在这个手稿的工作是由放射肿瘤学系和Huntsman癌症研究所和卫生拨款的国家研究所（R01-CA188520），以SB的资金支持。我感谢丹尼尔·约翰逊和史蒂芬·贝内特在我的实验室演示的协议和解释它的好处。我很感谢马赫什Chandrasekharan博士（Huntsman癌症研究所）对稿件批评意见。

1.染色质免疫沉淀（ChIP）以下BrdU标记<ol><li>培养NIH3T3细胞在DMSO或HDAC1,2选择性抑制剂的存在下如前面6所述。 </li><li>以下用DMSO或HDAC1,2选择性抑制剂治疗，在无菌组织培养罩添加BrdU的细胞中。孵育2×10 6个NIH3T3细胞接种在10ml的NIH3T3介质溴脱氧尿苷的20μM的最终浓度（BrdU）标记60分钟在无菌37℃组织培养箱中。 </li><li>交联细胞内蛋白质的DNA直接添加甲醛到培养基在1％的终浓度。孵育2×10 6的NIH3T3细胞与甲醛在室温在振荡器上10分钟。 </li><li>淬火通过加入甘氨酸至125 1mM的终浓度的交联。在室温下孵育5分钟，在摇动器上。 </li><li>取出纸张，收集细胞在PBS在15毫升离心管中。自旋CELLS在956 XG 5分钟，在4℃。用冰冷的PBS洗两次细胞沉淀。从管中取出PBS中。存储而不PBS（上清液）的交联的细胞沉淀在-80℃下直到使用。 </li><li>通过加入500微升FA140缓冲器准备从交联细胞沉淀的裂解物（50 HEPES的KOH毫pH为7.5，140 mM氯化钠的，1毫摩尔EDTA（pH8.0）中，1％的Triton-X-100和0.1％脱氧胆酸钠）补充有蛋白酶抑制剂混合物。 </li><li>通过超声处理样品5次35％的电力，并用15秒脉冲，0.9秒上和0.1秒关闭设定剪切染色质。保持在冰上的管中每一轮超声处理后，以避免样品的加热。 <ol><li>检查剪切的DNA凝胶作为剪切效率会不同sonicators之间变化上。要检查超声，反向交联加入NaCl至0.16米的浓度和执行步骤1.17.1。 - 1.22。运行洗脱10微升的DNA在1.5％琼脂糖凝胶上以检查是否剪切是之间的300 - 700个碱基，在500 bp的平均强度。 </li></ol></li><li>以下超声处理，离心样品10分钟，在17949×g离心在4℃下，将上清液转移到一个新试管中。 <ol><li>使用根据制造商的协议商用蛋白测定试剂盒进行蛋白估计。用裂解液等量的对照和实验样品的沉淀。一般情况下，使用1毫克每芯片的反应总裂解液。 </li></ol></li><li>准备使用含有蛋白酶抑制剂FA140缓冲蛋白A琼脂糖50％的浆液。加入1/10体积的蛋白酶抑制剂的鸡尾酒。计算所需珠基于在总的样本数的体积。 <ol><li>洗涤蛋白A琼脂糖珠FA140缓冲两次以除去存储缓冲器和等体积FA140缓冲器添加到珠子上，使50％的浆液。 </li><li>以除去蛋白质的非特异性结合的珠子，添加50μl的蛋白A琼脂糖的和在50％的淤浆cubate样品在4℃下以恒定的最终过端旋转旋转式烤架混合器30分钟。 </li></ol></li><li>旋转样品在956×g离心在4℃下进行一分钟。收集上清液，并通过添加任8微升H4K16ac抗体或5微升1mg / ml的SMARCA5抗体（5微克）在上清液进行免疫沉淀。孵育在4°CO / N在一个烤肉店搅拌机恒端过端的转动。 </li><li>保存提取物"输入"控制的1/10，并设置为预留在-20℃。输入需要反向以及IP样品交联的。 </li><li>使用等量兔IgG（5微升1mg / ml的）作为阴性对照免疫沉淀的样品。 </li><li>次日，添加50微升50％的蛋白A琼脂糖浆提取物，孵育1小时，在4℃下用旋转。 </li><li>在4℃颗粒琼脂糖珠离心956 XG 2分钟，小心地取出上清，WAsh的珠粒与顺序如下缓冲液。在缓冲制剂，使用前右侧添加蛋白酶抑制剂。 <ol><li>低盐免疫复合物的洗涤缓冲液洗（FA140; 50mM的HEPES的KOH pH为7.5的终浓度，140mM的氯化钠，1mM EDTA中（pH为8.0），1％的Triton-X-100和0.1％脱氧胆酸钠的），用于5分钟，在4℃下与最终过端旋转。 </li><li>用高盐免疫复合物的洗涤缓冲液洗（FA500; 50mM的HEPES的KOH pH为7.5的终浓度，500mM的氯化钠，1mM EDTA中（pH为8.0），1％的Triton-X-100和0.1％脱氧胆酸钠的），用于5分钟，在4℃下与最终过端旋转。 </li><li>同的LiCl免疫复合物的洗涤缓冲液进行5分（三Cl pH值8.0，250mM的氯化锂，0.5％NP40和0.5％脱氧胆酸钠的10毫摩尔终浓度）在4℃下与最终过端旋转清洗。 </li></ol></li><li>如上所述下列洗涤，加入200微升的洗脱溶液和incubat（1％SDS和100mM碳酸氢钠的终浓度）Ë在室温为15分钟，在室温月底环比端的转动。 </li><li>旋转样品在956×g离心在RT和洗脱液转移到一个新的1.5毫升管。额外的200微升洗脱液重复步骤1.15。 </li><li> 400微升洗脱液，氯化钠加入到了0.16米的浓度，搅拌均匀。 <ol><li>此外，新增400微升洗脱的解决方案被搁置10微升输入（步骤1.11），并添加氯化钠，以0.16米的浓度，以扭转交联输入样本为好。扭转孵育在65℃水浴中5小时交联的样品。 </li></ol></li><li>加入1毫升100％乙醇到反向交联洗脱液。拌匀，孵育在-80℃下2 - 3小时，或在-20°CO / N。旋转样品在17949×g离心15分钟并弃去乙醇。 </li><li>添加800微升70％乙醇洗涤沉淀。旋在17949×g离心15分钟，在4℃并丢弃乙醇。旋简要地，以去除任何残留的乙醇。空气干燥样品，在37℃下进行10分钟以去除残留的乙醇。 </li><li>溶解的DNA在90微升RNA酶和DNA酶的水和加入2微升10毫克/毫升RNA酶在0.2毫克/毫升的最终浓度。在水浴在37℃进行30分钟。 </li><li>添加10微升10X蛋白酶K缓冲液（100mM的三Cl pH为8.0，50毫摩尔EDTA pH为8.0，5％SDS）中。搅拌均匀，加入1μl蛋白酶K.孵育42℃1小时。 </li><li>使用根据商用PCR纯化试剂盒制造商的协议净化输入和沉淀的DNA并在50μl水中洗脱的DNA。商店的DNA在-20℃直至进一步使用。 </li></ol> 2.插槽的ChIP的DNA印迹分析<ol><li>测量输入和沉淀的DNA在50μl水中洗脱的产率作为用分光光度计在280 nm处，根据制造商的协议，在步骤1.22中描述。 </li><li>取输入的是检测的线性范围内的等体积（50微升）或免疫沉淀的DNA。 <ol><li>例如，如果输入DNA产量为1.5纳克/微升，然后取30微升（或45 ng总DNA），并卷至50微升水。对于要素沉淀，需要从步骤2.1整个50微升洗脱液，并继续执行步骤2.3。 </li></ol></li><li>变性50微升输入或免疫沉淀的DNA中加入2.5体积的0.4的1N NaOH，短暂涡旋，离心956×g离心10秒，并在室温下孵育30分钟。 </li><li>通过加入175微升的1M Tris-HCl中，pH值6.8，涡旋，离心956×g离心10秒中和变性的DNA，然后将样品在冰上。 </li><li>制备系列稀释的输入和免疫沉淀的DNA用于插槽印迹测定法，如下所述。 <ol><li>按照步骤2.3 - 2.4得到的350微升总样本量（ 即 50微升输入/芯片DNA，125微升0.4 1N氢氧化钠，175微升的1M的Tris-HCl，pH值6.8）。 注:对于上面的例子说明，45纳克的DNA模板，都会变成0.129纳克/微升。 </li><li>对于输入DNA，标签三管小号为100，50和25，并添加100微升，50微升和25微升变性和中和的DNA样品。使终体积为100微升用无核酸酶水。芯片的DNA，标记三个管200，100和50，并添加200微升，100微升和50微升变性和中和的DNA样品。使终体积为200微升用不含核酸酶的水。 </li></ol></li><li>切膜到达槽印迹装置的尺寸。预润湿的膜和吸油纸在20×SSC之前和之前添加的DNA。根据制造商的方案将它们组装成的槽印迹装置。 </li><li>吸取DNA步骤2.5.2为适当的插槽。适用于真空慢慢拉DNA到泽塔探头膜。停止真空后所有样品都通过装置。 <ol><li>拆卸该装置，并立即将DNA固定到使用UV交联剂膜。有该DNA的一面朝上在UV交联剂和使用盟tocrosslink在交联剂设置。按照制造商的协议。 </li></ol></li><li>检测的BrdU的插槽印迹通过执行印迹。 <ol><li>方框用5％无脂干牛奶的PBS含有0.1％Tween-20（PBST）中进行1小时，在室温，以防止抗体的非特异性结合到所述膜的膜。 </li><li>添加初级抗BrdU抗体（1:500稀释度）稀释于1％非脂肪干乳在PBST中孵育与第一抗体的印迹3小时在室温在振荡器上。第一抗体温育后用PBST洗膜三次。 </li><li>添加HRP偶联的抗小鼠二抗的印迹（1:5000稀释）和在室温下孵育1小时。第二抗体培养后用PBST洗膜三次。 注:使用足够量的抗体，以完全覆盖在初级和二级抗体孵育膜。 </li></ol></li><li>制备ECL混合并把它添加到印迹交流盘带制造商的协议。孵育用ECL混合膜5分钟，室温。 <ol><li>将膜在X射线盒，暴露在膜的X射线胶片和开发使用制造商的方案的印迹。 </li><li>量化使用根据制造商的协议的的Image J软件扫描印迹后所获得的输入和ChIPed DNA中的信号。用于定量，使用是检测的线性范围内，以确保测量的精确性的信号。 </li></ol></li></ol>

