes.
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影响因子：
16
作者：
Lo, WS;Trievel, RC;Berger, SL
通讯作者：
Berger, SL
DOI：
10.1073/pnas.1332608100
发表时间：
2003-06-24
影响因子：
11.1
作者：
de Boer, E;Rodriguez, P;Strouboulis, J
通讯作者：
Strouboulis, J
影响因子：
14.9
作者：
Drobic B;Pérez-Cadahía B;Yu J;Kung SK;Davie JR
通讯作者：
Davie JR
影响因子：
9.2
作者：
Fuchs, Stephen M.;Krajewski, Krzysztof;Baker, Richard W.;Miller, Victoria L.;Strahl, Brian D.
通讯作者：
Strahl, Brian D.
影响因子：
64.8
作者：
Sun, ZW;Allis, CD
通讯作者：
Allis, CD