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的10厘米的盘子，抬酶在第1.8-1.10所描述的细胞，并用血球计数。 <ol><li>在陈等人准备E8 培养基 。54 </li></ol></li><li>之前加入聚酰胺，具有连接到真空陷阱巴斯德吸管除去用过的培养基。加入新鲜的介质为erological移液器和分配器（每10厘米的培养皿8ml）中。 注意:介质添加到皿的一侧，以避免破坏细胞。从这点向前保护细胞免受光，以避免过早光交联。 </li><li>添加聚酰胺用吸管（8微升400μM的聚酰胺在DMSO，400nM的终浓度）直接向每个培养皿的培养基。涡流盘均匀地分散在聚酰胺到媒体。 注:浓度可以是多种多样的，但确保没有细胞毒性是观察所选的治疗。 </li><li>孵育细胞24小时，37℃，在5％CO 2的潮湿气氛中，并且确保它们避光。 注:培养时间可以变化，以测量聚酰胺跨越时间绑定。 </li><li>用4ml的PBS（1.05毫磷酸二氢钾，155.17毫氯化钠，2.97 mM磷酸氢二钠），使用血清学移液器和DIS清洗每个10-cm培养皿中penser。吸PBS和中加3毫升E8培养基。 </li><li>用灯光变暗，去掉5培养皿的盖子，然后将细胞在平面上的罩的外侧。放置在5培养皿的玻璃过滤器过滤出的光与λ&lt;300纳米。放置在过滤器的顶部的UV源。交联的样品30分钟，以365纳米的紫外线照射（2.4毫瓦/厘米2）。 注:交联时间应根据经验确定。 </li><li>转移培养皿回罩。与连接到一个真空陷阱巴斯德吸管吸媒体。清洗每个10-cm培养皿用4ml PBS中。吸出PBS。加3毫升酶细胞解离（见材料清单）每10厘米的菜以解离细胞。孵育5分钟，在37℃。 注意 :不要预先暖酶。 </li><li>淬火与每皿3 ml的E8媒体的酶。转移解离细胞向一个15毫升锥形管中。 注:将细胞在冰上从这个点开始。 </li><li> CENTRI夫格游离细胞5分钟，500×g离心在4℃下。吸去上清液以除去酶和介质。 注:暂停点 。细胞可以是快速冷冻在液氮中，储存在-80℃下进行后续处理，在以后的时间。 </li></ol> 2.隔离染色质<ol><li>添加1.2 ml的COSMIC缓冲液（20mM的Tris-HCl [pH值8.1]，2mM EDTA的，150毫摩尔NaCl，1％的Triton-X100，0.1％SDS）中，并移液管上下多次重悬细胞沉淀在每个样品1.2毫升COSMIC缓冲区。 <ol><li>加各100mM的苯基甲基磺酰氟（PMSF）133微升，100毫苄脒，和150μM的胃蛋白酶抑制剂蛋白酶抑制剂新鲜至1mM的PMSF和苯甲脒和1.5μM的胃蛋白酶抑制剂的终浓度。细胞裂解物的分裂溶液分成两个琥珀色1.7 ml微量离心管。 </li></ol></li><li>用超声处理超声仪35分钟（10秒上，10秒关闭，60％的功率），以片段的基因组之间100ND 500基点。 <ol><li>保持在平行的微量离心管中染色质溶液的水贮存器中的电平。确认此水平通过视觉检查。 注意:确认DNA的剪切用1.5％琼脂糖凝胶上55 </li><li>优化超声时间经验。使用冰的最小量在储来冷却的样品中，和确保冰不样品和杯角之间的干扰。 </li></ol></li><li>离心机样品12000×g离心10分钟。保存该水溶液含有水溶性染色质通过将其与吸液管转移到一个新的琥珀离心管中。弃沉淀。 </li><li>转移110微升样品（10％），到一个新的离心管中，贴上标签输入DNA。储存在-80℃。保存该染色质样品的其余部分在冰上在步骤3.3中使用。 </li></ol> 3.捕获配体DNA交联的<ol><li>用移液管来分配60微升streptavi在微量离心管嚣包被的磁珠。加入1毫升COSMIC缓冲和组合上的章动5分钟，在室温。