Name: a method to study de novo formation of chromatin domains
Uploaded: 2019-08-23T14:30:00
Description: Watch this Scientific Journal Video about A Method to Study de novo Formation of Chromatin Domains at JoVE.com

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。

产生诱导式EED救援mESC
1. 细胞培养
<ol><li>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。</li>
	<li>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...

<ol>
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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。
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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。

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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">(Z)-4-Hydroxytamoxifen (5 mg)</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">H7904-5MG</td>
			<td style="width:368px;">For induction of EED expression</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">16% Paraformaldehyde aqueous solution (10x10 ml)</td>
			<td style="width:317px;">Electron Microscopy Sciences</td>
			<td style="width:101px;">15710</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2-mercaptoethanol</td>
			<td style="width:317px;">LifeTechnologies</td>
			<td style="width:101px;">21985-023</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2% Gelatin Solution</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">G1393-100ml</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Accutase 500 ML</td>
			<td style="width:317px;">Innovative Cell Tech/FISHER</td>
			<td style="width:101px;">AT 104-500</td>
			<td>For mESCs culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:317px;">Jackson immunoresaerch</td>
			<td style="width:101px;">711-585-152</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Aqua-Mount Mounting Medium</td>
			<td style="width:317px;">FISHER/VWR</td>
			<td style="width:101px;">41799-008</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">CHAMBER SLD TC PRMA 8-CHM 16 PK</td>
			<td style="width:317px;">Fisher Sci</td>
			<td style="width:101px;">177445PK</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride) - 10 mg</td>
			<td style="width:317px;">Life Tech</td>
			<td style="width:101px;">D1306</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">ERK inhibitor, PD0325901</td>
			<td style="width:317px;">Stemgent</td>
			<td style="width:101px;">04-0006</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ESGRO Recombinant Mouse LIF Protein</td>
			<td>Millipore/Fisher</td>
			<td>ESG1107</td>
			<td>For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">FBS Stem Cell Qualified</td>
			<td style="width:317px;">Atlanta</td>
			<td style="width:101px;">S10250</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gibson Assembly Master Mix</td>
			<td style="width:317px;">NEB</td>
			<td style="width:101px;">E2611L</td>
			<td>For Donor template cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GSK3 inhibitor, CHIR99021</td>
			<td style="width:317px;">Stemgent</td>
			<td style="width:101px;">04-0004</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">H3K27me2 (D18C8) rabbit mAB</td>
			<td style="width:317px;">Cell Signaling</td>
			<td style="width:101px;">9728S</td>
			<td>Antibody for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">H3K27me3</td>
			<td style="width:317px;">Cell Signaling</td>
			<td style="width:101px;">9733S</td>
			<td>Antibody for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Histone H2Av antibody (pAb)</td>
			<td style="width:317px;">Active motif</td>
			<td style="width:101px;">39715</td>
			<td>Spike-in control for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Knockout DMEM</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:101px;">10829-018</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-glutamine</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">G7513</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Lipofectamine 2000</td>
			<td style="width:317px;">LifeTech</td>
			<td style="width:101px;">11668019</td>
			<td>For transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">MangoTaq DNA Polymerase</td>
			<td style="width:317px;">Bioline</td>
			<td style="width:101px;">BIO-21079</td>
			<td>For Genotyping PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Normal donkey serum (10 mL)</td>
			<td style="width:317px;">Jackson ImmunoResearch</td>
			<td style="width:101px;">017-000-121</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Penicillin-Streptomycin</td>
			<td style="width:317px;">Sigma/Roche</td>
			<td style="width:101px;">P0781</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">pSpCas9(BB)-2A-GFP (PX458)</td>
			<td style="width:317px;">Addgene</td>
			<td style="width:101px;">48138</td>
			<td style="width:368px;">For gRNA cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">QuickExtract DNA Extraction Solution</td>
			<td style="width:317px;">Lucigen</td>
			<td style="width:101px;">QE0905T</td>
			<td style="width:368px;">For Genotyping PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Triton X-100</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">T8787-250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td style="width:317px;">Thermo Fisher</td>
			<td>K270020</td>
			<td>For Donor template cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers/gBlocks</td>
			<td style="width:317px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-KO-gRNA-1</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: ctctggctactgtcaactag. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-KO-gRNA-2</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: TAGGCTATGACGCAGCTCAG. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-gRNA-inducible</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">i-WT-r Donor</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-gRNA-inducible</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:259px;">i-MT-r Donor</td>
			<td style="width:317px;"></td>
			<td style="width:101px;"></td>
			<td style="width:368px;">https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM. gRNA and Donor to generate i-MT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genotyping Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gnt-EED-KO-FW-1</td>
			<td></td>
			<td></td>
			<td>Sequence: ctgtaggctgccatctgtga. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gnt-EED-KO-REV-1</td>
			<td></td>
			<td></td>
			<td>Sequence: agccagggctacacagagaa. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-FW-1</td>
			<td></td>
			<td></td>
			<td>Sequence: tgcagtgaaacaaatttggaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-REV-1</td>
			<td></td>
			<td></td>
			<td>Sequence: gagaggggtggcactgtaaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-FW-2</td>
			<td></td>
			<td></td>
			<td>Sequence: ccccctctttctccttttct. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Inducible_Genotype-REV-2</td>
			<td style="width:317px;"></td>
			<td style="width:101px;"></td>
			<td>Sequence: atgcctgggtgaatgaaaaa. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.</td>
		</tr>
	</tbody>
</table>

a method to study de novo formation of chromatin domains

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。

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。?...

