Name: genome-wide mapping of drug-dna interactions in cells with cosmic (crosslinking of small molecules to isolate chromatin)
Uploaded: 2016-01-20T14:00:00
Description: Watch this Scientific Journal Video about Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin at JoVE.com

We thank members of the Ansari lab and Prof. Parameswaran Ramanathan for helpful discussions. This work was supported by NIH grants CA133508 and HL099773, the H.&#8197;I. Romnes faculty fellowship, and the W.&#8197;M. Keck Medical Research Award to A.Z.A. G.S.E. was supported by a Peterson Fellowship from the Department of Biochemistry and Molecular Biosciences Training Grant NIH T32 GM07215. A.E. was supported by the Morgridge Graduate Fellowship and the Stem Cell and Regenerative Medicine Center Fellowship, and D.B. was supported by the NSEC grant from NSF.

1.交联活细胞<ol><li>首先〜2.5×10 7的H1细胞或其它培养的细胞。 注意：此数的H1细胞的对应于5 10-cm的培养皿（约40％铺满）。 </li><li>生长的细胞在E8媒体上碗碟涂覆在支持多能干细胞的表面（参见材料列表），并培育它们在37℃下在5％CO 2的潮湿气氛。酶收获细胞（见材料清单）。注意：不要让H1细胞超过90％汇合;汇合诱导H1细胞的自发分化。要计算细胞，培养细胞的一个额外?...

One of the primary challenges with conventional ChIP is the identification of suitable antibodies. ChIP depends heavily upon the quality of the antibody, and most commercial antibodies are unacceptable for ChIP. In fact, the Encyclopedia of DNA Elements (ENCODE) consortium found only 20% of commercial antibodies to be suitable for ChIP assays.50 With COSMIC, antibodies are replaced by streptavidin. Because polyamides are functionalized with biotin, streptavidin is used in place of an antibody to capture polyam...

的信息，使在人体每个小区被编码在DNA中。有选择地利用这些信息用于调控细胞的命运。转录因子（TF）是结合特定的DNA序列，以表达在基因组中的基因的特定子集蛋白和转录因子的故障是与疾病各种各样的发作，包括发育缺陷，癌症和糖尿病1,2我们一直有兴趣开发分子，可以选择性地结合到基因组中并调节基因调控网络。 聚酰胺由 N个-methylpyrrole的和 N-甲基咪唑能合理设计的分子，可以靶DNA与特异性和亲和力，其竞争对手天然转录因子。3-6这些分子结合在DNA的小沟中的特异性序列。4,5,7 -11聚酰胺已采用两个再加压和激活的特定克的表达埃内斯。4,12-19他们也有有趣的抗病毒20-24和抗癌12,13,25-30性能。聚酰胺之一有吸引力的特点是它们的访问被甲基31,32和缠组蛋白9,10,33 DNA序列的能力。 为了测量DNA结合分子的全面结合特异性，我们实验室建立的同源站点标识符（CSI）的方法。34-39的基于体外的特异性（genomescapes）结合位点的预测的发生可以在基因组上被显示，因为在体外结合强度成正比缔合常数（K a）的 34,35,37，这些genomescapes洞察在整个基因组聚酰胺占用，但测量聚酰胺在活细胞中的结合一直是个难题。 DNA被紧紧包装在细胞核中，它可能会影响结合位点的可接近性。在一个这些chromatinized DNA序列聚酰胺ccessibility仍是一个谜。 最近，许多方法来研究已经出现小分子和核酸之间的相互作用。40-48的化学亲和捕获和大规模并行DNA测序（CHEM-SEQ）是一种这样的技术。的Chem-以次使用甲醛交联的小分子到感兴趣的基因组靶和小分子的感兴趣的生物素化的衍生物捕获配体-靶相互作用。48,49 甲醛的交联导致了可以产生假阳性的间接相互作用。50我们开发了一种新方法，小分子的交联以分离染色质（COSMIC），51与光交联剂，以消除这些所谓的“幻影”的峰。50首先，我们设计并合成聚酰胺的三官能的衍生物。这些分子含有一个DNA结合聚合酶yamide，光交联剂（补骨脂素），和亲和手柄（生物素， 图1）。用三官能聚酰胺，我们可以共价捕获与365nm的紫外线照射下，一个波长不损害DNA或诱导非补骨脂类交联剂聚酰胺-DNA相互作用。51接下来，我们片段的基因组中，净化捕获的DNA在严格的，半-denaturing条件以降低的DNA即非共价结合。因此，我们认为COSMIC作为与化学 - 以次的方法，但用的DNA的更直接读出定位。重要的是，弱（K 10 3 -10 4 M -1）补骨脂用于DNA的亲和力没有可检测的影响的聚酰胺特 ​​异性。51,52富集的DNA片段可以通过定量聚合酶链反应51待分析（COSMIC-qPCR的）或者通过新一代测序53（COSMIC-SEQ）。这些数据使能配体的一个不带偏见，全基因组指导下设计，跨充当与他们所希望的基因位点，并尽量减少脱靶效应。 <img alt="图1" src="/files/ftp_upload/53510/53510fig1.jpg"/> 图1.生物活性聚酰胺和宇宙方案 。 （一）夏萍聚酰胺1 - 2靶DNA序列5&#39;-WACGTW-3“。线性聚酰胺3 - 4靶 5&#39;- AAGAAGAAG-3&#39;。 N-甲基咪唑的环以粗体显示的清晰度。开放和充满圆圈表示 N -methylpyrrole 和 N甲基咪唑，分别。方块代表3氯噻吩和菱形代表β丙氨酸。补骨脂素和生物素是由P和B，分别表示。 （二）COSMIC计划。细胞用聚酰胺的三官能的衍生物。用365nm的UV照射交联后，将细胞是升ysed和基因组DNA被剪切。链亲和素包被的磁珠加入到捕获聚酰胺的DNA加合物。该DNA被释放，并可以通过定量PCR（定量PCR）或下一代测序（NGS）进行分析。 <a href="https://www.jove.com/files/ftp_upload/53510/53510fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td style="width:180px;">any source</td>
			<td style="width:172px;"></td>
			<td style="width:75px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benzamidine</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dynabeads MyOne Streptavidin C1</td>
			<td>Life Technologies</td>
			<td>65001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>Other sources can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">StemPro&#174; Accutase&#174; Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>We have tried other manufacturers of DNA columns with success.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TruSeq ChIP Sample Prep Kit</td>
			<td>Illumina</td>
			<td>IP-202-1012</td>
			<td>This Kit can be used to prepare COSMIC DNA for next-generation sequencing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td>Used to coat plates in order to grow H1 ESCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH paper</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">amber microcentrifuge tubes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microcentrifuge tubes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pyrex filter</td>
			<td>any source</td>
			<td></td>
			<td>Pyrex baking dishes are suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR master mix</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl (6 N)</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10-cm tissue culture dishes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Serological pipettes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pasteur pipettes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette tips</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15-mL conical tubes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">centrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microcentrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nutator</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic separation rack</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV source</td>
			<td>CalSun</td>
			<td>B001BH0A1A</td>
			<td>Other UV sources can be used, but crosslinking time must be optimized empirically</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Misonix Sonicator</td>
			<td>Qsonica</td>
			<td>S4000 with 431C1 cup horn</td>
			<td>Other sonicators can be used, but sonication conditions must be optimized empirically</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Humidified CO2 incubator</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biological safety cabinet with vacuum outlet</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide mapping of drug-dna interactions in cells with cosmic (crosslinking of small molecules to isolate chromatin)

