我们感谢拉斯 Dölken 为初级 T 细胞建立4sU 标记的建议;伊丽莎白. 格拉夫和托马斯 Schwarzmayr 在图书馆代和测序方面提供重要帮助;德克 Eick 和安德鲁弗拉特利提供 RNAPII 和 T 细胞抗体;n. 亨利埃特 Uhlenhaut 和 Franziska Greulich 帮助图书馆准备芯片序列;卡罗琳 c. 弗里德尔得到德意志 Forschungsgemeinschaft (DFG) 赠款 FR2938/7-1 和 CRC 1123 (Z2) 的支持;Elke Glasmacher 由德意志 Forschungsgemeinschaft (DFG) 和德国糖尿病研究中心 (DZD)、亥姆霍兹姆霍兹慕尼黑的赠款 GL 870/1-1 支持。

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</ol>

作者声明他们没有竞争的财政利益。

分析基因调控的整个过程是必要的, 以充分理解细胞适应反应特定的刺激或治疗。结合总 RNA 序列, 4 的 su 序列, 核糖体剖面和芯片序列在不同的时间点导致全面分析的主要过程中的基因调控的时间。对生物过程的深刻理解需要定义实验设置和最佳时间点。
由于研究基因调控的方法迅速改善, 这些协议可以适应快速变化。然而, 它们为研究任何类型细胞的基本基因调控机制提供了最重要的方法。在这里, 我们讨论在使用这些方法时必须考虑的一些缺陷和事实。
单元格:细胞必须是高度可行的, 如果使用主隔离细胞, 必须保证细胞数量的纯度 (例如, 主要 T 细胞的资产管制分析)。即使是轻微的压力细胞也会影响这些非常敏感的测序方法的结果, 并降低新转录或翻译的 RNA 的数量, 并导致在测序结果中不需要的应力响应读数。本协议中提到的离心速度对原 T 细胞进行了优化。因此, 根据细胞类型调整速度。
4sU 对细胞生理学的影响:除了上述的选择, 以验证最小摄动的细胞生理学后, 4sU 增加, 进一步和/或额外的分析可以执行, 特别是当细胞数是有限的。对细胞增殖的影响可以通过简单计数标记和未标记的细胞来验证细胞的加倍时间来测试。核仁应力诱导也可以通过免疫荧光染色 nucleolin 和核的细胞形态学来进行测试。为了进一步验证4sU 的影响, 可以通过将从标记的总 rna 到未标记的总 rna 的读计数关联来测量改变的全球基因表达。
单元格编号:对于体外生成的 T 单元格, 建议至少从Table 2中指示的单元格数量开始。根据单元格类型选择每个方法的适当编号。由于 T 细胞的细胞质和 RNA 比其他细胞少, 很可能是其他细胞数量的减少将是足够的。对于芯片序列, 细胞数高度依赖于所使用的抗体和细胞内感兴趣蛋白质的表达水平。对于组蛋白或 RNAPII 芯片, fewe 细胞可以使用, 而细胞数需要增加时, 转录因子的使用, 特别是如果他们是在低级别表达。
4sU 标记和 RNA biotinylation:使用粘附单元格时, 4sU 标记可以按 Rädle et al所描述的那样定向。19由于单元格非常快速地合并了 4sU, 所以可以直接添加到悬浮液、黏附剂或半黏附细胞的培养基中。
建议用 60-80 µg 的 RNA 开始 biotinylation。然而, 虽然我们没有测试少于30µg, 但可以使用较低数量的 rna. 添加一个 coprecipitant (例如, GlycoBlue), 如果颗粒很难看到, 在沉淀 RNA。达菲et也表明, methylthiosulfonate 活化生物素 (MTS-生物素) 比 HPDP-生物素31更有效地与 4 su 标记 RNA 反应。因此, 也许值得考虑改用 MTS-生物素, 特别是用于恢复小 rna, 后者往往有较少的苷残留 (参照达菲的 biotinylation 协议;参见纯化4个 su 标记的 RNA,实验程序)。
为了恢复新转录的 RNA, 有可能使用顺磁性珠或 RNA 清除珠的选择。总是考虑到这些套件可能或可能不会净化特定 rna。例如, 如果您对 miRNAs 感兴趣, 请考虑使用特定的工具包进行 miRNA 捕获和排序。
对新转录的 RNA 进行量化:要准确量化新转录的 RNA, 测量应由适当的荧光计执行 (请参阅材料表)。在1小时的4sU 曝光, 新转录 rna 代表约 1-4% 的总 RNA。新转录的 1 h 标记的 RNA, 活化 T 细胞由 ~ 90-94% rRNA11组成。
核糖体分析:在建立该方法时, 我们确定使用1.5x 的核酸酶量比原始协议中的建议保证了正确的消化。此外, 没有报告的不良影响, 增加了数量的核酸酶。由于 overdigest 的 RPFs 是相当困难的, 而它们是核糖体蛋白所束缚的 RNA 的一部分, 你仍然可以稍微增加滴定最佳核酸酶消化量。
如果在步进4.4.2 中恢复了不到500的爱国阵线 rna, 重复 rRNA 耗尽和池纯化 RPFs 与 rna 清洁 & Concentrator-5 柱。或者, 在胶体 (步骤 4.5.6) 的 RNA 洗脱过程中, 在凝胶 (步骤 4.5.3) 和池凝胶切片上分别加载两个相同的样本。
我们建议把 RPFs 上的凝胶尽可能紧密地切割到28和 30 nt 波段。这有助于从 rRNAs 和 tRNAs 中消除不需要的片段, 随后将成为库的一部分, 并减少对 RPFs 的排序读取。
