Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins.
Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins.
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DOI：
10.1038/nchem.2224
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发表时间：
2015-05
期刊：
影响因子：
21.8
通讯作者：
Muir TW
中科院分区：
文献类型：
--
作者：
David Y;Vila-Perelló M;Verma S;Muir TW
染色质中组蛋白的翻译后修饰在真核细胞DNA模板化过程的表观遗传控制中起着核心作用。因此，开发能够操纵组蛋白结构的方法对于理解基因组调控背后的生化机制是至关重要的。在这里，我们提出了一种合成生物学方法，通过蛋白质反式剪接来设计在细胞染色质上具有位置特异性修饰的组蛋白。我们在基因上将超快分裂内含子的N-末端片段与组蛋白H_2B的C-末端融合，组蛋白H_2B与互补的合成C-内含子反应生成标记的组蛋白。使用这种方法，我们结合了体内染色质的各种非天然化学修饰和时间控制。此外，在核中进行蛋白质反式剪接的时间和浓度依赖性使我们能够检查表观基因组常染色质和异染色质区域可及性的差异。最后，我们利用蛋白质反式剪接在分离的细胞核中半合成了一种天然的组蛋白修饰，即H2BK120泛素化，并表明这可以触发H3K79甲基化的下游表观遗传串扰。
Post-translational modification of the histone proteins in chromatin plays a central role in epigenetic control of DNA-templated processes in eukaryotic cells. Developing methods that enable the structure of histones to be manipulated is therefore essential to understand the biochemical mechanisms underlying genomic regulation. Here we present a synthetic biology method to engineer histones bearing site-specific modifications on cellular chromatin using protein trans-splicing. We genetically fused the N-terminal fragment of ultrafast split-intein to the C-terminus of histone H2B, which upon reaction with a complementary synthetic C-intein, generated labeled histone. Using this approach, we incorporated various non-native chemical modifications to chromatin in vivo with temporal control. Furthermore, the time and concentration dependence of protein trans-splicing performed in nucleo enabled us to examine differences in the accessibility of the euchromatin and heterochromatin regions of the epigenome. Finally, we used protein trans-splicing to semi-synthesize a native histone modification, H2BK120 ubiquitination, in isolated nuclei, and show that this can trigger downstream epigenetic cross-talk of H3K79 methylation.