DNP-enhanced solid-state NMR spectroscopy of chromatin polymers
DNP-enhanced solid-state NMR spectroscopy of chromatin polymers
复制标题
DOI：
10.1016/j.jmro.2022.100057
复制
发表时间：
2022-06-01
影响因子：
--
通讯作者：
Debelouchina, Galia T.
中科院分区：
文献类型：
--
作者：
Elathram, Nesreen;Ackermann, Bryce E.;Debelouchina, Galia T.
染色质是一种DNA-蛋白质聚合物，代表基因组的功能形式。染色质的主要构件是核小体，核小体结构包含147个碱基对的DNA和组蛋白H_2A、H_2B、H_3和H_4各两个副本。以前的工作表明魔角旋转(MAS)核磁共振光谱可以高分辨率地捕捉核小体，尽管由于低灵敏度、动态和刚性成分的存在以及染色质聚合物中核小体复杂的相互作用网络的存在，研究一直是具有挑战性的。在这里，我们使用动态核极化(DNP)来提高100K下核小体阵列的MAS核磁共振实验的灵敏度，并表明可以更有效地获得分辨率更好的13C-13C MAS核磁共振关联。我们评估了温度对光谱中化学位移和线宽的影响，并证明了变化相对最小，并且聚集在组蛋白DNA或组蛋白-组蛋白接触的区域。我们还比较了用和不用DNA制备的样品，表明低温13C-13C关联显示了足够的分辨率来检测形成DNA-组蛋白界面的残基的化学位移变化和线宽。另一方面，我们证明了DNP增强的核小体核心内15N-13C组蛋白-组蛋白相互作用的测量因样品中自然的13C丰度网络而变得复杂。然而，DNP提供的增强的灵敏度可以用于检测组蛋白残基和DNA之间的远程相关性。总体而言，我们的实验表明，染色质样品的DNP增强的MAS核磁共振谱产生了高分辨率和高灵敏度的谱，并可用于捕获功能相关的蛋白质-DNA相互作用，这些相互作用对基因调控和基因组组织具有重要意义。
Chromatin is a DNA-protein polymer that represents the functional form of the genome. The main building block of chromatin is the nucleosome, a structure that contains 147 base pairs of DNA and two copies each of the histone proteins H2A, H2B, H3 and H4. Previous work has shown that magic angle spinning (MAS) NMR spectroscopy can capture the nucleosome at high resolution although studies have been challenging due to low sensitivity, the presence of dynamic and rigid components, and the complex interaction networks of nucleosomes within the chromatin polymer. Here, we use dynamic nuclear polarization (DNP) to enhance the sensitivity of MAS NMR experiments of nucleosome arrays at 100 K and show that well-resolved 13C -13C MAS NMR correlations can be obtained much more efficiently. We evaluate the effect of temperature on the chemical shifts and linewidths in the spectra and demonstrate that changes are relatively minimal and clustered in regions of histoneDNA or histone-histone contacts. We also compare samples prepared with and without DNA and show that the low temperature 13C -13C correlations exhibit sufficient resolution to detect chemical shift changes and line broadening for residues that form the DNA-histone interface. On the other hand, we show that the measurement of DNP-enhanced 15N -13C histone-histone interactions within the nucleosome core is complicated by the natural 13C abundance network in the sample. Nevertheless, the enhanced sensitivity afforded by DNP can be used to detect long-range correlations between histone residues and DNA. Overall, our experiments demonstrate that DNP-enhanced MAS NMR spectroscopy of chromatin samples yields spectra with high resolution and sensitivity and can be used to capture functionally relevant protein-DNA interactions that have implications for gene regulation and genome organization.