还建议在凝胶纯化过程中避免紫外线照射。这会造成 RNA 片段中的缺口, 以及嘧啶脂肪酸, 最终会严重影响到图书馆的准备和测序结果。
库的准备和数据排序:核糖体分析协议能够生成适合于排序的 cDNA 文库。4 su 标签生成的样本可以直接用于图书馆准备与任何适当的 RNA 排序套件。由于新转录的 RNA, 特别是当使用短的标记时间, 可能还没有 polyadenylated, 不应进行聚一选择。相反, 我们强烈建议 rRNA 损耗, 以防止减少实际样品的测序深度。使用 T 细胞, 我们开始与 400 ng 新转录和总 RNA (取决于试剂盒, 见材料), 执行 rRNA 耗尽和减少周期 pcr 扩增, 以减少 pcr 偏倚。图书馆的准备工作可以用较少的起始材料进行。要考虑到库的复杂性, 应优化 PCR 循环的数量。
对于芯片序列, 还有许多工具包可供图书馆准备。在我们的手图书馆准备工作很好从 2 ng 芯片脱氧核糖核酸开始 (参见材料为建议在哪个套件使用)。确保在排序过程中检查颜色平衡的索引。我们建议≥40 x 106的测序深度分别读取4个 su 序列、总 RNA 序列和切屑样本, ≥80 x 106读取核糖体分析样本。测序深度取决于样品和下游生物信息学分析, 应慎重考虑。为了分析 cotranscriptional 拼接的内含子读数, 需要选择 100 bp 配对端测序。
排序偏差:在确定转录、翻译或转录因子结合的全球变化时, 测序已成为黄金标准。近年来, 现有的方法被推向极限, 或者开发出新的技术来测序越来越小的 RNA 起始量。这需要扩增 cDNA, 引入噪声或偏倚。最近, 开发了独特的分子标记 (UMIs), 以实验确定 PCR 引入的重复性。最近, 人们发现, UMIs 只是轻微地提高排序能力和错误发现率的差异基因表达式32。不过, 考虑使用独特的分子标识符 (UMIs) 来控制库的复杂性, 特别是从低数量的 RNA 开始, 以及当需要许多 PCR 周期时。
缓冲区和库存解决方案:所有4个 su 序列和核糖体剖面的缓冲器必须在严格的无 RNase 条件下使用核酸酶水来制备。建议购买预先制作的无核酸酶氯化钠, 三盐酸, EDTA, 柠檬酸钠和水。为确保核酸酶的条件, RNase 净化解决方案可用于清洁吸管或表面。所有芯片序列的缓冲器都需要至少 DNase, 并且可以在室温下存储。在使用和保持冰之前, 总是添加蛋白酶抑制剂, 或者是磷酸酶抑制剂。
生物信息学:分析所有测序数据 (即、芯片序列、RNA 序列和 ribsosome 分析) 涉及质量控制 (例如, 使用 FastQC, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), 适配器修整 (例如, 与cutadapt20), 然后映射到研究中的细胞的参考基因组。对于 rna 序列数据 (总共和4个 su 序列) 以及核糖体分析数据, 需要一个拼接的 RNA 序列映射器, 如 ContextMap 221。对于芯片序列数据 unspliced 对齐, 使用 BWA22是足够的。基因表达式可以使用 RPKM 模型 (每百万片段映射的外显子 Kilobase 读取)1, 在确定每个基因的读计数后使用一个程序,例如featureCounts23来计算。对于来自芯片序列数据的峰值调用, 有许多程序可用,例如、mac24或 GEM25。可以在 R26中执行进一步的下游分析, 特别是使用由 Bioconductor 项目27提供的工具。
在这里, 从核糖体分析中集成 4sU-和总 RNA 水平和平移活动的一个主要挑战是规范化的。解决这个问题的一个经典方法是规范化到保持家庭基因的水平。为了减少由于个体饲养基因随机波动而产生的噪音, 建议不要只使用少量的保留基因, 而是要在较大的集合中中位数,例如由艾森伯格和莱瓦诺编写的 > 3000 家保留基因28.对于从4sU 到总 rna 的比率计算 rna 更替率, 规范化是基于中位更替率 (例如, 假设 rna 半衰期为 5h)29。然而, 由于这不假设对保留基因的整体变化, 我们建议使用独立于规范化的分析方法,例如, 基于关联的不同数据类型的时间序列的聚类, 以确定基因组在激活过程中具有明显的转录和翻译行为。有关不同数据类型的 bioinformatical 集成的详细说明, 请参阅原始发布11。
营业额率和数据集成分析:最近发表的一篇论文33将多路复用基因控制 (MGC) 确定的半衰期与全球方法进行比较, 可以表明半衰期与其他方法 (如) 相比, 与代谢标记法获得的寿命最佳。药物转录的一般抑制)。但是, 应该指出, 半衰期计算之间的差异可能会出现, 并且已被描述为15、34。我们解释了由于长时间4sU 暴露引起的压力反应所引入的大多数问题和差异。因此, 排除 4 su 标记引入的应力响应是必不可少的。为了进一步验证周转率, 我们建议使用 MGCs。
此外, 此处生成的数据集还可用于更综合的数据分析 (例如, 对长的非编码 rna 的调节)35,36。

