Name: a cell free assay to study chromatin decondensation at the end of mitosis
Uploaded: 2015-12-19T13:00:00.000+00:00
Duration: 11 min 4 s
Description: Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

这项工作得到了德国研究基金会和ERC（AN377 / 3-2和309528 CHROMDECON到华盛顿州）和勃林格殷格翰全宗的博士研究生奖学金，以AKS图1和2是从的Developmental Cell 31（3），Magalska转载等人 ，RuvB样ATP酶在有丝分裂，305-318，2014年年底的功能染色质解聚，与爱思唯尔许可。

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The authors have nothing to disclose.

非洲爪蟾卵提取物是忠实地再现体外细胞过程一个非常有用的工具，并且该系 ​​统被成功地在细胞周期和细胞分裂事件2，3,5,6,17表征使用。由于隔离在卵子发生在卵核部件的大型商店，卵提取物的细胞成分的极好来源。相对于其他方法，如RNA干扰对哺乳动物组织的细胞系或遗传操作，它提供了几个优点:无细胞系统允许研究细胞过程中细胞生存力将是否则限制。此外单一步骤复杂过程可以用简单的测定法进行分析。在这里提出解聚法使学习有丝分裂后的解聚分子机制，没有从其他的有丝分裂事件的干扰，分别爪蟾卵提取物很容易就可以处理由特定蛋白质的耗尽和另外抑制剂或突变的蛋白质8。 例如 ，图2示出了添加非水解ATP或GTP类似物，ATPγS和GTPγS到解聚化验的结果。通过稀释和爪蟾卵组件，如膜和细胞溶胶可以分离16差速离心。 图3示出了在膜的存在或不存在进行解聚实验。最后，将无细胞试验也可用于鉴定新型因素例如，通过一个分馏的方法。利用我们已确定这种战略的AAA + -ATPases RuvBL1 / RuvBL2至关重要解聚因子8。 在体外系统基于X.蟾蛋已经采用不同的DNA模板:福布斯等人发现，注射λ噬菌体DNA导入未受精的X.蟾卵引起染色质上NAK大会EDλ噬菌体DNA。注射病毒DNA的活化蛋，染色质的组合件然后形成一个核状结构18和类似的λ噬菌体DNA的可组合使用卵提取物，以产生核状结构在体外 19。用DNA包被的磁珠已经被用于研究DNA 20的chromatinization一个核膜和招募核膜21以及装配和孔配合物22，虽然它仍保持打开在何种程度上这类似于一个真正的核重新组合过程。这里介绍的协议允许分离的有丝分裂染色体集群使用从非洲爪蟾激活卵子生成提取HeLa细胞解聚。它彻底重建导致的interphasic核8的改革事件。相比用于研究核膜的形成广泛应用核装配反应和核孔复合物在有丝分裂的端部，在解聚测定的HeLa有丝分裂染色体簇代替精DNA被使用。精子DNA可组装成有丝分裂染色质或之上孵育提取物未受精的和未激活的蛋3制备甚至单个染色体。我们决定使用的有丝分裂簇染色质源，以简化程序，避免从染色质缩合干扰。此外，卵提取物的制备是稍微修改:对于染色质解聚低速提取物通过在固定角转头2高速离心步骤清除被使用。低速提取物可以贮存长达6月液氮，而不会失去其活性。与此相反，在核装配反应，胞液和漂膜由低速提取物通过稀释和差的高速离心前可能冻结产生（参见，一个德泰艾森哈特等人 16导致协议）​​。在我们的测定系统，除了膜的允许的闭合核膜包括核孔复合物的形成。由此产生的细胞核胜任核进口和出口8。因此，该系统同时支持染色质解聚和核膜改革。有趣的是，染色质解聚，也可以在不存在膜（图3）。但是另外的膜产生稍大一点的细胞核。最有可能的，核膜的改革引起的二次解聚一步尚未定义的机制，它依赖于核进口。 对于有丝分裂染色体簇从HeLa细胞的分离中，使用由加塞和的Laemmli 15建立的协议的修改版本。同步有丝分裂细胞溶解于含有非离子去污剂毛地黄皂苷缓冲液和由技工的力。染色质分离为包含集群从一个细胞核所有染色体。相比单染色体分离方案的关键的区别是，该细胞不是hypotonically肿，但冷却至4℃裂解之前。这可以防止单个染色体15，23的断线。相比由JR保尔森23公布的协议识别谁整个染色质的簇的隔离的优点，加塞＆的Laemmli使用EDTA的含聚胺缓冲剂，代替的Mg 2+基于缓冲器来减少的激酶，核酸酶，蛋白酶和磷酸酶的活性和通过这种降低在分离过程15存在的蛋白质和DNA修饰的量。此外，在差速离心使用胶体二氧化硅粒子梯度高度降低细胞质污染。该协议也可以被用于分离的有丝分裂染色体簇从中国仓鼠卵巢和小鼠细胞15。 <p类="jove_content"&gt;总之，我们的协议忠实重新构成了染色质解聚，因为它发生在有丝分裂的末端。 ATP的体外染色质解聚的依赖性可以是至少部分地解释了RuvBL1 / 2的参与，而且其它类型的认证+ -ATP酶 ，P97，这消除从染色质有丝分裂激酶极光乙有丝分裂退出24中。为什么这个过程需要GTP水解是，我们打算使用此设置来回答开放性问题之一。