基因组是一些最有效的化学治疗剂的目标，但大多数这些药物​​缺乏DNA序列特异性，从而导致剂量限制性毒性和许多不良副作用。靶向序列特异性小分子的基因组中可使得分子具有增加的治疗指数和更少的脱靶效应。ñ-methylpyrrole / N甲基咪唑聚酰胺是分子，可以进行合理设计为靶向精致精度特定的DNA序列。不像最自然的转录因子，聚酰胺可以绑定到甲基化和chromatinized DNA无亲和力的损失。聚酰胺的序列特异性已被广泛研究在体外用同源站点识别（CSI），并用传统的生化和生物物理方法，但聚酰胺的细胞结合的基因组的目标的研究仍然难以捉摸。在这里，我们报告的方法，小分子交联伊索拉德染色质（COSMIC），标识在整个基因组聚酰胺结合位点。 COSMIC类似于染色质免疫沉淀，但不同之在两个重要方面：（1）一个光交联剂被用来使选择性的，在时间上控制捕获的聚酰胺结合事件，和（2）的生物素亲和手柄用于纯化聚酰胺半变性条件下-DNA偶联物降低的DNA即非共价结合。宇宙是可用于揭示聚酰胺和其他基因组靶向化学治疗剂的全基因组结合事件一般策略。

为了说明非均匀的基因组片段化和其他变量，纯化的DNA应该总是被规范化对输入DNA的参考。具体到感兴趣的基因座的引物都可以使用。是有帮助的同时分析的轨迹在未预期的分子结合，作为阴性对照。我们看到，在照射&gt; 100倍的聚酰胺占用365纳米的光（ 图2）。 富集的DNA也可以通过下一代测序。 DNA被制备用于测序中相同的方式的ChIP的DNA制备（例如，用一...

Watch this Scientific Journal Video about Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin at JoVE.com

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

识别基因组靶向分子的直接目标仍然是一个重大的挑战。要了解DNA结合分子如何参与基因组，我们开发了依靠小分子交联，染色质隔离的方法（COSMIC）。

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Research

JoVE Journal

Biology

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

科研

生物学

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...