Name: genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae
Uploaded: 2017-06-03T15:00:00.000+00:00
Duration: 10 min 43 s
Description: Watch this Scientific Journal Video about Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae at JoVE.com

我们感谢Moustafa Saleh和Jay Tourigny对手稿的批判性阅读，Steven Hahn和Steven Henikoff在开发ChEC-seq期间的指导和支持及其在Mediator综合体中的应用。 SG由NIH授权R01GM053451和R01GM075114支持，GEZ由印第安纳大学创业基金支持。

酵母菌的生成<ol><li> 产生带有MNase标记的感兴趣因子的酵母菌株。 <ol><li> PCR使用指定的反应混合物（ 表2 ）和循环条件（ 表3 ）从所需载体（ 表1 ）扩增MNase标记盒。混合5μLPCR反应和1μL6X DNA加载染料。在0.8V的琼脂糖凝胶上以120V的速度运行每个PCR等分试样40分钟。预期产品大小为〜2.3 kb。 </li><li>使用标准乙酸锂转化22将放大的标记盒转化为选择的菌株，并进行选择。 </li><li>验证标签盒与菌落PCR的正确整合。 <ol><li>为指定的反应混合物（ 表4 ）准备待筛选的菌落数。保持在冰上直到需要。 </li><li>选择每个殖民地的一部分是tes使用无菌牙签或移液管吸头进入PCR管的底部。 </li><li>微波挑选的殖民地在高功率1分钟。 </li><li>向每个微波菌落加入50μLPCR混合物（ 表4 ），上下移液管混匀。使用指定的循环条件进行PCR（ 表5 ）。 </li><li>在每个PCR管中直接混合50μLPCR反应和10μL6X DNA加载染料。在120℃下，在1％琼脂糖凝胶上运行10μL每个样品40分钟。预计产品大小约700 bp。 注意:如果需要，通过蛋白质印迹验证MNase融合蛋白的适当表达。预期标记蛋白的分子量约为20.6千道尔顿。 </li></ol></li></ol></li><li> 通过基因克隆将目的基因的启动子与pGZ136（表1）和转化和选择同步进行克隆，产生免费的MNase菌株，如步骤1.1.2所示。 <ol><li> Alternati将感兴趣的基因的启动子和3xFLAG-MNase-SV40 NLS组装到质粒载体中，如步骤1.1.2所示进行转化和选择，并在合适的选择条件下生长菌株以维持质粒。 </li></ol></li></ol> ChEC <ol><li>在ChEC实验前一天的下午/晚上，用含有MNase标记因子的菌株的单个菌落接种3mL酵母蛋白胨 - 葡萄糖（YPD）或合适的选择培养基。将该培养物孵育过夜（180RPM摇动，30℃）。 </li><li>在ChEC实验当天的早晨，将过夜培养物稀释到50mL的培养基中至600nm的光密度（OD 600 ）为0.2-0.3。孵育该培养物（180RPM摇动，30℃），直至达到0.5-0.7的OD 600 。当培养物接近适当的OD 600时，将热块或水浴置于30℃，开始解冻缓冲液A添加剂（ Ta6）。 </li><li>每个培养物准备5 mL缓冲液A加添加剂（ 表6 ）。在程序中保持缓冲液A在RT。 </li><li>准备收集每个时间点的含有90μL终止液（ 表7 ）和10μL10％SDS的微量离心管。对于分析的每个新因子，在0秒，30秒，1分钟，2.5分钟，5分钟和10分钟取样。 </li><li>将滗析培养物倒入50mL锥形管中，并以1,500×g和室温（RT）离心1分钟。 </li><li>将细胞重新悬浮在1mL缓冲液A中，并将悬浮的细胞转移到微量离心管中。将颗粒在1,500×g和RT中重悬浮于微量离心机中30秒。 </li><li>吸出上清液，并通过上下移液将细胞彻底重悬于1mL缓冲液A中。将颗粒在1,500×g和RT中重悬浮于微量离心机中30秒。重复此步骤一次。 </li><li>在570μL缓冲液A中彻底重悬细胞。