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I have no competing financial interests.

在这个手稿中描述的协议是一个相对快速的方法以证明对新复制或新生的DNA的蛋白质或它们的翻译后修饰的形式的存在。此外，这种技术允许一个以测量的蛋白质或它与新生的DNA修饰形式的关联-解离动力学。这种技术是相辅相成的优雅iPOND技术13。在iPOND技术，新合成的DNA标记用乙酸乙酯脱氧尿苷（EDU）。生物素结合物，然后用点击化学添加到EDU。生物素标记的新​​生DNA，然后用链霉珠和共纯化蛋白的免疫印迹检测免疫沉淀。另一方面，在我们的BrdU芯片插槽西方技术，用新生态的DNA结合的蛋白质或它的修饰形式是利用蛋白特异性或修饰形式特异性抗体和BrdU标记新生的DNA结合于量免疫沉淀蛋白质是个连接使用抗BrdU抗体定量测定按插槽-Western印迹。 还有在这个协议中，需要特别注意的关键步骤。以确保目标抗体工作良好标准ChIP实验以高效率是至关重要的。它执行的BrdU-CHIP插槽西方测定前测试中ChIP实验通过定量PCR检测的抗体的效率是重要的。为了使BrdU的片槽西方技术的定量是准确的BrdU标记的DNA的系列稀释应适用于狭槽印迹，并使用抗BrdU抗体西方分析应以最初执行以确定检测的线性范围。确定的ChIP的DNA浓度和输入的DNA使得人们能够确保BrdU标记的DNA的量是检测在Western印迹的线性范围内。例如，我们确定了50毫微克至12.5纳克输入DNA是在线性范围中的中试。而且，如果芯片的样品的浓度是非常高的，就必须在执行前槽以稀释的ChIP的DNA。该技术的限制是它的分辨率，这取决于染色质剪切的效率。染色质的通过超声剪切确定给定蛋白质或其修饰的形式向新合成的DNA的附近。 在过去，改变在哺乳动物细胞中的DNA复制过程中的染色质和染色质修饰酶的研究仍然是一个困难的问题，以解决由于适当的技术的不可用性。此外，由于Hdac1,2空细胞停滞于G1期，这是很难检查内S相对于Hdac1,2功能。在我们的研究中，我们使用的先入类选择性抑制剂的组合，以抑制内S相和新颖的BrdU的ChIP-S它们的活动结果显示这两个功能的HDAC DNA复制过程很多西方的技术。在将来，这种技术也可以用于研究DNA损伤应答和DNA修复蛋白的动力学的停滞叉除了研究组蛋白修饰和染色质修饰酶在交岔路口。而且，该技术不限于小鼠NIH3T3细胞。这种技术可以成功地用于在其他细胞系为了研究酶或蛋白质的其它抑制剂如何影响DNA复制和复制应激诱导染色质的变化，在复制叉。

