Name: chromatin immunoprecipitation assay for the identification of arabidopsis protein-dna interactions in vivo
Uploaded: 2016-01-14T16:00:00.000+00:00
Duration: 12 min 36 s
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo at JoVE.com

The authors would like to acknowledge The Plant Cell for allowing the use of some data published in this journal to elaborate the representative results described here in Figure 4. This work was supported by the EU 7FP Marie Curie-Initial Training Network EpiTRAITS (Grant Agreement 316965), and by the Spanish Ministerio de Econom&#236;a y Competitividad (grants BIO2010-15589 and BIO2013-43098-R).

1.交联的植物材料（1小时） <ol><li>生长在实验中使用的Arabidopsis品系（野生型 - WT - 与突变体，和/或线表达自己感兴趣的蛋白质的相对线表达该标记的标记版本不融合的任何蛋白质）12-18天上大培养皿（150毫米）与MS琼脂培养基（1L:1个的Murashige＆Skoog盐，10g的蔗糖，0.5g的MES，pH值5.7（KOH），1％琼脂）。另外，生长在含有3盆植物:1混合土和蛭石。 注意！甲醛是通过吸入，皮肤接触及吞食，并应在化学罩穿上适当的个人防护装备进行处理。步骤1.2 - 1.6应在通风橱下进行。 </li><li>使在50ml试管用实验室燃烧器加热的针的盖小的孔。加入40毫升的1×PBS缓冲液和1.08甲醛毫升至1％的终浓度（股票37％s的olution）。收集1.5克植物材料在这些50ml试管。请交联过程中冰上管。 </li><li>关闭50ml试管与盖。将管在干燥器和施加真空。真空渗入组织10分钟，然后小心地释放真空，以防止搅动起来的溶液。混合样本。重复两次。浸润后，观察植物材料，并确认它变得稍微半透明的，并趋向于下沉到管（图2） 的底部。 </li><li>添加2.5毫升2M的甘氨酸以达到0.125 M的最终浓度应用真空5分钟。甘氨酸作为甲醛交联的竞争性抑制剂。 </li><li>通过反转闭合50ml管中，在该盖的孔丢弃PBS甲醛甘氨酸溶液。 </li><li>与如在步骤1.5中所述的水弃去洗涤液冲洗小植株两次用1×PBS和一次。干他们在纸巾上并冷冻在液态nitrogeñ。 注:冷冻组织可保持在-80℃为周。 </li></ol> 2.制备的抗体的（1天） 注:对于免疫沉淀，利用经由蛋白质G或蛋白质与抗体重链的恒定结构域的高亲和性的连接物结合有抗体的磁珠被推荐。的DNA-蛋白复合物可以非特异性地连接到所述珠 - 抗体偶联物的表面上。出于这个原因，有必要进行控制，而不特异性抗体来定量非特异性背景结合。 <ol><li>对于每一个免疫沉淀，制备15微升磁珠在1.5ml微量离心管中。的小珠的量取决于用于免疫沉淀和也对抗体的染色质的量。 </li><li>洗，加入1 ml的染色质免疫沉淀（ChIP）稀释缓冲液至珠，通过旋转混合，然后将1.5 ml微量涂在一个磁性机架。等待约1分钟，以让所述珠附着到磁体。与1.5 ml微量离心管仍然在机架上，通过移液除去上清液。重复两次。 注:对于每个植物系，需要两个免疫组:1与珠子和抗体（抗体）和第二无抗体（无抗体）控制与仅磁珠。 </li><li>重悬在200微升的ChIP稀释缓冲液珠。添加所需的特异性Ab的量为每个植物系中制备的两个管中的一个（确切稀释不同于每个Ab和具有由实验优化，或在商业抗体的情况下，按照制造商的说明）。使用该样品进行免疫沉淀。