色质域新形成研究方法
Summary
该方法旨在跟踪细胞系中PRC2介导染色质域的形成，该方法可适用于许多其他系统。
Abstract
染色质域的组织和结构是单个细胞谱系所独有的。他们的管制不当可能导致细胞身份和/或疾病的损失。尽管作出了巨大努力，我们对染色质域的形成和传播的理解仍然有限。色质领域已在稳定状态条件下进行了研究，这不利于在建立过程中跟踪初始事件。在这里，我们提出了一种方法，以诱导地重建染色质域，并遵循其重新形成作为时间的函数。虽然，首先适用于PRC2介导的压制染色质域形成的情况，它可以很容易地适应其他染色质领域。该方法的修改和/或与基因组学和成像技术的结合，将为详细研究染色质域的建立提供宝贵的工具。我们相信，这种方法将彻底改变我们对染色质域如何形成和相互作用的理解。
Introduction
真核基因组组织性很强，染色质可及性的变化直接控制着基因转录1。基因组包含不同类型的染色质域，与转录活动和复制时间2，3相关。这些染色质域的大小范围从几个千碱（kb）到超过100kb，其特点是在独特的组蛋白修饰4中富集。核心问题是：这些域是如何形成的，如何传播？
一个最具特征的染色质域是通过 Polycomb 压制性复合体 2 （PRC2） 的活动培育的。PRC2 是一种多亚单位复合体，由蛋白质5、6 的 Polycomb 组 （PcG） 的子集组成，并催化组蛋白 H3 （H3K27me1/me2/me3）7、8 、 9，10.H3K27me2/me3与抑制性染色质状态有关，但H3K27me1的功能不清楚6，11。PRC2的核心部件之一，胚胎外阴体发育（EED），通过其芳香笼与PRC2催化的最终产物H3K27me3结合，这一特性导致PRC212，13的异体刺激。PRC2酶活性对于在发育过程中保持细胞特性至关重要，因为某些发育基因的不当表达，即特定血统的禁忌，将有害于5，6.因此，解开PRC2促进哺乳动物形成抑制性染色质域的机制，对于理解细胞特性至关重要。
所有过去旨在研究染色质域形成的实验系统，包括PRC2介导染色质域，都是在稳定状态条件下进行的，这些系统无法跟踪染色质域形成在细胞。在这里，我们提出了一个详细的协议，以生成一个诱导的细胞系统，监测染色质域的初始招募和传播。具体来说，我们专注于跟踪由H3K27me2/3组成的PRC2介导的压质染色质域的形成。该系统可以捕获染色质域形成的机械细节，可以调整以纳入其他染色质域，如广泛研究的域包括H2AK119ub或H3K9me。结合基因组学和成像技术，这种方法有可能成功地解决染色质生物学中的各种关键问题。
Protocol
产生诱导式EED救援mESC 1. 细胞培养 使用无进纸的C57BL/6小鼠胚胎干细胞（mESCs），具有稳定集成的CreERT2转基因，在给给4-羟基塔莫西芬（4-OHT）13时可转移到细胞核。 在传统的ESC介质14、15中生长mESC，辅以1000U/mL LIF、1μM ERK抑制剂PD0325901和3μM GSK3抑制剂CHIR99021。对于传统的ESC介质，使用含有…
Representative Results
有条件救助制度的一般方案图1显示了有条件地用WT或笼突变体（Y365A）EED从内源EED位点表达的EED KO细胞的靶向方案。在敲除对稳定性和酶活性至关重要的PRC2核心子单元EED后，引入EED外延9之后的内联磁带（图1）。该盒体由EED的剩余3’cDNA序列组成，与内源性基因序列相反。 盒内由异体倒置的 loxP 位点（lox66 和 lox71）18。?…
Discussion
在给定染色质域形成期间，理解机械细节的有力方法是首先破坏该域，然后跟踪其在细胞内正在进行的重建。在重建过程中，可以随时暂停该过程，以详细分析正在进行的事件。以前关于染色质域的研究无法解决在稳定状态条件下执行的事件（例如，比较野生型和基因挖空）。在这里，我们概述了一个系统，以评估染色质域的动态形成，这突出说明了在细胞中招募和传播PRC2介导的压制性域。
<…Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢L.Vales博士、D.Ozata博士和H.Mou博士对手稿的修订。D.R. 实验室由霍华德·休斯医学研究所和国家卫生研究院（R01CA199652和R01NS100897）提供支持。
Materials
| (Z)-4-Hydroxytamoxifen (5 mg) | Sigma | H7904-5MG | For induction of EED expression |
| 16% Paraformaldehyde aqueous solution (10×10 ml) | Electron Microscopy Sciences | 15710 | For immunofluorescence |
| 2-mercaptoethanol | LifeTechnologies | 21985-023 | For mESCs culture |
| 2% Gelatin Solution | Sigma | G1393-100ml | For mESCs culture |
| Accutase 500 ML | Innovative Cell Tech/FISHER | AT 104-500 | For mESCs culture |
| Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson immunoresaerch | 711-585-152 | For immunofluorescence |
| Aqua-Mount Mounting Medium | FISHER/VWR | 41799-008 | For immunofluorescence |
| CHAMBER SLD TC PRMA 8-CHM 16 PK | Fisher Sci | 177445PK | For immunofluorescence |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) – 10 mg | Life Tech | D1306 | For immunofluorescence |
| ERK inhibitor, PD0325901 | Stemgent | 04-0006 | For mESCs culture |
| ESGRO Recombinant Mouse LIF Protein | Millipore/Fisher | ESG1107 | For mESCs culture |