有缺陷的DNA修复和/或DNA复制是基因组不稳定性，这可以触发细胞死亡的一个主要原因。单个未修复的双链断裂足以导致细胞死亡1。染色质组织都复制期间瞬时改变和修2,3，和故障维护表观遗传信息在这些过程将导致威胁基因组的完整性。 HDAC3或HDAC1,2功能丧失阻碍S期的进展，DNA复制和修复，导致基因毒性应激（DNA损伤）和细胞死亡4-9。因此，它是一个实际的策略，利用选择性HDAC抑制剂破坏复制，修复和染色质在癌细胞和引起DNA损伤，这反过来又可以停止生长，诱导细胞死亡选择性地迅速增长的癌细胞。 期间DNA复制，染色质快速拆卸和重新组装下列DNA重复。新学年nthesized组蛋白H4设置在K5和K12残基（H4K5K12ac）和以下沉积乙酰到染色质，它们被迅速由组蛋白脱乙酰酶（HDACs）10,11脱乙酰。细胞未能保持由组蛋白去乙酰化新生染色质完整性可导致叉崩溃，这反过来可导致DNA损伤和细胞死亡。我们最近发现，HDAC1,2选择性抑制增加组蛋白乙酰化（H4K5ac，H4K12ac和H4K16ac）并抑制SMARCA5染色质remodeler活动上新生染色质，这与减少的复制叉进展，增加的叉崩溃和增加的复制压力诱导的DNA损伤6 。因此，HDAC抑制剂治疗可以改变新生染色质结构，以非常快速地触发DNA损伤和死亡癌细胞中，因为这些细胞周期迅速并通过S相通许多次。据了解的HDAC如何发挥作用来调节组蛋白乙酰化和蛋白质宾迪因此，重要纳克维持在哺乳动物细胞中DNA的复制。 定量地测量组蛋白乙酰化和DNA复制过程中与新生的DNA分子上SMARCA5染色质remodeler的量，我们设计了一种改性ChIP实验称为BrdU的芯片插槽西方技术。使用槽孔以下的所希望的蛋白质或组蛋白修饰染色质免疫沉淀（ChIP），新生的DNA的量的沉淀样品中可以使用的BrdU标记的ChIP的DNA印迹分析来检测（使用胸苷类似物，BrdU阳性）转移到膜印迹装置。使用这种技术，我们发现，H4K16ac（参与染色质包装的标记）和ISWI家族成员SMARCA5（染色质SWI / SNF相关的基质相关的肌动蛋白依赖调节剂）的染色质remodeler与在S期细胞新生DNA的关联6。我们还发现，新生H4K16ac标记通过HDAC1,2 DNA复制6中脱乙酰。 H4K16ac抑制在ISWI家族成员SMARCA5 12的染色质remodeler活动。因而，使用的BrdU片插槽西方技术中，我们可以连接为HDAC1,2的功能在DNA复制过程中调节染色质重塑。因此，尿嘧啶芯片插槽，Western印迹技术是一种有效的方法来定量测量绑定到新生的DNA或蛋白质的翻译后修饰的结合和解离动力学。