加入30μL的2％洋地黄皂苷（0.1％终浓度）以促进细胞的透化，并反转混合。 </li><li>在30℃的热封或水浴中使细胞渗透5分钟。 </li><li>上下反应以确保细胞的均匀分布。去除100μL等分的透化细胞作为阴性对照至含有终止溶液和SDS的微量离心管（步骤2.4）并短暂涡旋混合。 </li><li>向透化细胞中加入1.1μL1M CaCl 2 （2mM终浓度）以激活MNase并快速反转数次或短暂涡旋混合。立即在30°C返回混合反应并启动定时器。 </li><li>在每个时间点收集，上下移动反应以确保细胞的均匀分布，然后将100μL等渗透细胞的细胞除去含有终止液和SDS的微量离心管，并短暂涡旋混合。对于所分析的每个新因子，取0 s（不含CaCl 2 ），30 s，1分钟，2.5分钟，5分钟和10分钟时间点。 注意:可以在较低温度下进行在30℃快速切割DNA的因子的ChEC实验，以减缓MNase切割动力学。 </li><li>收集所有时间点后，向每个样品加入4μL20 mg / mL蛋白酶K，短暂旋转混匀，55℃孵育30 min。 </li><li>向每个样品中加入200μL25:24:1苯酚:氯仿:异戊醇，剧烈旋转混合，并在微量离心机中以最大速度和RT离心5分钟。 </li><li>去除每个水相（〜150μL）到一个新的管。加入30μg糖原和500μL100％乙醇。大量涡旋混合并在干冰上沉淀10分钟，或直到溶液粘稠。 </li><li>在微量离心机中以最大速度和4℃沉淀DNA沉淀10分钟。 </li><li>倾析上清液，并用1mL RT 70％乙醇洗涤沉淀。 </li><li>倾倒乙醇，通过倒置去除剩余物d轻轻地将管放在纸巾上。使用台式离心机简单旋转样品，并使用移液管去除残留的乙醇，注意不要打扰沉淀。空气干燥颗粒在室温下5分钟。 </li><li>当颗粒干燥时，制成主混合物，将干燥的沉淀物重悬于29μLTris，pH 8.0 +1μL的10mg / mL核糖核酸酶A中。在37℃下消化RNA 20分钟。 </li><li>将1μL6X DNA负载染料与5μL每个样品混合，并在120V下在1.5％琼脂糖凝胶上运行40分钟。 </li></ol> 3.尺寸选择注意:大小选择的目标是从待测序的样品中除去基因组DNA的多千碱基片段，并富集〜150bp的片段（大约为核小体DNA的大小）或更小。在测序数据中，富集了400bp的片段，在核小体DNA（〜150bp）的大小周围具有显着的峰值，并且亚核核糖体片段的峰或宽分布。 &lt;OL&gt; <li>向每个样品中加入75μL固相可逆固定（SPRI）珠粒在聚乙二醇（PEG）溶液23中 ，并在上下移液10次以混合。在室温孵育珠:DNA混合物5分钟。 </li><li>在SPRI珠孵育期间，准备每个样品含有96μL10 mM Tris，pH 8.0和4μL5 M NaCl的微量离心管。 </li><li>将管放在磁性架中以收集管侧。允许珠在管的一侧收集2分钟。 </li><li>将每个上清液转移到含有Tris和NaCl的新管中（步骤3.2）。 </li><li>向每个样品中加入200μL25:24:1苯酚:氯仿:异戊醇，涡旋混合，并在微量离心机中以最大速度和RT离心5分钟。 </li><li>将每个水相去除一个新的管。加入30μg糖原和500μL100％乙醇。在干冰上沉淀DNA 10分钟或溶液粘稠。 </li><li>颗粒preci在微量离心机中以最大速度和4℃排空DNA 10分钟。 </li><li>去除上清液并用1mL 70％乙醇洗涤沉淀。 </li><li>倾倒乙醇，通过翻转并轻轻敲打纸巾上的管，取出其余部分。使用台式离心机简单旋转样品，并使用移液管去除残留的乙醇，注意不要打扰沉淀。空气干燥颗粒在室温下5分钟。 </li><li>将干燥的沉淀物重悬于25μLTris，pH 8.0中。使用高灵敏度测定法确定样品浓度。对于TF ChEC实验，在大小选择后常规回收10-100ng DNA。 </li><li>准备测序库。如前所述，不依赖于大小选择的文库制备方案是期望的，以允许大范围片段大小（25-500bp）的测序。 </li><li>配对模式下的序列库。高质量阅读需要每端至少25个碱基序列映射。 1至2百万次读数为ChEC样品提供足够的覆盖率。 注意:ChEC-seq数据的分析是一个复杂的过程，超出了本文的范围。