| FBS Stem Cell Qualified | Atlanta | S10250 | For mESCs culture |
| Gibson Assembly Master Mix | NEB | E2611L | For Donor template cloning |
| GSK3 inhibitor, CHIR99021 | Stemgent | 04-0004 | For mESCs culture |
| H3K27me2 (D18C8) rabbit mAB | Cell Signaling | 9728S | Antibody for ChIP-seq |
| H3K27me3 | Cell Signaling | 9733S | Antibody for ChIP-seq |
| Histone H2Av antibody (pAb) | Active motif | 39715 | Spike-in control for ChIP-seq |
| Knockout DMEM | Invitrogen | 10829-018 | For mESCs culture |
| L-glutamine | Sigma | G7513 | For mESCs culture |
| Lipofectamine 2000 | LifeTech | 11668019 | For transfection |
| MangoTaq DNA Polymerase | Bioline | BIO-21079 | For Genotyping PCR |
| Normal donkey serum (10 mL) | Jackson ImmunoResearch | 017-000-121 | For immunofluorescence |
| Penicillin-Streptomycin | Sigma/Roche | P0781 | For mESCs culture |
| pSpCas9(BB)-2A-GFP (PX458) | Addgene | 48138 | For gRNA cloning |
| QuickExtract DNA Extraction Solution | Lucigen | QE0905T | For Genotyping PCR |
| Triton X-100 | Sigma | T8787-250ML | |
| Zero Blunt PCR Cloning Kit | Thermo Fisher | K270020 | For Donor template cloning |
| Primers/gBlocks | |||
| EED-KO-gRNA-1 | Sequence: ctctggctactgtcaactag. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems. | ||
| EED-KO-gRNA-2 | Sequence: TAGGCTATGACGCAGCTCAG. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems. | ||
| EED-gRNA-inducible | Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background. | ||
| i-WT-r Donor | https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI. gRNA and Donor to generate i-WT-r system in the EED-KO background. | ||
| EED-gRNA-inducible | Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background. | ||
| i-MT-r Donor | https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM. gRNA and Donor to generate i-MT-r system in the EED-KO background. | ||
| Genotyping Primers | |||
| Gnt-EED-KO-FW-1 | Sequence: ctgtaggctgccatctgtga. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp. | ||
| Gnt-EED-KO-REV-1 | Sequence: agccagggctacacagagaa. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp. | ||
| Inducible_Genotype-FW-1 | Sequence: tgcagtgaaacaaatttggaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp. | ||
| Inducible_Genotype-REV-1 | Sequence: gagaggggtggcactgtaaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp. | ||
| Inducible_Genotype-FW-2 | Sequence: ccccctctttctccttttct. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp. | ||
| Inducible_Genotype-REV-2 | Sequence: atgcctgggtgaatgaaaaa. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp. |
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Citer Cet Article
Oksuz, O., Reinberg, D. A Method to Study de novo Formation of Chromatin Domains. J. Vis. Exp. (150), e60039, doi:10.3791/60039 (2019).