Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors [Hi-C]. Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors [Hi-C]

Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although models of regulation have been proposed (e.g., loop extrusion), how cohesin and related factors collectively or individually regulate the hierarchical chromatin structure and gene expression remains unclear. We have depleted cohesin and related factors and then conducted a comprehensive evaluation of the resulting 3D genome, transcriptome and epigenome data. We observed substantial variation in depletion effects among factors at topologically associating domain (TAD) boundaries and on interTAD interactions, which were related to epigenomic status. Overall design: We used the in situ Hi-C protocol as described in Rao et al. (Rao et al., Cell, 2014, 10.1016/j.cell.2014.11.021). In brief, ~3 × 10^6 RPE cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by an additional 5 min with 200 mM glycine in phosphate-buffered saline (PBS). Fixed cells were permeabilized in Hi-C lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA630; 1× protease inhibitor cocktail [Sigma]) on ice. The cells were treated with 100 U of MboI (New England Biolabs) for chromatin digestion, and the ends of digested fragments were labeled with biotinylated nucleotides followed by ligation. After DNA reverse crosslinking and purification, ligated DNA was sheared to a size of 300–500 bp using a Covaris S2 focused-ultrasonicator (settings: Duty Cycle, 10%; Intensity, 4; Cycles per Burst, 200; Duration, 55 sec). The ligated junctions were then pulled down with Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific). The pulled-down DNA was end-repaired, ligated to sequencing adaptors, amplified on beads and purified using Nextera Mate Pair Sample Preparation Kit (Illumina) and Agencourt AMPure XP (Beckman Coulter). DNA was then sequenced to generate paired-end 150-bp reads using the Illumina HiSeq-2500 or X Ten system.

黏连蛋白（cohesin）在染色质折叠与基因表达调控中发挥至关重要的作用，可与黏连蛋白加载因子、卸载因子以及绝缘因子CTCF等各类因子协同行使功能。尽管已有相关调控模型被提出（如环挤出模型），但黏连蛋白及其相关因子如何协同或单独调控层级染色质结构与基因表达，目前仍未明确。
本研究通过敲除黏连蛋白及相关因子，对由此获得的三维基因组、转录组与表观基因组数据开展了全面评估。我们观察到，不同因子在拓扑关联结构域（topologically associating domain, TAD）边界及TAD间相互作用上的敲除效应存在显著差异，且该差异与表观基因组状态密切相关。
整体实验设计：我们采用Rao等人（Rao et al., Cell, 2014, 10.1016/j.cell.2014.11.021）报道的原位Hi-C实验流程。简言之，将约3×10^6个RPE细胞在室温下用1%甲醛交联10分钟，随后用含200 mM甘氨酸的磷酸盐缓冲液（phosphate-buffered saline, PBS）淬灭交联反应5分钟。固定后的细胞在冰上用Hi-C裂解缓冲液（10 mM Tris-HCl，pH 8.0；10 mM NaCl；0.2% Igepal CA630；1×蛋白酶抑制剂混合物[Sigma]）透化。随后用100 U MboI（New England Biolabs）对染色质进行酶切，酶切片段的末端用生物素标记的核苷酸进行补平标记，再进行连接反应。完成DNA去交联与纯化后，使用Covaris S2聚焦超声破碎仪将连接产物剪切至300–500 bp（参数：占空比10%；强度4；每脉冲循环数200；持续时间55秒）。随后使用Dynabeads MyOne Streptavidin T1磁珠（Thermo Fisher Scientific）富集带有生物素标记的连接位点。富集得到的DNA经末端修复、连接测序接头后，在磁珠上进行扩增，再使用Nextera Mate Pair Sample Preparation Kit（Illumina）与Agencourt AMPure XP（Beckman Coulter）进行纯化。最终使用Illumina HiSeq-2500或X Ten测序平台进行双端150 bp测序。