有关数据分析的详细信息可以在以前的出版物1,21中找到，用于分析的自定义脚本可在GitHub（https://github.com/zentnerlab/chec-seq）上获得。 HOMER 25 （http://homer.salk.edu）也可用于ChEC-seq数据的命令行分析。 </li></ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocortin+receptor-mediated+mobilization+of+intracellular+free+calcium+in+HEK293+cells.">Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.</a> Physiol Genomics. 5 (1), 11-19 (2001).</li><li>Seurin, D., Lombet, A., Babajko, S., Godeau, F., Ricort, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-Like+Growth+Factor+Binding+Proteins+Increase+Intracellular+Calcium+Levels+in+Two+Different+Cell+Lines.">Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines.</a> PLoS ONE. 8 (3), e59323 (2013).</li><li>Grienberger, C., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Calcium+in+Neurons.">Imaging Calcium in Neurons.</a> Neuron. 73 (5), 862-885 (2012).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotech. 32 (4), 347-355 (2014).</li><li>Filion, G. 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=Systematic+Protein+Location+Mapping+Reveals+Five+Principal+Chromatin+Types+in+Drosophila+Cells.">Systematic Protein Location Mapping Reveals Five Principal Chromatin Types in Drosophila Cells.</a> Cell. 143 (2), 212-224 (2010).</li><li>van Bemmel, J. G., 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=A+Network+Model+of+the+Molecular+Organization+of+Chromatin+in+Drosophila.">A Network Model of the Molecular Organization of Chromatin in Drosophila.</a> Mol Cell. 49 (4), 759-771 (2013).</li><li>Izzo, A., 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=The+Genomic+Landscape+of+the+Somatic+Linker+Histone+Subtypes+H1.1+to+H1.5+in+Human+Cells.">The Genomic Landscape of the Somatic Linker Histone Subtypes H1.1 to H1.5 in Human Cells.</a> Cell Rep. 3 (6), 2142-2154 (2013).</li></ol>

