Genome-wide profiling of nucleosome sensitivity and chromatin accessibility to MNase in D. melanogaster [RNA-seq]

Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect.

核小体（nucleosome）的结构与定位在真核细胞的基因调控、DNA修复及其他关键生命过程中发挥核心作用。此前学界普遍认为，核小体结合的DNA对DNA结合因子与加工因子（如微球菌核酸酶（MNase））均呈完全不可接近的状态。但本研究证实，核小体的可及性以及对MNase的敏感性并非均一，而是存在差异。我们使用两种不同浓度的MNase消化果蝇染色质，发现了两类核小体：敏感型与抗性型。MNase抗性型核小体阵列对低浓度MNase的可及性更低，而敏感型核小体阵列则会在高浓度MNase作用下被降解。抗性型核小体组装于A/T碱基缺失、且富含G/C二核苷酸的序列区域。与之相反，敏感型核小体则形成于以转录起始位点、转录终止位点、增强子以及DNase超敏感位点（DNase hypersensitive sites）为代表的富A/T序列区域。将细胞生长温度降至约10℃可稳定敏感型核小体，这表明核小体对MNase的敏感性差异，或与染色质纤维的热波动有关，或与酶促系统的活性相关。在活跃基因与DNase超敏感位点附近，核小体排布为同步的周期性阵列。此类排布模式大概率由DNA结合因子形成的潜在屏障所介导的核小体“相位排布”所导致，本研究还提供了一套全面的生物物理框架来解释这一现象。