Active regulatory elements recruit cohesin to establish cell-specific chromatin domains [ChIP-seq]. Active regulatory elements recruit cohesin to establish cell-specific chromatin domains [ChIP-seq]

The genome adopts varied and specific 3D conformations in different cell types. The loop extruding factor, cohesin, is thought to play a key role in the formation of this 3D structure, however it is unknown how cell-type specificity is achieved. One proposed mechanism is that cis-regulatory elements, which are active in unique combinations in different cell types, recruit cohesin and therefore direct the formation of specific 3D conformation. We have identified genome-wide natural variation in enhancer sites in healthy human donors, by performing cohesin ChIP-seq in these cells we show that cohesin occupancy is directly correlated with enhancer activity. Identification of the genetic variants associated with the gain or loss of enhancer-like elements and associating this with the recruitment of cohesin provides evidence of a direct link between widespread genetic variation in the non-coding genome and cell-specific changes in 3D genome organization. Therefore, enhancer elements have evolved not only the ability to upregulate transcription at promoters, but also the ability to stimulate the mechanisms that insure robust interactions between them. Next, using enhancer KO models in the well characterised alpha globin locus, we show how cohesin recruitment and 3D genome organization correlates with changes in the local regulatory landscape. We then selected the strongest of the alpha globin enhancers and inserted it into a neutral region of the genome, to test if a single active enhancer was capable of recruiting cohesin and initiating the formation of a 3D chromatin domain when isolated from its native context. Our results show that an enhancer is able to direct the recruitment of cohesin when in an active state and that this is correlated with changes in the local chromatin environment, giving rise to open chromatin sites and de novo chromatin interactions in an otherwise “neutral” region of the genome. These results support the hypothesis that tissue-specific recruitment of cohesin is facilitated by enhancers and that this plays a role in establishing cell-specific changes in 3D genome conformation. Overall design: ChIP-seq was performed on aliquots of 3-10x10^6CD71+ cells. For calibrated ChIP-seq, a 1% or 4% spike-in of WT HEK293 cells were added prior to sonication. Cells were double cross-linked using disuccinimidyl glutarate (DSG, Sigma) and 1% formaldehyde (Sigma) for a total fixation time of 1 hour. Fixed chromatin samples were fragmented using the Covaris sonicator (ME220) for 10 minutes (75 power, 1000 cycles per burst, 25% duty factor) at 4°C. 50μL per sample was removed as an input control. Sonicated samples were pre-cleared to remove background signal through incubation with a 1:1 mix of Protein A/G Dynabeads (InVitrogen). Antibody was added at a concentration of 1 μg/μL per sample and immunoprecipitated at 4°C overnight. Immunoprecipitated samples were incubated with a 1:1 mix of Protein A/G Dynabeads for 5 hours at 4°C. Beads were then washed 4x in RIPA buffer on a magnetic stand, followed by 1x wash with TE (Sigma Aldridge) + 50mM NaCl (ThermoFisher). Chromatin was eluted from the beads using elution buffer and incubated for 30 minutes at 65°C with shaking. Input samples were diluted 1:1 with elution buffer. Samples and input controls were incubated at 65°C overnight for de-crosslinking, before RNase (Roche) and proteinase K (BioLabs) treatment. DNA fragments were purified using the Zymo ChIP DNA Clean & Concentrator kit (Zymo Research) and eluted in water. DNA concentration was quantified using the Qubit dsDNA HS assay (Invitrogen) as per manufacturers protocol. To assess sonication efficiency the D1000 Tapestation (Agilent) assay was performed on input sample. An approximately equal mass of input and IP DNA was used for indexing (0.5 ng – 1 μg). NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) was used to prepare indexed sequencing libraries following the manufacturer supplied protocol. PCR amplification was performed for 7-11 cycles (depending on input DNA concentration) using NEBNext Mulitplex Oligos (New England Biolabs). Indexed sample concentration was quantified using the KAPA Library Quantification Complete Kit (Universal)(Roche). Samples were pooled as a 4 nM library and sequenced with a high-Output v2 75 cycle kits on the Illumina NextSeq platform.