非洲爪蟾卵提取物是一个强大的和广泛应用的工具，研究在无细胞的测定法的简单复杂细胞事件。由于通过Lohka＆升井1首次描述它们已经被广泛地用于研究的有丝分裂过程，如染色质缩合2，主轴组件3，核膜破裂4，而且核质运输5或DNA复制6。发生在有丝分裂的末端的事件，所必需的interphasic核的改造如核膜改革与核孔复合重组小得多理解相比早期有丝分裂事件，但可使用爪蟾卵提取物7被类似研究。最近，我们建立了基于爪蟾卵提取物研究染色质解聚在有丝分裂8月底的检测，欠调查过程，等待着我TS细致的刻画。 在后生动物，染色质是高度会聚在进入有丝分裂，以便执行的遗传物质的忠实偏析。以确保该染色质是用于基因表达和DNA复制访问间期，它需要被解压缩在有丝分裂的末端。在脊椎动物中，染色质是高达50倍相比相间9有丝分裂期间更紧密，而相比之下，酵母，其中有丝分裂压实通常要低得多， 例如，只有两个倍S.酵母 10。脊椎动物的染色质解聚已大部分研究精子DNA重组的卵子受精后的环境。的分子机制，其中nucleoplasmin，一种丰富的卵母细胞的蛋白质，交换精子特异性精蛋白组蛋白H2A和H2B存储在蛋。这个过程也阐明用爪蟾卵提取11，12。然而，表达nucleoplasmin的仅限于卵母细胞13和有丝分裂的染色体不包含这些精子特异性鱼精蛋白。因此，在有丝分裂的结束染色质解聚为nucleoplasmin 独立的8。 对于体外解聚反应，我们采用的激活爪蟾卵和染色质集群从同步的HeLa细胞中分离产生的提取物。鸡蛋与钙离子载体治疗模仿的钙释放到受精过程中精子的进入所产生的卵母细胞。钙波触发细胞周期的恢复和卵子在减数分裂的第二个中期逮捕，发展到第一相间14。因此，准备形式激活卵卵提取物所代表的有丝分裂退出/间期状态，并有能力来诱导特异性的有丝分裂的退出状染色质解聚，核膜事件和孔复合体改造。对于有丝分裂通道的隔离romatin簇我们使用加塞＆的Laemmli 15，其中染色体簇从HeLa细胞中有丝分裂的同步和通过梯度离心分离在含有多胺缓冲器释放裂解发布的协议的一个略加修改的版本。