<table><tbody><tr><td>Anti-BrdU (westerns)</td><td>BD Biosciences</td><td>B555627</td><td></td></tr><tr><td>Anti-SMARCA5</td><td>Abcam</td><td>ab3749</td><td></td></tr><tr><td>Anti-H4K16ac</td><td>Active Motif</td><td>39167</td><td></td></tr><tr><td>Zeta-Probe GT Membrane</td><td>Bio-Rad</td><td>162-0197</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>Protein A agarose</td><td>Millipore</td><td>16-156</td><td></td></tr><tr><td>Rnase A</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P4850</td><td></td></tr><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>BrdU</td><td>Sigma</td><td>B9285</td><td></td></tr><tr><td>Rabbit IgG&amp;nbsp;</td><td>Millipore</td><td>12-370</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S-3014</td><td></td></tr><tr><td>Triton-X-100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>NP40</td><td>USB Corporation</td><td>19628</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>D6750</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S-4019</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>04-355-222</td><td></td></tr><tr><td>DEPC-treated water</td><td>Sigma</td><td>95284</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>BP-152-5</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>SDS</td><td>Ambion</td><td>AM9820</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Mallinckrodt GenAR&amp;nbsp;</td><td>MAL7772-06</td><td></td></tr><tr><td>20x SSC</td><td>Life Technologies</td><td>15557-036</td><td></td></tr><tr><td>Non-fat dry milk</td><td>Lab Scientific Inc</td><td>M0841</td><td></td></tr><tr><td>ECL</td><td>Thermo Fisher</td><td>80196</td><td></td></tr><tr><td>X-ray film</td><td>Genesee Sci</td><td>30-101</td><td></td></tr><tr><td>Developer</td><td>Konica Minolta</td><td>SRX-101A</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>XLE-1000</td><td></td></tr><tr><td>Sonicator</td><td>Branson Sonifier&amp;nbsp; Digital Ultrasonic Cell Disruptor</td><td>Model: 450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Model: 5810R</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific Isotemp</td><td>Model: 2322</td><td></td></tr><tr><td>NanoDrop 1000 spectrophotometer</td><td>ThermoScientific</td><td>Model: 1000</td><td></td></tr><tr><td>Slot Blot apparatus&amp;nbsp;</td><td>Schleicher and Schuell Minifold II</td><td>44-27570</td><td></td></tr><tr><td>Tissue Culture Incubator</td><td>Thermo Scientific</td><td>Series II 3110 Water-Jacketed CO2 Incubators</td><td></td></tr></tbody></table>

examination of proteins bound to nascent dna in mammalian cells using  brdu-chip-slot-western technique

组蛋白脱乙酰1和2（HDAC1,2）定位于DNA复制的位点。在以前的研究中，使用的选择性抑制剂和遗传击倒系统，我们在复制叉进展和在哺乳动物细胞中染色质新生维护表明新颖功能HDAC1,2。此外，我们使用了结合的溴脱氧尿苷（尿嘧啶）标记的DNA染色质免疫沉淀（ChIP）与狭槽印迹和西方分析定量测量与新生的DNA结合蛋白或组蛋白修饰的BrdU芯片插槽西方的技术。 活跃分裂细胞用HDAC1,2选择性抑制剂或转染的抗HDAC1和 HDAC2的siRNA，然后新合成的DNA进行标记与胸苷类似物溴脱氧尿苷（BrdU）标记。 BrdU标记做在一个时间点没有显著细胞周期阻滞或凋亡因HDAC1,2功能的丧失。用L细胞用BrdU的组蛋白乙酰化的痕迹，染色质免疫沉淀或染色质remodeler的abeling用特异性抗体进行。 BrdU标记输入DNA和免疫沉淀（或ChIPed）DNA随后点样到使用狭缝印迹技术的膜和使用UV固化。新生DNA的每个时隙的数量，然后定量地使用Western分析用抗BrdU抗体进行评估。对新合成的DNA相关的组蛋白乙酰化的标记和染色质remodeler水平HDAC1,2功能损耗的效果然后通过从处理过的样品，以对照样品中获得的BrdU的芯片信号标准化来确定。

