Long-distance association of topological boundaries through nuclear condensates (ChIP-Seq). Long-distance association of topological boundaries through nuclear condensates (ChIP-Seq)

Chromatin is partitioned into distinct topological domains in an activity-dependent manner, with topological boundaries limiting the interaction between adjacent domains. Recent studies support the concept that several well-established nuclear compartments are assembled as ribonucleoprotein condensates. Here we ask whether the physical processes driving the assembly of the nuclear condensates play any role in three-dimensional chromatin architecture. We report that the insulation of approximately 20% of topological boundaries in human embryonic stem cells is substantially weakened following brief treatment with 1,6-hexanediol, a chemical known to disrupt several nuclear condensates. The disrupted boundaries are characterized by a high level of transcription, striking spatial clustering, and the augmented presence of transcription units widely expressed in diverse cell types. These topological boundary regions tend to be spatially associated, even inter-chromosomally, and segregate with nuclear speckles. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring widely-expressed transcription units and associated transcriptional condensates. Overall design: Human RUES1 cells were treated with either 7% 2,5-Hexanediol or 7% 1,6-Hexanediol for 5 minutes, washed with media, and incubate with fresh media for additoinal time points at 37 deg C as indicated. Cells were subsuqently prccesed for in situ Hi-C, ChIp-seq, GRO-seq and PRO-seq. For transcription inhibition experiments, RUES1 cells were treated with either 5uM flavopiridol hydrochloride (F3055 Sigma) or 1uM triptolide (T3652 Sigma), for 15 minutes, 1 hour and 3 hours. Cells were then washed with PBS, cross-linked with 1% formaldehyde in PBS at room temperature for 10 min and neutralized with 0.125M glycine, and further proccessed for in situ Hi-C.

染色质以活性依赖的方式被分区为不同的拓扑结构域，拓扑边界可限制相邻结构域之间的相互作用。近期研究证实，多个已被充分表征的细胞核区室是以核糖核蛋白凝聚体（ribonucleoprotein condensates）的形式组装而成的。本研究旨在探究驱动细胞核凝聚体组装的物理过程，是否对染色质的三维架构具有调控作用。
我们发现，人类胚胎干细胞中约20%的拓扑边界，在经过已知可破坏多种细胞核凝聚体的化学物质1,6-己二醇（1,6-hexanediol）短暂处理后，其绝缘能力显著减弱。受破坏的拓扑边界具有以下特征：转录水平较高、呈现显著的空间聚集，且广泛表达于多种细胞类型的转录单元丰度显著升高。这些拓扑边界区域往往存在空间关联，甚至可发生跨染色体的关联，并与核斑点（nuclear speckles）共定位。上述观测结果揭示了一种此前未被认知的基因组组织模式：该模式由携带广泛表达转录单元的保守边界元件及其相关的转录凝聚体所介导。
实验整体设计：将人类RUES1细胞分别用7% 2,5-己二醇或7% 1,6-己二醇处理5分钟，随后用培养基洗涤，并按指示在37℃下用新鲜培养基继续培养至指定时间点。之后收集细胞，分别进行原位Hi-C（in situ Hi-C）、染色质免疫共沉淀测序（ChIP-seq）、全局运行测序（GRO-seq）及精确运行测序（PRO-seq）实验。
在转录抑制实验中，将RUES1细胞分别用5μM 盐酸黄酮哌啶醇（F3055，Sigma）或1μM 雷公藤内酯（T3652，Sigma）处理15分钟、1小时及3小时。随后用磷酸盐缓冲液（PBS）洗涤细胞，于室温下用含1%甲醛的PBS交联10分钟，再用0.125M甘氨酸终止交联，最后进一步开展原位Hi-C实验。