DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [damC]

Mammalian chromosomes are folded into an intricate hierarchy of structural domains, within which topologically associating domains (TADs) and CTCF-associated loops partition the physical interactions between regulatory sequences. Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after crosslinking of chromatin. To measure chromosome structure in vivo, quantitatively and without relying on crosslinking and ligation, we have implemented a new method named damC. DamC combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and a biophysical model of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of TADs and CTCF loops, confirms 3C-based measurements of the scaling of contact probabilities within TADs, and provides evidence that mammalian chromatin in vivo is essentially rigid below 5 kilobases. Combining damC with transposon-mediated genomic engineering shows that new loops can be formed between ectopically introduced and endogenous CTCF sites, which alters the partitioning of physical interactions within TADs. This establishes damC as a crosslinking- and ligation-free framework to measure and modify chromosome interactions combined with a solid theoretical background for rigorous data interpretation. This orthogonal approach to 3C validates the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated. Overall design: Mouse ESC lines #94.1_2.7 (carrying random insertions of 50x TetO arrays spanning approx. 2.7kb each) and #94.1_216_C3 (carrying different random insertions of the same 50x TetO cassette flanked by 3 CTCF sites faced outwards) were generated starting from an X0 clone of the PGKT2 line in Masui et al (Cell 145:447-458, 2011). The remaining X chromosome additionally carries the deletion of the Linx promoter within the X inactivation center (Nora et al., Nature 485:381-385, 2012). Random insertions were generated using the piggyBac transposon system. Both lines stably express rTetR-EGFP-Dam-ERT2 under the control of an ectopic Rosa26 promoter. Cells were cultured on gelatin-coated culture plates in Dulbecco Modified Eagle's medium (Sigma) in the presence of 15% foetal calf serum (Eurobio Abcys), 100 µM ß-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) and 250 µg/mL hygromycin in 8% CO2 at 37°C.To induce nuclear translocation of the rTetR-Dam fusion protein to the nuclei, mESC were trypsinized and directly seeded in culture medium containing 4-hydroxy-tamoxifen (4-OHT) at various concentration as indicated in the sample titles. Binding of the Dam fusion protein to the TetO arrays was induced by simultaneously adding 2.5 µg/ml doxycycline (Dox). After 18 hours, 3x106 cells were harvested using trypsin. Genomic DNA was extracted using the Qiagen blood and tissue kit adding RNaseA in step 1. Genomic DNA was eluted in 80ul ddH2O. DNA concentration was measured using the Qbit DNA Broad Range kit. Genomic DNA (100ng input) was treated with Shrimp Alkaline Phosphatase treatment (NEB, 1U), followed by DpnI digestion (ThermoFisher Scientific, 10U), A-tailing (0.6mM final dATP, 5U Klenow exo-, ThermoFisher Scientific), and UMI adapters ligation (30U T4 DNA ligase, PEG4000, ThermoFisher Scientific) performed within the same tube and buffer (Tango 1X, ThermoFisher Scientific) by heat inactivating each enzymatic step followed by adjustment with the reagents required for the next step. UMI adapters were made by annealing the following oligos: 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T and 5'-pGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT. Ligation reactions were treated with Exonuclease I (20U, ThermoFisher Scientific ) then purified using AMPureXP beads (1:0.8 ratio, Agencourt) and the second sequencing adapter (5' TGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNN*N 3', IDT) was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (10 cycles) using 1U of Phusion polymerase, 2µl 10 µM DAM_UMIindex_PCR (5' AATGATACGGCGACCACCGAGATCTACA*C 3'), and 2µl 10µM NEBnext indexed primer (NEB). Final libraries were purified AMPure XP beads (1:1 ratio) and QCed using Bioanalyser and Qbit. DamC libraries were sequenced on a NextSeq500 (75 cycles single-end) with a custom sequencing protocol (dark cycles at the start of read1 to "skip" the remaining DpnI site TC sequence). Samples index were determined using index1 read, and UMI sequence using index2 read. UMIs are contained in the readname of each read in each fastq file Please note that processed data was generated from multiple samples (as indicated in the corresponding sample description field and in the README.txt) and is linked as Series supplementary file.