放在磁分离磁珠机架2分钟捕捉到珠。除去COSMIC缓冲用吸管。 注意:不要让小球变干。立即进入下一个步骤。 </li><li>添加染色质样品（〜1毫升）珠（60微升），重悬珠子。孵育染色质用链霉亲包被的磁珠至少4小时在旋转，摇动混合机在4℃。如果需要孵育样品O / N。 </li></ol> 4.分离亲和纯化的DNA的<ol><li>用7分钟时间在RT用下面的洗涤缓冲液洗珠以除去非特异性相互作用。使用磁分离架上每次洗涤后拍照珠。在洗涤液每次更改后重悬珠。 </li><li>在使用前制备洗涤缓冲液在蒸馏去离子水和过滤器（0.2微米）。商店洗涤缓冲液1和2在4℃下对于several个月。每天准备洗涤液3清新。添加和用吸管除去样品中的洗涤缓冲液。为2和4，加1 和 3（5毫摩），分别在洗涤。样品还可用COSMIC缓冲器下面列出的洗涤（一次12小时，一次4小时）之前，洗涤两次。 <ol><li>用洗涤缓冲液1（10mM的三Cl [pH 8.0的]，1毫摩尔EDTA，3％SDS）中洗一次。用洗涤缓冲液2（10毫摩尔Tris-氯[pH为8.0]，250mM的氯化锂，1mM的EDTA，0.5％NP40，1％脱氧胆酸钠）洗一次。用洗涤缓冲液洗3次（4M尿素，10毫摩尔Tris-氯[pH为7.5]，1毫摩尔EDTA，0.1％NP-40）。用Tris EDTA（TE）缓冲液（10mM三Cl [pH 8.0的]，1mM EDTA）中洗涤两次。 </li></ol></li><li>重悬在200μlTE缓冲液珠用吸管。捕获的DNA的该样品被称为亲合纯化的（AP）的DNA。 </li><li>补充输入和AP的DNA具有10交联的逆转缓冲液（100mM的三Cl [pH 8.0的]，1M的KOH，4毫EDTA）的1倍终浓度。56,57孵育30分钟，在90℃。 注:254纳米紫外线照射也可以用扭转补骨脂交联，但58环丁基嘧啶二聚体可以从照射而形成在该波长和干扰DNA的下游加工。 </li><li>对于AP的DNA，将装有磁分离样品的离心管架在室温2分钟，分离液体（DNA），用吸管。传送的AP的DNA（其已被释放的珠）到一个新的琥珀离心管中。 注意:所希望的AP的DNA在液体，不再附着于珠。 </li><li>通过用移液管将约1μl的6N HCL中每100微升样品中和的输入和AP的DNA用浓HCl至pH 7。确认样品中加入〜0.5微升至pH试纸用吸管警告中和:将浓HCl是强腐蚀性的酸。根据你的机构处理217; S准则相应的个人防护装备。 </li><li>加入RNA酶A（100毫克/毫升），以0.2微克/微升的最终浓度，以两者的输入和AP的DNA。孵育1小时，在37℃。添加蛋白酶K（20毫克/毫升），以0.2微克/微升的最终浓度，以两者的输入和AP的DNA，加。孵育1小时，在55°C。 </li><li>净化用DNA柱净化试剂盒的DNA（见材料清单）。55洗脱DNA的58微升DNA级H 2 O.存储输入和AP样品在-20℃或-80℃ </li><li>分析美联社DNA的定量聚合酶链反应（qPCR的）与基因座特异性的引物，和/或通过新一代测序。对于定量PCR，采用每个位点2微升AP DNA以下参数:95℃10分钟1个循环和95℃20秒40个循环，54℃或56℃20秒，72℃40秒。 注:退火温度应根据所使用的引物对的熔化温度进行修改。选择一个退火temperatu重，最大限度地减少了定量周期没有非特异性扩增。 注:对于通过新一代测序分析，目的是为至少有1000万映射读取。的映射读取数可以通过增加的AP DNA的量与由较少的样本组合成用于测序运行。更读取提高灵敏度。标准封装的芯片起分析 （例如，荷马，59 MACS，60和最高人民检察院61）与宇宙序列数据进行工作。作为与依靠新一代测序等方法，包括基因组测序和芯片起，重复区域创建对齐和组装含糊不清，因此仍然是一个技术挑战。 </li></ol>