添加非特异性IgG的相同量的其他管，其将被用作阴性对照。 </li><li>孵育O / N上的旋转轮，在4​​℃下，让该抗体附着到珠子上。 </li></ol> 3.染色质萃取（4小时） </p&gt; <ol><li>使用研钵和杵直到粉末变得均匀和浅绿色研磨彻底冷冻植物材料在液氮中。粉末转移到一个新50ml管中。 </li><li>对于步骤3.2 - 3.6，工作通风柜下。加入30毫升提取缓冲液1（EXB 1），拌匀浸泡组织粉末。从在此步骤中，保持所述样品在4℃下在任何时候。冷冻组织和液氮剩菜将导致缓冲器冻结。确保反相管4-5次，每次2分钟完全解冻冷冻组织。 </li><li>通过在4℃下使其通过一过滤组织具有22-25微米孔径到一个新的50ml管中，并离心它在1000×g离心20分钟清除浆料。 注:在这一步骤中，细胞核，所有的细胞器将沉淀。 EXB 1含有足够的蔗糖，以提供高密度，防止细胞器结构的破坏。 </li><li>轻轻倾析去除上清。在这站GE，观察约2毫升生球。 </li><li>重悬在5ml EXB的粒料2.再悬浮颗粒将难以在这一点上。离心机在1000×g离心在4℃下10分钟。 注:EXB 2中含有1％的Triton X-100不由一阵叶绿体开并取下叶绿素。 </li><li>重悬在300微升提取液3（EXB 3）的颗粒。 </li><li>在一个干净的离心管中，加入600微升预算外3.取悬浮颗粒从步骤3.6和仔细的清洁预算外3的顶部层吧。 </li><li>自旋为1小时，在16000×g离心在4℃下。 注:此步骤有助于除去从样品的洗涤剂。 </li><li>通过用移液管抽吸除去上清液。通过缓慢上下抽吸，以避免形成气泡，这可能会影响声处理效率重悬在300微升超声缓冲液的核沉淀。认真吸取所有的悬挂出来，转移到一个干净的1.5 ml离心管。 注:此缓冲液含有SDS，以裂解细胞核和释放染色质。 </li><li>超声处理该悬浮液以裂解细胞核和随机剪切染色质成小碎片。使用该渲染染色质富集200-800碱基对之间的片段（20-30个周期的设置30秒开/ 30秒关，在4℃下可利用的材料/试剂的表中所描述的超声处理装置）超声处理的条件。检查超声处理将所得到的DNA片段的电泳分离的效率在琼脂糖凝胶上13，加载2微升超声染色质（图3）。 小心！确保期间中的情况下超声装置不包含在一个隔音柜内超声处理步骤到穿耳朵保护设备。 关键的一步！染色质碎片在很大程度上取决于超声设备。超声处理条件应进行调整，以实现富集的合适的DNA尺寸。看到图3为如何优化染色质片段化的一个例子。 </li><li>旋溶液一次以12,000rpm离心10分钟，在4℃。上清液通过移液枪转移到一个干净的1.5 ml离心管。弃沉淀。取上清的1/10到一个新的1.5 ml离心管，将其标记为输入并冷冻在-20℃。 </li><li>稀释10倍的染色质与沉淀稀释缓冲液稀释的SDS达到0.1％的最终浓度。 注意:样品可以冷冻并储存在-20℃下几个星期。 </li></ol> 4.免疫沉淀利益的蛋白质和DNA的救援（1天，3小时） <ol><li>从步骤用1ml的ChIP稀释缓冲液洗涂覆有抗体的珠子的2.4三倍如步骤2.2中所述，以除去过量的抗体。重悬在200微升的ChIP稀释缓冲液。加入50微升孤立的染色质。孵育O / N旋转轮上，在4℃。 注:检查concentrati在微量紫外可见分光光度计的染色质。 50微升孤立染色质应包含超过10个的DNA微克。 </li><li>对于在4℃的步骤4.2-4.5工作。如在步骤2.2中描述的1ml低盐洗涤缓冲液洗两次的珠。 </li><li> 1毫升高盐洗涤缓冲液洗一次的珠子。 </li><li>在1ml的LiCl洗涤缓冲液洗一次珠。 </li><li>在1ml TE缓冲液洗两次的珠。最后一次洗涤后，请务必通过抽吸完全删除TE缓冲留在管吸管。 </li><li>取出输入样本（步骤3.9）。转印5微升输入到一个新的1.5 ml离心管，并继续作为与芯片的样品。反向加入200微升5％的螯合离子交换树脂的交联，并孵育10分钟，在95℃下振荡，每2-3分钟。降速在16000×g离心30秒，小心地通过吸液将上清液转移到一个新的离心管中。 