Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes
Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes
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DOI：
10.1371/journal.pone.0057018
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发表时间：
2013-04-18
期刊：
影响因子：
3.7
通讯作者：
Langowski, Joerg
中科院分区：
文献类型：
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作者：
Gansen, Alexander;Hieb, Aaron R.;Langowski, Joerg
核小体的结构和稳定性通过改变局部染色质形态影响遗传可及性。最近的FRET核小体实验提供了宝贵的洞察到它们可以采用的结构转换。然而，即使在看似相同的条件下进行，由于每种技术的局限性，在批量和单分子水平上进行的实验也给出了混合的答案。然而，为了比较这些实验，它们必须在相同的条件下进行。在这里，我们开发了一个实验框架，克服了传统的限制，每种方法：单分子FRET实验进行批量浓度通过添加未标记的核小体，而批量FRET实验进行微孔板中的浓度接近用于单分子检测。此外，微孔板可以同时探测许多条件，然后再花费宝贵的仪器时间进行单分子实验。我们通过探索组蛋白H3的选择性乙酰化对核小体结构和稳定性的作用来突出这种实验策略;在散装中，H3-乙酰化的核小体比非乙酰化的核小体显着不稳定。单分子FRET分析进一步揭示，组蛋白H3的乙酰化促进了额外构象状态的形成，该构象状态在较高的核小体浓度下被抑制，并且可能是核小体调节中的重要结构中间体。
Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.