Watch this Scientific Journal Video about A Method to Study de novo Formation of Chromatin Domains at JoVE.com

A Method to Study de novo Formation of Chromatin Domains

色质域新形成研究方法

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。

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

我们概述了一个系统，以评估染色质域的动态形成。我们利用这个系统跟踪在细胞中招募和传播 PRC2 中分染色质域。可随时暂停该过程以分析当前事件。该系统可以用于监控任何给定细胞过程的动态变化，因此不限于跟踪染色质域的形成。首先设计指南RNA，使用CRISPR设计工具删除小鼠胚胎干细胞中的胚胎外生胚芽发育（EED）的外生拷贝10和11。根据手稿指示将指南 RNA 克隆到 CRISPR/Cas9 质粒中。在六井板格式中，根据制造商的说明，转染20万只小鼠胚胎干细胞（或mESCs）与每个引导RNA的1微克使用转染试剂。转染后 24 小时更换介质。转染两天后，使用FACS分离GFP阳性细胞。转染效率范围从10%到30%不等，因此对大约50万个细胞进行排序，以捕获足够的GFP阳性细胞进行电镀。将分离的 GFP 阳性细胞板板镀入 15 厘米的板中，这些板预涂有 0.1% 的明胶，用于聚落采摘。在ESC介质中生长细胞约一周，直到单个菌落可见，确保每两天更换一次介质。使用 20 微升移液器尖端在吸气时刮过菌落，至少选择 48 个菌落。在不将菌落分解成单细胞的情况下，将其转移到含有高分酶的96井板中。在37摄氏度下孵育菌点10分钟，分离细胞。然后通过多通道移液器将 200 微升 ESC 介质添加到每井中。混合好，将电池板分成两个独立的96孔板。使用其中一个板进行基因分型，并保持另一个生长，直到基因分型结束。使用商业试剂和制造商的指示从板中提取脱氧核糖核酸。使用跨越删除站点的基因分型底转，使用 Taq DNA 聚合酶执行基因分型 PCR。观察与野生型细胞相对同源缺失的细胞中分子量较低的DNA产品。设计指南RNA使用CRISPR设计工具在EED的外向9之后引入内继的切口，并按照手稿指示克隆到CRISPR/Cas9质粒中。设计一个供体模板DNA，包括 exon 9 之后的 EED cDNA 序列和 T2A-GFP 序列上游的 C-终端标志-HA 标记，所有参数均与内源基因序列相反。用拼接接受器和聚亚二聚亚化序列在异质倒置 loxP 位点之间，从两端至少包含 500 个同源臂碱基对。将捐赠者模板拆分为 gBlocks 基因片段的两部分，然后按照制造商的指示使用吉布森克隆将它们组装成 PCR Blunt 载体。在六井板格式中，具有一微克的引导RNA和一微克的供体模板，可转染20万个MC。然后使用 FACS 分离 GFP 阳性细胞。根据手稿方向分离用于基因分型的单个菌落。使用流细胞学确认mESC没有GP的泄漏表达，如果它们有，在开始实验之前通过FACS分离GFA阴性细胞。将细胞扩大为5个15厘米的板块，通过施用5微摩尔4-羟基-莫西芬，在5个不同的时间持续时间内从零小时到8天来诱导野生型或笼状突变EED的表达。12 小时后更换介质，进行持续时间超过该时间的治疗。使用GFP分离FACS成功重组的细胞，对H3K27me2和H3K27me3执行ChIP-seq，以调查其与染色质的沉积，以响应野生型或笼状突变EED的重新表达。为了验证可诱导野生型救援和诱导突变体救援系统，在诱导野生型或笼状突变EED后，对整体提取物进行了4-羟基-莫西芬治疗。利用流细胞学分析确认4-WT-r细胞T2A-GFP表达在4-羟基-莫西芬治疗后的效率，证明倒置盒的成功翻转。H3K27me3的ChIP-seq是在野生型或笼状突变EED的重新表达后进行的。H3K27me3的出现是在离散核位野生型EED表达后12小时观察到的，然后在远离初始成核点的区域24小时观察到的。ChIP-seq还用于跟踪H3K27me2的时态沉积，该沉积似乎先于H3K27me3沉积。H3K27me3 foci 及其生长也通过荧光显微镜进行可视化。经过12小时的野生型EED表达，有H3K27me3 foci形成的证据。这些物质的数量和大小增加了24小时，最终扩散到核的大区域36小时。DNA构造的精确设计对于生成所需的可致性救援系统具有十分重要的作用。该方法可对小鼠蛋白质突变进行时间和特殊控制。这样，就可以确定突变在特定组织中或特定发育阶段的影响。我们目前使用这个系统来监控另一个染色质域，Polycomb压制复合物1的动态建立。

Research

JoVE Journal

Genetics

A Method to Study de novo Formation of Chromatin Domains

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

利用CyVerse资源<em&gt; De Novo</em&gt;不合格（非模型）生物的比较转录组学

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

科研

遗传学

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

逆转录病毒扫描：映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

血栓烷 A2 受体基因 C924T 多态性的研究方法

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...