作者没有什么可以披露的。

我们已经表明，ChEC可以将不同类型的酵母蛋白质映射到染色质上，并且预计它将广泛适用于酵母中TF和其他染色质结合因子的不同家族。 ChEC-seq的优点在于它不需要交联，染色质溶解或抗体。因此，ChEC避免了X-ChIP-seq中可能存在的伪像，例如超级ChIPable伪影3,4和原生ChIP，例如由于不完全的蛋白质溶解引起的假阴性14 。与所有酶融合方法一样，ChEC-seq的主要缺点是...

映射转录因子（TF），染色质重组体和其他染色质相关调控因子的结合位点是理解所有基于染色质的过程的关键。虽然染色质免疫沉淀和高通量测序（ChIP-seq）方法已被用于获取对基因组生物学的许多重要见解，但它们具有显着的局限性。我们最近引入了一种替代方法，称为染色质内源性切割和高通量测序（ChEC-seq） 1 ，以克服这些缺点。 最常使用初始甲醛交联步骤（X-ChIP-seq）进行ChIP-seq以保护蛋白质-DNA相互作用。然而，一些最近的研究表明，X-ChIP-seq捕获瞬时或非特异性蛋白质 - DNA相互作用2，3，4，5 ，sup&gt;， 6，7，8 ，导致假阳性结合位点。此外，通常用于在X-ChIP-seq实验中分离染色质的超声处理，优先剪切开放染色质区域，导致片段从这些区域偏向恢复9,10。超声处理还产生片段长度的不均匀混合物，最终限制了结合位点分辨率，尽管加入外切核酸酶消化步骤可以极大地提高分辨率11,12 。来自亲和纯化的天然分离染色质（ORGANIC） 13的基因组的占据区域的本地ChIP方法不使用交联和片段染色质与微球菌核酸酶（MNase），减轻与甲醛交联和超声处理相关的潜在偏差。然而，许多染色质结合蛋白在天然染色质提取所需的相对温和条件下的溶解性差，可能导致动态范围和/或假阴性减少14 。 虽然ChIP-seq的各种迭代最常用于蛋白质-DNA相互作用的全基因组映射，但是还已经实现了基于目的蛋白质融合到各种DNA修饰酶的几种测绘技术。一种这样的方法是DNA腺嘌呤甲基转移酶鉴定（DamID） 15 ，其中感兴趣的染色质结合蛋白遗传融合到Dam，并且该融合在细胞或动物中表达，导致接近蛋白质结合位点的GATC序列的甲基化。 DamID的优点在于它不依赖于免疫沉淀，因此避免交联，抗体或染色质溶解。它也在体内进行 。然而，DamID的分辨率限于千碱基量表，Dam融合蛋白的甲基化活性是组成型的。基于酶融合的第二种方法是Calling Card-seq 16 ，其使用感兴趣因子融合转座酶，引导位点特异性整合转座子。像DamID一样，Calling Card-seq不是基于免疫沉淀，因此具有类似的优点，增加分辨率的附加益处。然而，呼叫卡-seq可能受到转座酶的序列偏差的限制，并且还依赖于接近转座子插入位点的限制性位点的存在。 在Laemmli实验室开发的第三种酶融合方法是染色质内源性切割（ChEC） 17 。在ChEC中，染色质相关蛋白和MNase之间的融合物在细胞中表达，并且通过钙添加来活化MNase，DNA被切割到接近标记的结合位点因子（ 图1 ）。结合Southern印迹，ChEC已被用于表征17,18号酵母中多个单个位点的染色质结构和蛋白结合，并结合低分辨率微阵列分析来探测核孔组分与酵母的相互作用基因组19 。 ChEC提供类似DamID和Calling Card-seq的优点，当通过引物扩展19进行分析时，其分辨率几乎为单碱基对。 ChEC也是可控的:MNase的强力DNA切割取决于加入毫摩尔钙，确保MNase在活酵母细胞中观察到的低游离钙浓度下是无活性的。 以前，我们假设将ChEC与高通量测序（ChEC-seq）相结合将提供TF结合位点的高分辨率图谱。事实上，ChEC-seq生成跨基因组1的出芽酵母总调控因子（GRFs）Abf1，Rap1和Reb1的高分辨率图 。我们还成功地将ChEC-seq应用于模块化Mediator复合体，这是一种保守的，重要的全局转录共激活因子21 ，扩大了ChEC-seq对不直接接触DNA的巨大体细胞复合物的适用性，可能难以通过ChIP-基于方法。 ChEC-seq是独立验证ChIP-seq结果和产生对染色质定位过程调节的新见解的有力方法。在这里，我们提出了一种在芽殖酵母中实施该方法的分步骤方案。