注意:当TRA用于交联的逆转ditional方法涉及的O / N培养用NaCl，与螯合离子树脂在95℃下温育允许有效解交联和DNA在10分钟洗脱，因此使得协议之一天缩短。 </li><li>加入2微升蛋白酶K（10毫克/毫升），并在37℃下30分钟以消化蛋白质并释放DNA。 </li><li>停止反应，在95℃下孵育10分钟。 </li><li>快速旋转，在16000×g离心30秒，并通过移液转移上清至新管。 </li><li>清洁用标准的DNA片段纯化试剂盒的DNA分离出来。 注:根据制造商的说明进行操作。 </li><li>从试剂盒中的DNA结合柱转移到新的1.5ml微量管中。通过加入20微升DNA酶的水，以塔的中心，并在16000×g离心纺丝60秒洗脱纯化的DNA。停止点:样品可以贮存于-80℃下几个星期。 </li></OL&gt; 5.测量结合位点的免疫沉淀DNA通过qPCR的丰度（4小时） 注:脱氧核糖核酸从沉淀的染色质中分离已被分析，以确定哪些DNA片段已沉淀-编从总染色质，由于它结合到感兴趣的蛋白质。 <ol><li>设计的引物用于以60℃的熔融温度（Tm）为30％-80％的GC含量感兴趣的基因组区域。染色质通过超声处理片段化，扩增序列的长度不应该比150-200个核苷酸（表1）长。 注:引物设计的一个有用的工具是底漆3程序（http://bioinfo.ut.ee/primer3-0.4.0/）。对于引物设计的附加 ​​准则在以前的报告中8，14，15。 </li><li>使用BLAST程序，以确保引物是特定的（特别是启动子可以共享相似TF结合序列）和测试引物效率使用设克1点10稀释在输入脱氧核糖核酸（步骤4.9）的水中。基因组的特定区域将被净化比别人做得更好。因此，重要的是设计PCR引物，并检查它们的稀释输入的DNA。 </li><li>稀释纯化的DNA以1:10的水和吸管在PCR管为每个反应取5μl稀释的DNA。 注的稀释:仅在需要的量，因为DNA可以连接到1.5 ml微量离心管的壁。多个解冻冷冻循环增加附着的DNA分子的数量。对于每个引物对，扩增在输入，抗体芯片和无抗体的ChIP已经被分析。 </li><li>要完成PCR混合物，SYBR绿PCR主混合物添加到1倍的终浓度，及0.5每种引物μM。填充至20微升用DNase的水。 </li><li>执行定量PCR反应后的的SYBR Green PCR主混合物制造商的说明。 </li></ol> 6.数据分析注:其中前isting分析的ChIP-数据的方式，两个是最常用的。的其中第一个是在倍富集方法，也命名为"相对富集"，"信号超过背景"或"相对于无抗体对照&#39;。第二种方法被命名为"％输入的"。 <ol><li>通过倍富集法数据分析。 <ol><li>创建与已定量PCR反应后获得的样品名称和原始Ct值的电子表格。 </li><li>对于每个样品，从它的原始Ct值减去用于相应无抗体对照获得的原始Ct值。注:本数学运算后，对于无抗体样品Ct值将等于0。 </li><li>通过求幂运算计算倍富集与基座2和指数（功率）等于在步骤6.1.2（表2）中获得的负的余数。 倍浓缩= 2 -数（Ct（样品） -的Ct（无抗体）） 注:在此实现方法具D中的ChIP-ED试样中的特定DNA片段的丰度与该片段中无抗体对照的丰度进行比较。该方法的前提是，背景信号的电平是可重复的不同的引物组，样品，并重复实验之间。然而，这种&#39;noise&#39;信号电平做引物组，样品和实验之间变化。 </li></ol></li><li>由％输入法的数据分析。 <ol><li>创建与样品名称和qPCR反应后已获得他们的原始Ct值的电子表格。 </li><li>通过从总提取染色质的被取作输入的分数中减去对数底数2的值调整原始Ct值对输入样本。 注:在本协议中的减值等于3.32，占总染色质的10％被作为一个输入在这个协议。日志2（10）等于3.32。 </li><li>通过乘以100求幂运算的用b值计算％输入的酶2和指数（功率）等于从样品（见表3）的原始Ct值中减去调整后的输入值的剩余部分。 ％输入= 100 * 2 （调整后的输入-的Ct（样品）） 注意:对于这个过程，以总孤立染色质（输入取样）特异性DNA丰度的片编样品之间的关系来计算。芯片数据分析的进一步细节，可以在哈林等人 8中找到。 </li></ol></li></ol>