近年来, rna 测序 (rna 序列) 已成为分析细胞或生物体内所有表达 rna 的标准工具1。然而, 为了了解适应特定刺激/药物的细胞的整个过程, 有必要充分确定所有的基础过程, 从 mRNA 转录到加工、更替和翻译。rna 转录的短期变化几乎不能用总 RNAseq 来衡量, 因为总 rna 的变化取决于因素例如rna 半衰期和转录活动, 这是一个不良的模板, 以反映细胞的适应环境影响2,3。事实上, 已经制定了各种新的测序技术, 允许对基因调节过程中的不同步骤进行分析,4当以正确的方式组合时。该协议描述了如何结合一些相当容易应用的排序技术, 以比较的方式跟踪对 mRNA 的基本层的调节。为了分析转录活动, 我们描述了多种方法, 如基因表达式的 Cap 分析 (笼)5, 本机伸长率转录序列 (净序)6, 以及全基因组的核运行 (的序列)7,8, 以及 bromouridine 序列 (老兄) 和 4-thiouridine 序列 (4 su), 它使用在新转录的 RNA9,10中合并的代谢产物, 仅举几个。当笼子识别准确的转录起始点时, 网络序列和增加序列提供了关于阅读方向的更准确的信息, 4 的 su 序列 (这里描述的方法) 只检测新转录的 RNA。然而, 4 的 su 序列是高度敏感的, 可以应用于不同的时间框架来测量积极变化细胞中的定量转录活性, 以及 mRNA 处理的数量变化 (在几分钟内发生)9, 11,12。此外, 4 su 是理想的结合 rna 序列计算 rna 更替率的基因9。mRNA 的转录是由 RNA 聚合酶 II (RNAPII) 进行的, 这反过来受到多种因素的影响, 如转录因子、组蛋白修饰和一般的激活剂/阻遏, 甚至可以是转录的一部分。复杂.为了测试有多少基因/启动子/增强器区域受一个因素的约束, 芯片序列已被开发, 这是现在的标准方法为此目的, 由于许多商业上可用的抗体13。但是, 尽管 ChIPseq 提供了有关调节因素绑定的明确信息, 但它并不反映它是否确实导致了转录14的变化。因此, 用4的 su 序列进行芯片序列是这类生物问题的理想组合。基因表达调控也可能在以后的阶段发生, 因为 mRNA 和蛋白质水平不一定关联15,16, 表明有可能对平移或后转化的重要调节级别, 具体取决于上下文。在 2011, 核糖体的分析首先与 RNA 序列结合, 现在是选择的方法来量化迅速发生的蛋白质变化, 因为仍然有一些敏感性限制与质谱17。事实上, 从这些方法获得的笔译率已证明对蛋白质水平的变化 (至少是长期变化的衡量) 提供了相对较好的估计, 并允许对翻译过程有更详细的看法,例如确定起始端和可选翻译框架17。所有四方法的组合使用可以在稳定状态、不同单元格类型之间或在快速变化的单元格11的时间序列实验中使用。这一用途提供了一个全基因组范围内的变化转录因子结合影响 RNA 转录, 处理和翻译。

<table><tbody><tr><td>&lt;strong&gt;4sU-labeling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4-thiouridine (100 mg)</td><td>Carbosynth</td><td>13957-31-8</td><td>Prepare 50 mM stock in sterile H2O/PBS; store at &amp;ndash;20&amp;deg;C in aliquots of 50-500 &amp;micro;l; do not refreeze.</td></tr><tr><td>1.5 ml safe-lock tubes</td><td>Eppendorf</td><td>30121589</td><td>Optional</td></tr><tr><td>1.5 ml screw-top polypropylene tubes</td><td>Sarstedt</td><td>72692005</td><td>Compatible with Dimethylformamide</td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td>Compatible with Dimethylformamide</td></tr><tr><td>2.0 ml screw-top polypropylene tubes</td><td>Sarstedt</td><td>72694005</td><td>Compatible with Dimethylformamide</td></tr><tr><td>50 ml tubes</td><td>BD Falcon</td><td>352070</td><td>Compatible with Dimethylformamide</td></tr><tr><td>Agencourt RNAClean XP</td><td>Beckman Coulter</td><td>A63987</td><td>We recommend to use these paramagnetic beads. Aliquot and store at 4&amp;deg;C</td></tr><tr><td>Chloroform</td><td>Sigma Aldirch</td><td>372978</td><td>WARNING &amp;ndash; HAZARDOUS TO HEALTH</td></tr><tr><td>Dimethylformamide</td><td>Sigma Aldrich</td><td>D4551</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Roth</td><td>6908.1</td><td>Prepare