基因组在不同细胞类型中呈现出多样且特异的三维（3D）构象。环挤出因子黏连蛋白（cohesin）被认为在该三维结构的形成中发挥关键作用，但目前尚不清楚细胞类型特异性是如何实现的。一种已被提出的机制认为，在不同细胞类型中以独特组合形式激活的顺式调控元件（cis-regulatory elements），可招募黏连蛋白，进而指导特异三维构象的形成。
本研究通过对健康人类供体的增强子（enhancer）位点开展全基因组自然变异鉴定，并在这些细胞中进行黏连蛋白染色质免疫共沉淀测序（ChIP-seq），结果显示黏连蛋白的结合占有率与增强子活性直接相关。鉴定与增强子样元件得失相关的遗传变异，并将其与黏连蛋白的招募情况相关联，可为非编码基因组（non-coding genome）中广泛存在的遗传变异与三维基因组组织的细胞特异性改变之间的直接关联提供证据。因此，增强子元件不仅进化出了上调启动子处转录的能力，还进化出了促进自身之间稳定相互作用的机制。
随后，我们在特征明确的α-珠蛋白基因座（alpha globin locus）中使用增强子敲除（KO）模型，揭示了黏连蛋白的招募情况与三维基因组组织如何随局部调控景观的改变发生变化。接着，我们选取了活性最强的α-珠蛋白增强子，并将其插入基因组的中性区域，以验证单个活性增强子脱离原生环境后，是否能够招募黏连蛋白并启动三维染色质结构域（chromatin domain）的形成。本研究结果表明，处于激活状态的增强子能够指导黏连蛋白的招募，且这一过程与局部染色质环境的改变相关，可在基因组原本的“中性”区域形成开放染色质位点并产生新生染色质相互作用（de novo chromatin interactions）。上述结果支持以下假说：增强子可促进黏连蛋白的组织特异性招募，且该过程在建立细胞特异性的三维基因组构象改变中发挥作用。

整体实验设计：
本研究对3~10×10^6个CD71+细胞的等分样本开展染色质免疫共沉淀测序（ChIP-seq）。对于校准型ChIP-seq，在超声破碎前加入1%或4%的野生型HEK293细胞作为内参。使用戊二酰二亚胺酯（DSG，Sigma）与1%甲醛（Sigma）对细胞进行双重交联，总固定时长为1小时。固定后的染色质样本于4℃下使用Covaris超声破碎仪（ME220）进行10分钟的片段化处理（功率75，每爆发1000个循环，占空比25%）。每份样本取50μL作为输入对照。将超声后的样本与Protein A/G磁珠（Invitrogen）按1:1比例混合液孵育，以去除背景信号完成预清除。按照每样本1μg/μL的浓度加入抗体，于4℃下孵育过夜进行免疫沉淀。将免疫沉淀后的样本与Protein A/G磁珠按1:1比例混合液于4℃下孵育5小时。随后在磁力架上使用RIPA缓冲液洗涤磁珠4次，再使用TE缓冲液（Sigma Aldrich）+50mM氯化钠（ThermoFisher）洗涤1次。使用洗脱缓冲液从磁珠上洗脱染色质，并于65℃下振荡孵育30分钟。将输入样本与洗脱缓冲液按1:1比例稀释。将样本与输入对照于65℃下过夜孵育以完成去交联，随后分别用核糖核酸酶（RNase，罗氏）与蛋白酶K（BioLabs）进行处理。使用Zymo ChIP DNA纯化与浓缩试剂盒（Zymo Research）纯化DNA片段，并用纯水洗脱。采用Qubit dsDNA高灵敏度检测试剂盒（Invitrogen）按照制造商规程对DNA浓度进行定量。为评估超声破碎效率，对输入样本开展D1000 TapeStation检测系统（安捷伦）检测。取质量大致相等的输入DNA与免疫沉淀DNA用于索引建库（0.5ng~1μg）。使用NEBNext Ultra II DNA文库制备试剂盒（New England Biolabs）按照制造商提供的规程构建索引测序文库。使用NEBNext多重寡核苷酸（New England Biolabs）进行7~11个循环的PCR扩增（循环数取决于输入DNA浓度）。采用KAPA文库定量完整试剂盒（通用型，罗氏）对索引样本的浓度进行定量。将样本混合为4nM的文库，使用Illumina NextSeq测序平台的高输出v2 75循环试剂盒进行测序。