School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853 USA.
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 USA.
Nucleic Acids Res. 2018 Jun 1;46(10):4978-4990. doi: 10.1093/nar/gky206.
Chromatin remodelers are ATP-dependent motors that reorganize DNA packaging by disrupting canonical histone-DNA contacts within the nucleosome. Here, we show that the Chd1 chromatin remodeler stimulates DNA unwrapping from the edge of the nucleosome in a nucleotide-dependent and DNA sequence-sensitive fashion. Nucleosome binding, monitored by stopped flow, was complex and sensitive to nucleotide, with AMP-PNP promoting faster binding than ADP·BeF3-. Nucleosome unwrapping by Chd1, examined by bulk FRET, occurred in the presence and absence of nucleotide and did not require the Chd1 DNA-binding domain. In AMP-PNP conditions, Chd1 unwrapped one side of the Widom 601 DNA more easily than the other, consistent with previous observations of 601 asymmetry and indicating that Chd1 amplifies intrinsic sequence properties of nucleosomal DNA. Using small angle X-ray scattering (SAXS) with contrast variation, we found distinct DNA conformations depending on the nucleotide analog bound to Chd1: with AMP-PNP, DNA primarily unwrapped in-plane with the nucleosomal disk, whereas with ADP·BeF3-, a significant fraction showed distinctive out-of-plane unwrapping as well. Taken together, our findings show tight coupling between entry/exit DNA of the nucleosome and the Chd1 ATPase motor, suggesting that dynamic nucleosome unwrapping is coupled to nucleosome binding and remodeling by Chd1.
染色质重塑因子是一类 ATP 依赖性的马达蛋白，通过破坏核小体中经典组蛋白-DNA 接触来重新组织 DNA 包装。在这里，我们展示了 Chd1 染色质重塑因子以核苷酸依赖性和 DNA 序列敏感性的方式刺激核小体边缘的 DNA 解缠绕。通过停流监测到的核小体结合非常复杂且对核苷酸敏感，AMP-PNP 比 ADP·BeF3- 促进更快的结合。通过 bulk FRET 检测到 Chd1 对核小体的解缠绕在有无核苷酸的情况下都能发生，并且不需要 Chd1 的 DNA 结合结构域。在 AMP-PNP 条件下，Chd1 更容易解开 Widom 601 DNA 的一侧，而不是另一侧，这与 601 不对称性的先前观察结果一致，并表明 Chd1 放大了核小体 DNA 的固有序列特性。使用带有对比度变化的小角度 X 射线散射 (SAXS)，我们发现结合到 Chd1 上的不同核苷酸类似物会导致不同的 DNA 构象：与 AMP-PNP 结合时，DNA 主要在核小体盘内解缠绕，而与 ADP·BeF3- 结合时，相当一部分 DNA 显示出明显的出平面解缠绕。综上所述，我们的研究结果表明核小体进出 DNA 与 Chd1 ATP 酶马达之间存在紧密的偶联，表明动态核小体解缠绕与 Chd1 结合和重塑相偶联。
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