哺乳动物染色体折叠形成复杂的结构域层级体系，其中拓扑关联结构域（topologically associating domains, TADs）与CTCF结合环将调控序列间的物理相互作用进行分区。当前学界对染色体折叠机制的认知，大多基于染色体构象捕获（chromosome conformation capture, 3C）类实验——这类实验通过交联染色质后检测连接产物，实现染色体相互作用的检测。为了在不依赖交联与连接的前提下，实现体内染色体结构的定量检测，我们开发了一种名为damC的全新实验方法。damC将基于DNA甲基化的染色体相互作用检测技术、下一代测序（next-generation sequencing, NGS）技术与甲基化动力学生物物理模型相结合。在小鼠胚胎干细胞中开展的damC实验，首次在体内验证了TADs与CTCF结合环的存在，证实了基于3C实验得到的TAD内部接触概率缩放规律，并证明了哺乳动物体内染色质在5kb以下的区域本质上具有刚性。将damC与转座子介导的基因组工程技术结合后，研究人员可在异位引入的CTCF位点与内源CTCF位点之间形成新的结合环，从而改变TAD内部物理相互作用的分区模式。这一成果确立了damC作为一种无需交联与连接的染色体相互作用检测与调控技术框架的地位，同时为严谨的数据解析提供了坚实的理论基础。这种与3C技术正交的实验方法，验证了哺乳动物染色体关键结构特征的存在，并为TAD内部染色体结构的调控机制提供了全新的研究视角。实验整体设计如下：研究人员从Masui等人2011年发表于《Cell》（145卷：447-458）的PGKT2细胞系的X0克隆出发，构建了两种小鼠胚胎干细胞（ESC）株系：#94.1_2.7（携带约2.7kb的50x TetO阵列随机插入位点）与#94.1_216_C3（携带相同的、侧翼带有3个向外朝向的CTCF位点的50x TetO盒的不同随机插入位点）。剩余的X染色体还携带着X失活中心内Linx启动子的缺失突变（Nora等人2012年发表于《Nature》，485卷：381-385）。随机插入位点通过piggyBac转座子系统构建。两种细胞株系均在异位Rosa26启动子的调控下稳定表达rTetR-EGFP-Dam-ERT2融合蛋白。细胞培养于明胶包被的培养板中，培养体系为含15%胎牛血清（Eurobio Abcys）、100 μM β-巯基乙醇、20 U/ml白血病抑制因子（Miltenyi Biotec，优质级）与250 μg/mL潮霉素的达尔伯克改良伊格尔培养基（Sigma），培养条件为37℃、8% CO₂。为诱导rTetR-Dam融合蛋白向细胞核转位，将小鼠胚胎干细胞用胰酶消化后，直接接种于添加了不同浓度4-羟基他莫昔芬（4-OHT）的培养基中，具体浓度详见样本标题。同时添加2.5 μg/ml多西环素（Dox）以诱导Dam融合蛋白与TetO阵列的结合。18小时后，收集3×10⁶个细胞，使用胰酶消化完成收集。使用Qiagen血液与组织试剂盒提取基因组DNA，步骤1中添加RNaseA。基因组DNA用80 μL ddH₂O洗脱。采用Qbit DNA宽范围试剂盒检测DNA浓度。取100 ng基因组DNA作为起始样本，依次进行以下处理：虾碱性磷酸酶处理（NEB，1 U）、DpnI酶切（ThermoFisher Scientific，10 U）、A尾加尾反应（终浓度0.6 mM dATP，5U Klenow exo-，ThermoFisher Scientific）与UMI接头连接（30 U T4 DNA连接酶、PEG4000，ThermoFisher Scientific），所有反应均在同一管同一缓冲液（1× Tango缓冲液，ThermoFisher Scientific）中进行，每一步酶促反应后通过热灭活终止，再添加下一步反应所需的试剂即可进行后续反应。UMI接头由以下寡核苷酸退火制成：5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T 与 5'-pGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT。连接反应产物用核酸外切酶I（20 U，ThermoFisher Scientific）处理，随后采用AMPureXP磁珠（1:0.8比例，Agencourt）纯化。使用热变性与第二链合成反应（5 U T4 DNA聚合酶，ThermoFisher Scientific）对第二个测序接头（5' TGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNN*N 3', IDT）进行标记，标记反应采用AMPure XP磁珠（1:1比例）纯化。最后使用1U Phusion聚合酶、2 μL 10 μM DAM_UMIindex_PCR引物（5' AATGATACGGCGACCACCGAGATCTACA*C 3'）与2 μL 10 μM NEBnext索引引物（NEB）进行10轮循环的文库扩增。最终文库采用AMPure XP磁珠（1:1比例）纯化，并通过生物分析仪与Qbit进行质量控制。damC文库采用NextSeq500测序平台（75个循环单端测序），使用自定义测序流程（read1起始处设置暗循环以“跳过”剩余的DpnI识别位点TC序列）。样本索引通过index1 read确定，UMI序列通过index2 read确定。每个fastq文件中每条reads的readname均包含UMI信息。请注意，处理后的数据来自多个样本（详见对应样本描述字段与README.txt文件），并作为系列补充文件提供。