<table><tbody><tr><td>spermine tetrahydrochloride</td><td>Fluka analytical</td><td>85610-25G</td><td></td></tr><tr><td>spermidine trihydrochloride</td><td>Sigma&amp;nbsp;</td><td>S2501-5G</td><td></td></tr><tr><td>high-purity digitonin</td><td>Millipore</td><td>300410-1GM</td><td>toxic</td></tr><tr><td>PMSF</td><td>Applichem</td><td>A0999,0100</td><td>toxic</td></tr><tr><td>thymidine</td><td>Calbiochem</td><td>6060</td><td></td></tr><tr><td>nocodazole</td><td>Calbiochem</td><td>487928</td><td>toxic</td></tr><tr><td>37% formaldehyde solution</td><td>Roth</td><td>7398-1</td><td>toxic</td></tr><tr><td>trypan blue solution (0.4%)</td><td>Sigma</td><td>T8154</td><td>toxic</td></tr><tr><td>1,4-dithiothreitol (DTT)</td><td>Roth</td><td>6908.2</td><td></td></tr><tr><td>AEBSF hydrochloride</td><td>Applichem</td><td>A1421,0001</td><td></td></tr><tr><td>pepstatin</td><td>Roth</td><td>2936.1/2/3</td><td></td></tr><tr><td>leupeptin</td><td>Roth</td><td>CN334</td><td></td></tr><tr><td>aprotinin</td><td>Roth</td><td>A162.3</td><td></td></tr><tr><td>Percoll (colloidal silica particles solution)</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>75 cm&amp;sup2; tissue culture flasks</td><td>Greiner Bio-one</td><td>658175</td><td></td></tr><tr><td>heat-inactivated fetal bovine serum (FBS)</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>Homogenizer (40 ml tissue grinder)</td><td>Wheaton</td><td>357546</td><td></td></tr><tr><td>Neubauer chamber</td><td>Assistent</td><td>441/1</td><td></td></tr><tr><td>&amp;nbsp;Oak Ridge Centrifuge Tubes, polycarbonate (50 ml)</td><td>Nalgene</td><td>3118-0050</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer, nylon</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>cytochalasin B</td><td>Applichem</td><td>A7657,0010</td><td>toxic</td></tr><tr><td>cycloheximide</td><td>Roth</td><td>8682.3</td><td>toxic</td></tr><tr><td>L-cystein</td><td>Merck</td><td>1,028,381,000</td><td></td></tr><tr><td>hCG available as Ovogest</td><td>MSD</td><td>1431593</td><td></td></tr><tr><td>PMSG available as Intergonan</td><td>MSD</td><td>1431015</td><td></td></tr><tr><td>A23187 (mixed calcium-magnesium-salt)</td><td>Enzo</td><td>ALX-450-002-M010</td><td>toxic</td></tr><tr><td>syringe needles (1.20 x 40 mm, 18 G x 1 1/2&quot;)</td><td>Braun</td><td>4665120</td><td></td></tr><tr><td>ATP</td><td>Serva</td><td>10920.03</td><td></td></tr><tr><td>GTP, 2 Na x 3 H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Roth</td><td>K056.1/2/3/4</td><td></td></tr><tr><td>creatine phosphat disodium salt</td><td>Calbiochem</td><td>2380</td><td></td></tr><tr><td>creatine phosphokinase</td><td>Sigma</td><td>C3755-35KU</td><td></td></tr><tr><td>DMAP</td><td>Sigma</td><td>D2629-1G</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P-6148</td><td>toxic</td></tr><tr><td>centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.)</td><td>Beckman Coulter</td><td>343775</td><td></td></tr><tr><td>&quot;Cell-Saver&quot; (tips with wide opening, 1,000 &amp;micro;l)</td><td>Biozym</td><td>729065</td><td></td></tr><tr><td>50% glutaraldehyde solution, grade I</td><td>Sigma alderich</td><td>G7651-10 ml</td><td>toxic</td></tr><tr><td>0.1% (w/v) poly-L-lysine solution</td><td>Sigma</td><td>P8920-100 ml</td><td></td></tr><tr><td>flat-bottom tubes (6 ml, 16.0/55 mm)</td><td>Greiner Bio-one</td><td>145211</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector laboratories</td><td>H1000</td><td></td></tr><tr><td>tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.)</td><td>Beckman Coulter</td><td>343778</td><td></td></tr><tr><td>&quot;Cell-Saver&quot; (tips with wide opening, 200 &amp;micro;l)</td><td>Biozym</td><td>729055</td><td></td></tr><tr><td>12 mm coverslips</td><td>Thermo Scientific</td><td>0784 #1</td><td></td></tr></tbody></table>

a cell free assay to study chromatin decondensation at the end of mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

在解聚反应的时间依赖性 图1显示了测定解聚的典型时间进程。可见的染色体在反应开始的簇decondenses并合并成一个单一的，圆和光滑的核。当卵提取物被替换蔗糖缓冲染色体簇仍稠，这表明，解聚活性存在于卵提取物。 染色质解聚是一个能源依赖的过程 在体外解聚反应可方便地操纵，例如，通过加入抑制剂。在关于图2所示的实验中，非水解ATP或GTP类似物，ATPγS或GTPγS的，加入到反应中。既抑制解聚有显示，这是一个ATP和GTP依赖，活动过程（ 图2）。 在存在或不存在的膜（图3）中进行的解聚法。请注意，在这两种条件下染色质经历解聚，但是另外的膜会导致更大的细胞核。最可能的是，核膜改革导致由另外的机制依赖于核运输辅助解聚的一步。 <img alt="图1" src="/files/ftp_upload/53407/53407fig1.jpg" /> 对前作 在体外 解聚反应。有丝分裂染色质团从HeLa细胞图1.时间当然孵育interphasic 爪蟾卵提取物。样品固定在指定的时间点，用4％的PFA和0.5％戊二醛，用DAPI染色，并通过共分析<html> nfocal显微镜。重新印刷从Magalska 等 。8。比例尺为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig1large.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/53407/53407fig2.jpg" /> 图2.染色质解聚需要ATP和GTP水解。在10mMATPγS，10毫GTPγS或对照缓冲液的存在下进行解聚的染色质。样品固定用4％的PFA和0.5％戊二醛在指定的时间点，并通过共聚焦显微镜分析。重新印刷从Magalska 等 。8。比例尺为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3"SRC ="/文件/ ftp_upload / 53407 / 53407fig3.jpg"/&gt; 在不存在（A）或120分钟浮选纯化膜的存在下（B）的，进行图3染色质解聚在存在和不存在的膜。染色质解聚。样品固定用4％的PFA和0.5％戊二醛并通过共聚焦显微镜分析。染色质用DAPI染色，膜与介质隔离集成电路18（1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- tetramethylindocarbocyanine高氯酸盐）。比例尺为5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

a-cell-free-assay-to-study-chromatin-decondensation-at-end

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K 次观看.  Max Planck Society. The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

视频: A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

科研

遗传学

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

癌症研究

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

发育生物学

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

生物学

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

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

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

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

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

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

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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

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

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

具体的基因组区域及相关分子鉴定enChIP隔离

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

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.