Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module.
Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module.
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DOI：
10.1002/anie.201600638
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发表时间：
2016-04-11
期刊：
影响因子：
--
通讯作者：
Brik A
中科院分区：
文献类型：
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作者：
Jbara M;Maity SK;Morgan M;Wolberger C;Brik A
组蛋白H2B的单泛素化在转录激活中起核心作用，并且是下游组蛋白甲基化事件所必需的。Spt-Ada-Gcn5乙酰转移酶（SAGA）共激活因子复合物对H2B的去泛素化作用受到最近发现的组蛋白标记磷酸化H2AY57 （H2AY57p）的调控，该标记抑制SAGA复合物对H2B的去泛素化作用，并限制H3的去甲基化并增加其乙酰化作用。然而，H2AY57p作用的证据是间接的，并且通过监测Tyr激酶CK2的化学抑制作用或通过突变磷酸化位点在体内进行了研究。我们采用全化学合成的方法高效制备了H2AY57p，并研究了其调控的分子细节。该类似物与半合成制备的泛素化H2B使我们能够直接证明这两个标记之间的串扰以及H2AY57p对SAGA活性的抑制。蛋白质的全化学合成使得通过一锅合成高效地制备Tyr57位点均匀磷酸化的H2A。磷酸化的H2A和泛素化的H2B与H3和H4组装成核小体，为抑制已知从H2B中切割泛素的SAGA复合物的活性提供了直接证据。
Monoubiquitination of histone H2B plays a central role in transcription activation and is required for downstream histone methylation events. Deubiquitination of H2B by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex is regulated by a recently discovered histone mark, phosphorylated H2AY57 (H2AY57p), which inhibits deubiquitination of H2B by the SAGA complex as well as restricting demethylation of H3 and increasing its acetylation. Evidence for the effect of H2AY57p however, was indirect and was investigated in vivo by monitoring the effects of chemical inhibition of Tyr kinase CK2 or by mutating the phosphorylation site. We applied total chemical synthesis of proteins to prepare H2AY57p efficiently and study the molecular details of this regulation. This analogue, together with the semisynthetically prepared ubiquitinated H2B enabled us to provide direct evidence on the cross talk between those two marks and the inhibition of SAGA activity by H2AY57p. Total chemical synthesis of proteins enabled the efficient preparation of homogeneously phosphorylated H2A at Tyr57 via one-pot synthesis. The assembly of phosphorylated H2A and ubiquitinated H2B with H3 and H4 into nucleosome, provided direct evidence for the inhibition of the activity of SAGA complex known to cleave ubiquitin from H2B.