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HISTONE+DEACETYLASE6+interacts+with+FLOWERING+LOCUS+D+and+regulates+flowering+in+Arabidopsis.">HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis.</a> Plant Physiology. 156 (1), 173-184 (2011).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiling+of+histone+H3+lysine+9+acetylation+and+dimethylation+in+Arabidopsis+reveals+correlation+between+multiple+histone+marks+and+gene+expression.">Genome-wide profiling of histone H3 lysine 9 acetylation and dimethylation in Arabidopsis reveals correlation between multiple histone marks and gene expression.</a> Plant Mol Biol. 72 (6), 585-595 (2010).</li><li>Lafos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+H3K27+trimethylation+during+Arabidopsis+differentiation.">Dynamic regulation of H3K27 trimethylation during Arabidopsis differentiation.</a> PLoS Genet. 7 (4), e1002040 (2011).</li><li>Barcala, M., Fenoll, C., Escobar, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+cells+and+isolation+of+high-quality+RNA+after+cryosectioning.">Laser microdissection of cells and isolation of high-quality RNA after cryosectioning.</a> Methods Mol Biol. 883, 87-95 (2012).</li><li>Carter, A. D., Bonyadi, R., Gifford, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescence-activated+cell+sorting+in+studying+plant+development+and+environmental+responses.">The use of fluorescence-activated cell sorting in studying plant development and environmental responses.</a> Int J Dev Biol. 57 (6-8), 545-552 (2013).</li><li>Deal, R. B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INTACT+method+for+cell+type-specific+gene+expression+and+chromatin+profiling+in+Arabidopsis+thaliana.">The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.</a> Nat Protoc. 6 (1), 56-68 (2011).</li><li>Zeng, P. Y., Vakoc, C. R., Chen, Z. C., Blobel, G. A., Berger, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dual+cross-linking+for+identification+of+indirect+DNA-associated+proteins+by+chromatin+immunoprecipitation.">In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation.</a> Biotechniques. 41 (6), 694-698 (2006).</li><li>Das, P. M., Ramachandran, K., vanWert, J., Singal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay.">Chromatin immunoprecipitation assay.</a> Biotechniques. 37 (6), 961-969 (2004).</li><li>Kaufmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+genes+of+the+MADS+transcription+factor+SEPALLATA3:+integration+of+developmental+and+hormonal+pathways+in+the+Arabidopsis+flower.">Target genes of the MADS transcription factor SEPALLATA3: integration of developmental and hormonal pathways in the Arabidopsis flower.</a> PLoS Biol. 7 (4), e1000090 (2009).</li></ol>

