Histone variant H2A.Z regulates nucleosome unwrapping and CTCF binding in mouse ES cells

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 genome-wide unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z is enriched with unwrapped nucleosomes. Interestingly, the function of +1 H2A.Z nucleosome in transcriptional regulation is correlated with the unwrapping states. We further showed that H2A.Z nucleosomes adjacent the CTCF binding sites (CBS) may adopt an open conformation. We confirmed the unwrapping state of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in increased CTCF binding, indicating dynamic competition between the unwrapped H2A.Z nucleosomal intermediates and CTCF at the CBS. Taken together, our results showed that histone variant H2A.Z regulates transcription and CTCF binding through modulating the nucleosome unwrapping. Chromatin was first crosslinked with formaldehyde, then digested with MNase and solubilized by minimal sonication. After Chromatin immunoprecipitation (ChIP), the ChIPed DNA was subjected to paired-end sequencing without size selection. The read pairs were mapped to the mouse reference genome (mm9) using Bowtie2, and only read pairs with mapping quality higher than 10 (mapq > 10) were retained for further analyses. We counted the frequency of the length of ChIPed DNA fragments per base pair from 0 to 200 bp to analyze the fragment length profiles of histones.

核小体（Nucleosome）是染色质的基本结构单元，其动态变化在基因组功能的调控中扮演关键角色。然而，组蛋白变体在体内如何调控核小体结构，目前仍未得到充分阐释。本研究通过对交联染色质实施微球菌核酸酶（Micrococcal nuclease, MNase）消化，随后结合染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）与双端测序（paired-end sequencing）构建了MNase-X-ChIP-seq实验方法，并在小鼠胚胎干细胞（embryonic stem, ES cells）中绘制了携带组蛋白变体H2A.Z的核小体全基因组解旋状态图谱。我们发现H2A.Z在解旋核小体中富集。有趣的是，+1核小体中H2A.Z在转录调控中的功能与其解旋状态密切相关。我们进一步证实，紧邻CCCTC结合因子（CCCTC-binding factor, CTCF）结合位点（CBS）的H2A.Z核小体可能呈现开放构象。我们通过在小鼠ES细胞中对CTCF进行CUT&RUN后再对H2A.Z实施重免疫共沉淀（re-ChIP），验证了天然条件下H2A.Z核小体的解旋状态。值得注意的是，我们发现H2A.Z的耗竭会导致CTCF结合增强，这表明在CBS处，解旋的H2A.Z核小体中间产物与CTCF之间存在动态竞争关系。综上，本研究结果证实，组蛋白变体H2A.Z通过调控核小体解旋过程，参与转录调控与CTCF结合。实验中首先将染色质用甲醛进行交联，随后经MNase消化并通过温和超声处理实现增溶。染色质免疫共沉淀（ChIP）完成后，无需进行片段大小筛选，即可对所富集的DNA开展双端测序。我们使用Bowtie2将测序读段对比对至小鼠参考基因组mm9，仅保留比对质量值大于10（mapq>10）的读段对用于后续分析。我们统计了0至200 bp范围内每一个碱基位点处经ChIP富集的DNA片段的长度频率，以此分析组蛋白的片段长度分布特征。