<table><tbody><tr><td>dNTPs</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>Q5 high-fidelity DNA polymerase</td><td>NEB</td><td>M0491L</td><td>Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.</td></tr><tr><td>TrackIt 1 Kb Plus DNA ladder</td><td>ThermoFisher Scientific</td><td>10488085</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>NEB</td><td>M0273L</td><td></td></tr><tr><td>cOmplete Mini EDTA-free protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>11836170001</td><td>It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit MNase cleavage by chelation of Ca&lt;sup&gt;2+&lt;/sup&gt;.</td></tr><tr><td>PMSF</td><td>ACROS Organics</td><td>AC215740010</td><td></td></tr><tr><td>Digitonin, High Purity</td><td>EMD Millipore</td><td>300410-250MG</td><td>Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.</td></tr><tr><td>Proteinase K, 20 mg/mL</td><td>Invitrogen</td><td>25530049</td><td></td></tr><tr><td>RNase A, 10 mg/mL</td><td>ThermoFisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Ampure XP beads</td><td>Beckman Coulter</td><td>A63880</td><td>Ampure-like beads can be generated using a published protocol (ref 24).</td></tr><tr><td>MagneSphere magnetic rack</td><td>Promega</td><td>Z5342</td><td></td></tr></tbody></table>

genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae

蛋白质 - DNA相互作用的全基因组映射对于了解基因调控，染色质重塑和其他染色质定位过程至关重要。甲醛交联随后进行染色质免疫沉淀和高通量测序（X-ChIP-seq）已被用于获得许多对基因组生物学的宝贵见解。然而，X-ChIP-seq具有与交联和超声处理有关的显着的局限性。原生ChIP通过省略交联来避免这些缺点，但通常导致染色质结合蛋白的恢复不良。此外，所有基于ChIP的方法均受抗体品质的考虑。用于测定蛋白质-DNA相互作用的酶学方法，其涉及将目的蛋白质融合到DNA修饰酶中，也用于测定蛋白质-DNA相互作用。我们最近将一种这样的方法，染色质内源性切割（ChEC）与高通量测序合并为ChEC-seq。 ChEC-seq依赖于染色质相互融合将微生物核酸酶（MNase）感兴趣的蛋白质在活细胞中的钙存在下产生靶向的DNA切割。 ChEC-seq不是基于免疫沉淀，因此避免了交联，超声，染色质溶解和抗体质量的潜在问题，同时以最小的背景信号提供高分辨率图谱。我们设想ChEC-seq将成为ChIP的强大对手，提供独立的手段来验证ChIP-seq结果并发现对基因组调控的新见解。

在成功的ChEC实验的情况下，通过琼脂糖凝胶电泳分析DNA将显示DNA片段化随时间的钙依赖性增加，如通过涂抹和最终完全消化基因组DNA所指示的。在一些情况下，在扩展消化后观察到类似于传统MNase消化所观察到的条带。 Reb1的ChEC分析就是这种情况，Reb1是结合核小体耗尽区域（NDR）的一般调控因子（ 图2 ）。我们发现，在GRF的情况下，延长的消化导致...

Watch this Scientific Journal Video about Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae at JoVE.com

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

我们描述了染色质内源性切割与高通量测序（ChEC-seq），染色质免疫沉淀（ChIP） - 正交法，用于在全基因组范围内用微球菌核酸酶（MNase）融合蛋白测定蛋白质结合位点。

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

genome-wide-mapping-protein-dna-interactions-with-chec-seq

Research

JoVE Journal

Genetics

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

11.1K 次观看.  Fred Hutchinson Cancer Research Center. 我们描述了染色质内源性切割与高通量测序（ChEC-seq），染色质免疫沉淀（ChIP） - 正交法，用于在全基因组范围内用微球菌核酸酶（MNase）融合蛋白测定蛋白质结合位点。 

视频: Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

科研

遗传学

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

生物化学

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

生物学

从酵母细胞中分离基因座特异性染色质

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

作者聚焦：使用高富集位点特异性染色质分离方案推进染色质研究并克服限制 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

酿酒酵母的单拷贝基因位点染色质纯化

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

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微阵列分析FOR酿酒酵母

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用