as 100 mM DTT in nuclease-free H&lt;sub&gt;2&lt;/sub&gt;O; always prepare fresh</td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.00983.1000</td><td></td></tr><tr><td>EZ-Link Biotin-HPDP (50 mg)</td><td>Pierce</td><td>21341</td><td>Prepare 1 mg/ml stock solution by dissolving 50 mg Biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4&amp;deg;C in aliquots of 1 ml. DMF dissolves some plastic materials. We recommend to use glass pipettes to transfer DMF from ist stock glass bottle to 50 ml Falcon tubes.</td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>Isopropanol</td><td>Merck</td><td>1.09634.1011</td><td></td></tr><tr><td>NaCl (5M)</td><td>Sigma Aldrich</td><td>71386</td><td>Stock solution</td></tr><tr><td>nuclease-free EDTA (500 mM ), pH 8.0</td><td>Invitrogen</td><td>15575-020</td><td>Stock solution</td></tr><tr><td>Nuclease-free H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>W4502</td><td>Stock solution</td></tr><tr><td>nuclease-free Tris Cl (1M), pH 7.4</td><td>Lonza</td><td>51237</td><td>Stock solution</td></tr><tr><td>Phase Lock Gel Heavy tubes (2.0 ml)</td><td>5Prime</td><td>2302830</td><td>Use in step 1.3.4.</td></tr><tr><td>Polypropylene 15&amp;nbsp;ml centrifuge tubes</td><td>Greiner Bio-One</td><td>188271</td><td>Or equivalent; they have to tolerate up to 15,000 &amp;times; g</td></tr><tr><td>QIAzol Lysis Reagent (200 ml)</td><td>Qiagen</td><td>79306</td><td>Use this or equivalent TRI reagent for RNA isolation, WARNING &amp;ndash; CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol)</td></tr><tr><td>Qubit RNA HS assay kit</td><td>Life Technologies</td><td>Q32852</td><td>Use this kit for quantifying RNA quantity in step 1.4.11</td></tr><tr><td>RNeasy MinElute Kit</td><td>Qiagen</td><td>74204</td><td>Optional; includes Buffer RLT</td></tr><tr><td>Sodium citrat</td><td>Sigma Aldrich</td><td>C8532</td><td>Prepare 1.6 M stock solution using nuclease-free water</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>&amp;micro;Macs Streptavidin Kit</td><td>Miltenyi</td><td>130-074-101</td><td>Store at 4&amp;deg;C, includes &amp;micro;Macs columns used in step 1.4.6. (store at RT)</td></tr><tr><td>&lt;strong&gt;Cell viability and stress assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PE Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences</td><td>559763</td><td>Optional</td></tr><tr><td>Thapsigargin</td><td>Sigma-Aldrich</td><td>T9033</td><td>Optional</td></tr><tr><td>p53</td><td>abcam</td><td>ab26</td><td>Optional</td></tr><tr><td>p-EIF2a (Ser51)</td><td>Cell Signaling</td><td>9721</td><td>Optional</td></tr><tr><td>BH3I-1</td><td>Sigma-Aldrich</td><td>B 8809</td><td>Optional</td></tr><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4sU Washing Buffer</td><td></td><td>store at RT</td><td>100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Biotinylation Buffer (10x)</td><td></td><td>store at 4 &amp;deg;C</td><td>100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water; make aliquots of 1 ml; store at 4&amp;deg;C</td></tr><tr><td>RNA precipitation buffer</td><td></td><td>store at RT</td><td>1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2100 Bioanalyzer instrument</td><td>Agilent</td><td>G2939BA</td><td></td></tr><tr><td>RNA 6000 Nano Kit</td><td>Agilent</td><td>5067-1511</td><td>Use this kit to verify RNA integrity in step 1.3.10</td></tr><tr><td>RNA 6000 Pico Kit</td><td>Agilent</td><td>5067-1513</td><td>Optional</td></tr><tr><td>UV/VIS spectrophotometer</td><td>Thermo Scientific</td><td>NanoDrop 1000</td><td>Or equivalent. Use in step 1.2.2/3.1.8/3.3.3/3.4.3</td></tr><tr><td>High-speed centrifuge</td><td>Thermo Scientific</td><td>Heraeus Multifuge X3R</td><td>Or equivalent equipment capable of reaching 13,000&amp;nbsp;&amp;times;&amp;nbsp;g</td></tr><tr><td>High-speed rotor</td><td>Thermo Scientific</td><td>Fiberlite F15-6 x 100y</td><td></td></tr><tr><td>Adaptors for 15 ml tubes</td><td></td><td></td><td></td></tr><tr><td>Refrigerated table-top centrifuge</td><td>Eppendorf</td><td>5430 R</td><td>Or equivalent.</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>Thermomixer C</td><td>Or equivalent.</td></tr><tr><td>Magnetic stand</td><td>Miltenyi Biotec</td><td>130-042-109</td><td>One stand holds 8 &amp;micro;Macs columns.</td></tr><tr><td>Ultra-fine scale</td><td>Mettler Toledo</td><td>ML204T</td><td>Or equivalent.</td></tr><tr><td>E-Gel iBase Power System</td><td>Invitrogen</td><td>G6400UK</td><td>For RNA gels; or equivalent.</td></tr><tr><td>E-Gel EX 1% agarose precast gels</td><td>Invitrogen</td><td>G4020-01</td><td>For RNA gels; or equivalent.