National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
University of Chinese Academy of Sciences, Beijing, China.
Nucleic Acids Res. 2020 Jun 19;48(11):5939-5952. doi: 10.1093/nar/gkaa360.
Nucleosome is the basic structural unit of chromatin, and its dynamics plays critical roles in the regulation of genome functions. However, how the nucleosome structure is regulated by histone variants in vivo is still largely uncharacterized. Here, by employing Micrococcal nuclease (MNase) digestion of crosslinked chromatin followed by chromatin immunoprecipitation (ChIP) and paired-end sequencing (MNase-X-ChIP-seq), we mapped unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z nucleosomes are more enriched with unwrapping states compared with canonical nucleosomes. Interestingly, +1 H2A.Z nucleosomes with 30-80 bp DNA is correlated with less active genes compared with +1 H2A.Z nucleosomes with 120-140 bp DNA. We confirmed the unwrapping of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z and H2A after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in decreased unwrapping of H3.3 nucleosomes and increased CTCF binding. Taken together, through MNase-X-ChIP-seq, we showed that histone variant H2A.Z regulates nucleosome unwrapping in vivo and that its function in regulating transcription or CTCF binding is correlated with unwrapping states of H2A.Z nucleosomes.
核小体是染色质的基本结构单位，其动力学在调节基因组功能方面起着关键作用。然而，组蛋白变体如何在体内调节核小体结构在很大程度上仍未被描述。在这里，我们通过微球菌核酸酶（MNase）消化交联染色质，然后进行染色质免疫沉淀（ChIP）和配对末端测序（MNase-X-ChIP-seq），在小鼠胚胎干细胞（ES 细胞）中绘制了含有组蛋白变体 H2A.Z 的核小体的展开状态。我们发现 H2A.Z 核小体比经典核小体更富集展开状态。有趣的是，与含有 120-140bp DNA 的 +1 H2A.Z 核小体相比，具有 30-80bp DNA 的 +1 H2A.Z 核小体与活性较低的基因相关。我们通过 CTCF CUT&RUN 在小鼠 ES 细胞中重新进行 H2A.Z 和 H2A 的 ChIP，证实了在天然条件下 H2A.Z 核小体的展开。重要的是，我们发现 H2A.Z 的耗竭导致 H3.3 核小体的展开减少和 CTCF 结合增加。总之，通过 MNase-X-ChIP-seq，我们表明组蛋白变体 H2A.Z 调节体内核小体的展开，其在调节转录或 CTCF 结合中的功能与 H2A.Z 核小体的展开状态相关。