A.Z.A. is the sole proprietor of Vista Motif, LLC and WINStep Forward.

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 polyamide&#8211;DNA crosslinks. The interaction between biotin and streptavidin is one of the strongest known in nature and one thousand to one million times stronger than the interaction of an antibody with its ligand.62 We harness this robust interaction, and the intrinsic stability of streptavidin, by subjecting captured polyamide&#8211;DNA crosslinks to harsh washes that would denature an antibody. These washes reduce the background signal of COSMIC. Furthermore, biotinylated proteins such as histones unfold and dissociate from DNA under these harsh conditions.63,64
The addition of the biotin moiety increases the size of the molecule, which could be reduced by substitution with a &#8216;clickable&#8217; handle such as an alkyne.43-46 This handle could be used to introduce the biotin moiety after the cells have been lysed. Taken together, COSMIC avoids one of the major sources of unreliability that plagues many ChIP experiments.
Although formaldehyde will crosslink protein&#8211;DNA interactions, formaldehyde also crosslinks protein&#8211;protein interactions.65,66 Thus, formaldehyde can lead to the misidentification of DNA-binding events that are indirect or transient.65,66 This issue is especially prominent in highly transcribed regions.67 COSMIC uses a photocrosslinker (psoralen) instead of formaldehyde to avoid the issues of formaldehyde-based crosslinking. Because a light-sensitive crosslinker is used, the sample must be shielded from light to prevent premature crosslinking.
We have performed COSMIC in multiple conditions and cell types. The cell type, concentration of polyamide, and duration of treatment can all be varied, but it is important to confirm that polyamide treatment does not induce cellular toxicity. It is also critical to empirically determine a sonication time that produces sheared DNA with a size between 100 and 500 bp. If the COSMIC signal is low, optimize the number of cells, incubation time, concentration of polyamide, and UV crosslinking time.
The bioinformatic methods to analyze ChIP-seq data are compatible with COSMIC-seq data. Similar to ChIP, COSMIC requires normalization to a reference sample of fragmented DNA that has not been enriched with an antibody or streptavidin. This sample, normally called input DNA, is designed to control for many possible sources of bias including sonication efficiency, PCR bias, sequencing bias, and mappability of DNA fragments. We have found success with the ENCODE guidelines to identify peaks in our COSMIC-seq datasets.50,68
Some of the most successful therapeutic agents are molecules that bind to DNA and interfere with an array of genomic transactions.43,69,70 COSMIC can be readily applied to small molecules other than polyamides. The crosslinker can be UV active, as we have chosen, or it could also be DNA-reactive.69,70 In summary, COSMIC reveals the binding sites of polyamides and other ligands throughout the genome and uncovers new strategies to selectively target DNA.

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.remotexs.ntu.edu.sg/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，用一个商业样品制备试剂盒，见材料清单）。即使新一代测序，我们还是用定量PCR来确认DNA的富集在基因位点，我们预计聚酰胺的约束。一旦测序，原始的DNA读取被对准以在基因组和密度迹线制备设计成分析芯片起数据（图3）相同的软件。巴sed的我们在细胞聚酰胺分布进行分析，我们发现，聚集的结合位点，涵盖范围广泛的亲和力，最佳预测占用的细胞。我们开发了一种算法得分整个基因组与我们的体外 CSI数据（图4A）的结合。这种评分方法表明，不同的基因组位点有类似预测的结合分数表现出不同的亲和力（ 图4B）的多个站点的不同集群。 <img alt="图2" src="/files/ftp_upload/53510/53510fig2.jpg" /> 图 365纳米的紫外线照射宇宙的qPCR的影响对 AP /输入来自HEK293细胞的比例2的结果 。 AP时，亲和纯化。结果绘制在对数尺度和表示平均±SEM <img alt="图3" src="/files/ftp_upload/53510/53510fig3.jpg"/> 网络连接古尔COSMIC-SEQ的H1细胞3.结果。线性聚酰胺 4设计目标AAG重复宇宙序列标记密度轨道。标签密度标准化为10 7的标签和综合基因组查看器中显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53510/53510fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53510/53510fig4.jpg" /> 聚酰胺图4.模型绑定。（A）地块小提琴预测分数的整个基因组的2和4的结合。还示出了 4代表genomescapes。用生物信息学评分法采用，基因位点可以归纳到相同预测得分以不同的方式。 （B）位点与多个低收入和中等亲和力序列翔W¯¯类似的聚酰胺入住基因座与几个高亲和力的序列。授权转载。51 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53510/53510fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 ...

genome-wide-mapping-drug-dna-interactions-cells-with-cosmic

Nanyang Technological University

Research

JoVE Journal

Biology

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

8.2K 次观看.  University of Wisconsin–Madison. 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）。 

视频: 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

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

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

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

（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

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

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

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

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

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

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

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

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

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

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

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

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

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

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

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

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 ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

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

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

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

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

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

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

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

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

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 ...