为了确定HDAC1,2选择性抑制剂的特异性，Hdac1,2 FL / FL和 HDAC3 FL / FL纤维肉瘤细胞。 Cre重组酶（AD-CRE）含有腺病毒是用来删除Hdac1,2和HDAC3在这些细胞中。以下广告Cre重组感染Hdac1,2 FL / FL的 细胞中，强劲增长H4K5ac进行了观察。 Hdac1,2敲除细胞用233或898的治疗不导致确认233和898抑制Hdac1,2和不HDAC3在这些细胞中的任何进一步增加在H4K5ac。与此相反，在加入233或898至HDAC3敲除细胞导致在H4K5ac一个显著相比增加的增加见于HDAC3敲除细胞由于对H4K5ac水平抑制Hdac1,2和3的结果累加效应。因此， 233和898是HDAC1，2选择性抑制剂。这些结果示于图1。 为了验证BrdU的芯片隙技术，进行BrdU的脉冲大通分析看PCNA装载的动力学上的HeLa细胞新生的DNA。我们的研究结果表明，新生的DNA PCNA协会迅速发生在15分钟和30分钟的追逐同意与先前公布的结果，13后消失。这些结果示于图2。 的BrdU-H4K16ac沉淀槽西方技术被用于确定与在不存在HDAC1,2功能的新生DNA分子上H4K16ac的量。相比时兔IgG对照（ 图3A和3B）的鲁棒富集H4K16ac与新生的DNA分子进行了观察。在以下的抑制HDAC1,2活动或Hdac1,2击倒新生DNA的相关H4K16ac的增加示于图3C中 </STRONG&gt;，3D和3E。 使用SMARCA5染色质remodeler对新生DNA水平确定的BrdU SMARCA5沉淀槽西方的技术。我们的结果表明，SMARCA5同伙与在哺乳动物细胞新生的DNA。此外，HDAC1,2抑制或Hdac1,2击倒没有改变的新生DNA相关SMARCA5染色质remodeler量，如图4。 <img alt="图1" src="/files/ftp_upload/53647/53647fig1.jpg" /> 图1. 确认的HDAC1,2选择性抑制剂的特异性。之后的Ad-Cre的感染和治疗898或HDAC1 FL / FL HDAC2 FL / FL或HDAC3 FL / FL纤维肉瘤细胞制备的全细胞裂解液西方分析233.细胞用3μM898或233 24小时以下一个48小时的Ad-Cre的感染。这个数字是从我们以前发表的作品6得出的 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53647/53647fig2.jpg" /> 图 PCNA协会与新生DNA在HeLa细胞 2. 动态。海拉细胞用溴脱氧尿苷（尿嘧啶）30分钟。然后将细胞洗涤以除去未掺入的BrdU和在培养基中培养无的BrdU的时间指示的时段（追）。染色质免疫沉淀，用抗增殖细胞核抗原（PCNA）抗体进行的。 BrdU的存在于输入DNA和那些与PCNA相关联标记的DNA分别在狭槽印迹分析使用抗BRD评估如图2 U抗体。这个数字是从我们以前发表的作品6得出的 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53647/53647fig3.jpg" /> 在HeLa和NIH3T3细胞中进行图3损失蛋白去乙酰化酶1和2增加H4K16ac上新生的DNA。（AB）溴脱氧尿苷（BrdU）标记脉冲追踪的 ，以确定与在指定的时间点新生DNA H4K16ac的关联。 （CD）的 NIH3T3细胞用DMSO或一个HDAC1,2选择性抑制剂（898或233）24小时。 （ 五）NIH3T3细胞，转任非靶向（NT）或Hdac1,2（H12）的siRNA。 选自 （C - E）的细胞被BrdU标记和使用芯片具有抗H4K16ac随后插槽印迹。将膜用抗BrdU抗体。数字表明，以高，中批量芯片DNA的发现平均的BrdU信号的Image J定量。这个数字是从我们以前发表的作品6得出的 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig3large.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/53647/53647fig4.jpg" /> 图4. SMARCA5协会与新生的DNA。显示SMARCA5对新生的染色质下列HDAC1,2功能的丧失量。 NIH3T3细胞，无论是处理与HDAC1,2选择性抑制剂（898）或转非靶向（NT）或Hdac1,2（H12）的siRNA。细胞用BrdU标记按照上述处理和沉淀用抗SMARCA5或兔IgG（负c-ONTROL）进行。的ChIP的DNA增加量被发现在槽印迹和将膜用抗BrdU抗体。这个数字是从我们以前发表的作品6得出的 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique at JoVE.com

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

examination-proteins-bound-to-nascent-dna-mammalian-cells-using-brdu

Nanyang Technological University

Research

JoVE Journal

Biology

9.1K 次观看.  University of Utah School of Medicine. In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA. 

视频: Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

科研

遗传学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

生物化学

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

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 ...

生物学

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

该芯片外型方法:确定蛋白质-DNA相互作用与近基部精密配对

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

使用BrdU-DNA标记结合细胞周期鉴别成像评估全球DNA双链末端切除术

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

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此视频中的章节

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

RNA 结合区质量谱鉴定的样品制备

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

采用芯片识别亚型特定的靶基因的全基因组分析

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

该芯片外型方法:确定蛋白质-DNA相互作用与近基部精密配对

使用BrdU-DNA标记结合细胞周期鉴别成像评估全球DNA双链末端切除术