The authors declare that they have no competing financial interests.

此处所描述的沉淀协议是一个可再现的和强大的技术来分析蛋白质和特定的DNA序列之间的相互作用在体内在拟南芥植物。一个成功的鉴定结合位点的目的蛋白质需要适当的选择植物器官或发育阶段，其中相关的相互作用实际上是正在发生的。此外，重要的是，获得的植物材料的适当的固定和染色质的通过超声处理的最佳剪切。是对于所选择的蛋白质/标签的有效免疫沉淀的高度特异性抗体也...

近年来广泛的遗传，分子和基因组工具被开发在模型物种拟南芥。该技术促进了极大的进展，了解如何植物生长发育的调节。在使用拟南芥为模式研究的发展过程中，开花时间的基因控制已经被广泛分析。这些研究表明，植物开花响应于内源性线索非常精确的时间调制如激素和植物的年龄，并且还认为则同步开花时间随着季节1的自然循环环境信号诸如光周期和温度， 2。拟南芥突变体的分离和鉴定与改建开花的时候已经决定在公布的基因，调节开花时间响应内源性因素和环境因素的复杂网络。这些遗传电路集成在充当开花交换机的几个主基因水平和花芽分化的确切时间取决于促进开花，压制这项工作的花卉积分基因1,3上游活动的平衡。 确定了其在开花开始的控制作用的基因的功能特性，由最近使用基因组学方法的帮助下，已揭示转录调控中的开花时间的调制的中心作用。事实上，许多的开花编码转录的主控基因因子4。此外，一些染色质重塑蛋白复合物影响开花的主基因的表达。许多拟南芥突变体中分离得到的改变的开花时间的横空出世，携带突变基因编码的各种染色质修饰的。不同的染色质remodelers的引入华宏翻译后修饰Ë尾巴，重新定位相对于DNA核或组蛋白变体交换规范的组蛋白所必需的开花拟南芥5,6的适当调节。其中的一些染色质重塑活动催化沉积或除去的共价修饰，如乙酰化或甲基化的组蛋白的特定残基。这些组蛋白标记由该招募其他染色质重塑复合物，转录因子或转录机制的成分以调节开花的基因的转录活性专门效应特异性识别。 染色质免疫沉淀（ChIP）允许在体内的DNA结合位点的感兴趣的蛋白质（图1）的识别。这个过程利用了某些化学品的能力交联的蛋白质的DNA。得到的DNA蛋白质复合物可以通过使用特异性抗体来阿加然后免疫沉淀研究所转录因子，染色质结合蛋白，或特定变型和异源表位（通常被​​称为"标签"）附连到所选择的蛋白质。从这些免疫沉淀物进行纯化的DNA可以用作定量PCR（qPCR的）反应的模板，以评估所关注的特定序列的富集。以这种方式，转录因子的结合位点或在特定的基因组蛋白标记的分布可以建立7,8。此外，结合新一代测序（NGS），使大规模平行测序，芯片技术已经成为可能的转录因子结合位点，以及组蛋白修饰揭幕表观景观的全基因组的鉴定。此外，基因表达的同时分析可以监控转录调节如何结合或特定组蛋白标记的沉积相关的转录活性状况TY基因9。 利用拟南芥的ChIP协议允许评估的影响，多种转录调节因子有开花的主控基因的染色质组织，如何将这些结构变化影响基因表达5,6。以前的结果表明，拟南芥基因先期抽薹在短日照 （EBS）作为开花突变体中该基因节目开花和开花的FT的主基因的上调的加速度的阻遏。此外，丧失功能的突变的FT充分抑制EBS突变植物的早期开花表型，这表明FT需要EBS突变体的过早开花和EBS是必要的这个主开花10的基因的压制，11。EBS编码含有PHD-蛋白可以特异性结合组蛋白H3的二和三甲基在第Ë赖氨酸残基4（H3K4me2 / 3），这表明对EBS在FT 12的染色质介导的抑制的作用。使用该芯片的做法表明，拟南芥含有PHD蛋白EBS 10,11结合花卉积分FT基因的调控区，以抑制其表达12。通过使用芯片技术所获得的额外数据表明，该蛋白质是必需的，以保持在拟南芥发展初期低水平组蛋白乙酰化，活性转录的标志的，在这个主开花的基因的染色质。这些观察，连同遗传和基因表达数据，证明该拟南芥含有PHD-蛋白具有在开花时间的微调通过调制花香积分基因FT 12的表达的中心作用。这里提出的工作提供了优化的方法不仅对组蛋白的分析，但也可用于其他有用染色质相关蛋白质，和以更高的效率和降低的实验时间。此外，该报告说明了如何使用的ChIP协议提供了新的见解中染色质修饰改变和植物基因的转录状态之间的关系，以及如何将这些染色质介导的基因表达的控制影响的机制上开花拟南芥的发作。

