German Cancer Research Center, Division Biophysics of Macromolecules, Heidelberg, 69120, Germany.
Heidelberg University, BioQuant and IPMB, Biomedical Computer Vision Group, Heidelberg, 69120, Germany.
Sci Rep. 2019 Sep 4;9(1):12788. doi: 10.1038/s41598-019-49163-4.
DNA compaction and accessibility in eukaryotes are governed by nucleosomes and orchestrated through interactions between DNA and DNA-binding proteins. Using QuantAFM, a method for automated image analysis of atomic force microscopy (AFM) data, we performed a detailed statistical analysis of structural properties of mono-nucleosomes. QuantAFM allows fast analysis of AFM images, including image preprocessing, object segmentation, and quantification of different structural parameters to assess DNA accessibility of nucleosomes. A comparison of nucleosomes reconstituted with and without linker histone H1 quantified H1's already described ability of compacting the nucleosome. We further employed nucleosomes bearing two charge-modifying mutations at position R81 and R88 in histone H2A (H2A R81E/R88E) to characterize DNA accessibility under destabilizing conditions. Upon H2A mutation, even in presence of H1, the DNA opening angle at the entry/exit site was increased and the DNA wrapping length around the histone core was reduced. Interestingly, a distinct opening of the less bendable DNA side was observed upon H2A mutation, indicating an enhancement of the intrinsic asymmetry of the Widom-601 nucleosomes. This study validates AFM as a technique to investigate structural parameters of nucleosomes and highlights how the DNA sequence, together with nucleosome modifications, can influence the DNA accessibility.
真核生物中的 DNA 压缩和可及性受核小体调控，并通过 DNA 与 DNA 结合蛋白之间的相互作用进行协调。我们使用 QuantAFM（一种自动分析原子力显微镜 (AFM) 数据的方法），对单核小体的结构特性进行了详细的统计分析。QuantAFM 允许快速分析 AFM 图像，包括图像预处理、对象分割和不同结构参数的定量，以评估核小体的 DNA 可及性。我们比较了含有和不含有连接组蛋白 H1 的核小体，定量了 H1 已经描述的压缩核小体的能力。我们进一步使用在组蛋白 H2A 的位置 R81 和 R88 处带有两个电荷修饰突变的核小体 (H2A R81E/R88E)，在不稳定条件下表征 DNA 的可及性。在 H2A 突变后，即使存在 H1，入口/出口处的 DNA 开口角度也增加了，并且围绕组蛋白核心的 DNA 缠绕长度减少了。有趣的是，在 H2A 突变后观察到 DNA 较不易弯曲侧的明显开口，表明 Widom-601 核小体的固有不对称性增强。这项研究验证了 AFM 作为一种研究核小体结构参数的技术，并强调了 DNA 序列以及核小体修饰如何影响 DNA 可及性。
