A Chemical Genetic Method for Monitoring Genome-Wide Dynamics of O-GlcNAc Turnover on Chromatin-Associated Proteins
A Chemical Genetic Method for Monitoring Genome-Wide Dynamics of O-GlcNAc Turnover on Chromatin-Associated Proteins
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DOI：
10.1021/acscentsci.9b00044
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发表时间：
2019-04-24
影响因子：
18.2
通讯作者：
Vocadlo, David J.
中科院分区：
文献类型：
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作者：
Liu, Ta-Wei;Myschyshyn, Mike;Vocadlo, David J.
DNA测序的进步使研究染色质的新的实验模式成为可能。一个新兴的领域是使用高通量DNA测序来监测染色质发生的动态变化。O-连接N-乙酰氨基葡萄糖(O-GlcNAc)是一种在许多染色质相关蛋白上发现的可逆蛋白质修饰。O-GlcNAc调控基因转录的机制引起了人们极大的兴趣。在这里，我们使用DNA沉淀法来监控与染色质相关的O-GlcNAc修饰蛋白的随时间变化的情况。利用一种无抗体的化学报告策略将O-GlcNAc定位到基因组，我们对野生型果蝇幼虫和缺乏O-GlcNAc水解酶(OGA)的幼虫进行了时程代谢喂养实验，这些幼虫因此无法清除OGlcNAc。对得到的下一代DNA测序数据的分析表明，在大多数基因组基因座上，染色质相关蛋白上的O-GlcNAc的半衰期为几个小时。值得注意的是，失去OGA只会使这个半衰期增加大约3倍。有趣的是，有一小部分基因组座位对OGA的丢失特别敏感。除了这些观察结果和允许监测O-GlcNAc在染色质上周转的新策略外，我们还详细介绍了样本编码盲化的方法以及新的归一化策略，以实现使用化学遗传学方法进行时间分辨的全基因组分析。我们设想这些通用的方法将适用于不同的蛋白质和核酸修饰。
Advances in DNA sequencing are enabling new experimental modalities for studying chromatin. One emerging area is to use high-throughput DNA sequencing to monitor dynamic changes occurring to chromatin. O-Linked N-acetylglucosamine (O-GlcNAc) is a reversible protein modification found on many chromatin-associated proteins. The mechanisms by which O-GlcNAc regulates gene transcription are of high interest. Here we use DNA precipitation methods to enable monitoring time-dependent turnover of O-GlcNAc modified proteins associated with chromatin. Using an antibody-free chemical reporter strategy to map O-GlcNAc to the genome, we performed time course metabolic feeding experiments with wild-type Drosophila larvae alongside larvae lacking O-GlcNAc hydrolase (OGA), which are accordingly unable to remove OGlcNAc. Analysis of resulting next-generation DNA sequencing data revealed that O-GlcNAc on chromatin-associated proteins at most genomic loci is processed with a half-life in hours. Notably, loss of OGA only increases this half-life by similar to 3-fold. Interestingly, a small set of genomic loci are particularly sensitive to loss of OGA. In addition to these observations and new strategies to permit monitoring turnover of O-GlcNAc on chromatin, we also detail methods for coded blinding of samples alongside new normalization strategies to enable time-resolved, genome-wide analyses using chemical genetic methods. We envision these general methods will be applicable to diverse protein and nucleic acid modifications.