<table><tbody><tr><td>MES</td><td>Sigma</td><td>M8250</td><td></td></tr><tr><td>MS (Murashige and Skoog Basal Salt Mixture)</td><td>Sigma</td><td>M5524</td><td></td></tr><tr><td>Formaldehyde 37%&amp;nbsp;</td><td>Sigma</td><td>F8775-25</td><td>Use under the fume hood</td></tr><tr><td>Protease inhibitor mix cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack</td><td>Roche</td><td>5892791001</td><td></td></tr><tr><td>Bioruptor Standard sonication device</td><td>Diagenode</td><td>B01010002 (UCD200TO)</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>50046</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Dynabeads magnetic beads coupled with protein A or protein G</td><td>Life Technologies</td><td>10003D/10001D</td><td>Check manufacturer&amp;rsquo;s manual for antibody affinity</td></tr><tr><td>Miracloth</td><td>Merck Millipore</td><td>475855</td><td></td></tr><tr><td>Triton X-100 Surfact-Amps Detergent Solution</td><td>Life Technologies</td><td>85112</td><td></td></tr><tr><td>(mouse, rat, rabbit&amp;hellip;)-IgG</td><td>Diagenode</td><td>C15400001, C15420001, C15410206</td><td></td></tr><tr><td>Magnetic rack - DynaMag-2</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>H3K9/14ac polyclonal antibody - Premium&amp;nbsp;</td><td>Diagenode</td><td>C15410200-10</td><td></td></tr><tr><td>Chelex 100 Resin</td><td>Bio-Rad</td><td>142-2832</td><td></td></tr><tr><td>Proteinase K</td><td>Life Technologies</td><td>17916</td><td></td></tr><tr><td>Anti-Myc Tag Antibody, clone 4A6</td><td>Millipore</td><td>05-724</td><td></td></tr><tr><td>LightCycler 480 SYBR Green I Master</td><td>Roche</td><td>4707516001</td><td></td></tr></tbody></table>

chromatin immunoprecipitation assay for the identification of arabidopsis protein-dna interactions in vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

八个主要步骤可以单挑在此芯片协议用 ​​于体内蛋白-DNA相互作用，包括生长和收获的植物材料，交联的染色质的，染色质隔离，染色质片段化，DNA和之间的复合物的选择性分离的的识别通过免疫沉淀，蛋白质消化，DNA纯化，和qPCR分析（图1）感兴趣的蛋白质。在芯片协议的一个关键步骤是DNA-蛋白质相互作用的交联反应进行固定。典型地，甲醛交联是用在植物沉淀。甲醛穿透...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo at JoVE.com

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

染色质免疫沉淀是一 ​​个功能强大的技术的DNA结合蛋白拟南芥的体内位点的鉴定。此过程包括染色质交联和碎裂，免疫沉淀与针对感兴趣的蛋白的选择性抗体，和结合的DNA的定量PCR分析。我们描述了拟南芥植物优化的一个简单的ChIP实验。

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

chromatin-immunoprecipitation-assay-for-identification-arabidopsis

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

20.3K 次观看.  Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid. 染色质免疫沉淀是一 ​​个功能强大的技术的DNA结合蛋白拟南芥的体内位点的鉴定。此过程包括染色质交联和碎裂，免疫沉淀与针对感兴趣的蛋白的选择性抗体，和结合的DNA的定量PCR分析。我们描述了拟南芥植物优化的一个简单的ChIP实验。 

视频: Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

科研

生物化学

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

检测组蛋白修饰在植物叶片

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

生物学

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

基于 TurboID 的接近标签，用于蛋白质-蛋白质相互作用网络的植物中识别

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

免疫与感染

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

基于AirID的植物蛋白质-蛋白质相互作用的邻近标记

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells

Source: Fuchs, A. R.,et al. <a href="https://www.jove.com/t/58270">A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia.</a> J. Vis. Exp. (2018).
This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.

染色质免疫沉淀测定:一种研究细胞中蛋白质-DNA 相互作用的技术

Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. ...

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定

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