Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries.
Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries.
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DOI：
10.1038/nmeth.3022
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发表时间：
2014-08
期刊：
影响因子：
48
通讯作者：
Muir, Tom W.
中科院分区：
文献类型：
--
作者：
Nguyen, Uyen T. T.;Bittoval, Lenka;Mueller, Manuel M.;Fierz, Beat;David, Yael;Houck-Loomis, Brian;Feng, Vanessa;Dann, Geoffrey P.;Muir, Tom W.
阐明染色质相关因子如何沉积、移除和识别组蛋白翻译后修饰(‘PTM’)信号的分子细节在表观遗传学领域仍然是一项艰巨的任务。在这里，我们介绍了一个通用的平台，它极大地加速了染色质识别和信号的生化研究。这项技术是基于DNA条形码核小体文库的简化半合成和不同的PTM组合。用纯化的染色质效应物或结合核蛋白的染色质识别和修饰活性处理的这些文库进行染色质免疫沉淀，然后进行多重DNA条形码测序。这一超灵敏的工作流程使我们能够收集数千个生化数据点，揭示各种核因素对PTM模式的结合偏好，以及预先存在的PTM如何单独或协同通过串扰机制影响进一步的PTM沉积。我们预计，该技术的高通量和高灵敏度将有助于加速解密在染色质水平上操作的各种分子控制。
Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.
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作者：
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通讯作者：
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