</td></tr><tr><td>DynaMag-2 Magnet-1 each</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>RNaseZap</td><td>Sigma</td><td>R2020</td><td>Optional</td></tr><tr><td>TruSeq stranded total RNA library prep kit</td><td>Illumina</td><td>RS-122-2201</td><td>Or equivalent. For T cells we used 400 ng 4sU and Total RNA with 11 cycles for PCR amplification. rRNA depletion is included in this kit</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td></td><td>use a Nanodrop or equivalent instrument to measure RNA concentration</td></tr><tr><td>&lt;strong&gt;Ribosome Profiling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TruSeq Ribo Profile kit (Mammalian or Yeast)</td><td>Illumina</td><td>RPYSC12116 (Yeast)</td><td></td></tr><tr><td>TruSeq Ribo Profile kit (Mammalian or Yeast)</td><td>Illumina</td><td>RPHMR12126 (Mammalian)</td><td></td></tr><tr><td>Illustra MicroSpin S-400 HR Columns</td><td>GE Healthcare</td><td>27-5140-01</td><td></td></tr><tr><td>RNA Clean &amp;amp; Concentrator-25 kit</td><td>Zymo Research</td><td>R1017</td><td></td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5 kit</td><td>Zymo Research</td><td>R1015</td><td></td></tr><tr><td>Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat)</td><td>Illumina</td><td>MRZG126 or MRZG12324</td><td></td></tr><tr><td>(High Sensitivity DNA Kit)</td><td>Agilent Technologies</td><td>5067-4626</td><td>Already needed for 4sU-seq</td></tr><tr><td>&lt;strong&gt;All other consumables and equipment are listed in the User guide&lt;/strong&gt;</td><td>!!!</td><td></td><td>Carefully read the user guide and order required consumables in advance (consider a long delivery time for some consumables e.g. gels)</td></tr><tr><td>&lt;strong&gt;ChIP&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-HCl (pH 8.0)</td><td>gereral lab supplier</td><td></td><td></td></tr><tr><td>100 bp Plus Marker</td><td>Thermo Fisher</td><td>SM0323</td><td></td></tr><tr><td>16 % Formaldehyde</td><td>Thermo Fisher</td><td>28908</td><td>Add to a final concentration of 1 %</td></tr><tr><td>70% EtOH</td><td>gereral lab supplier</td><td></td><td>Always prepare fresh</td></tr><tr><td>Agarose</td><td>gereral lab supplier</td><td></td><td></td></tr><tr><td>Agencourt RNAClean XP beads</td><td>Beckman Coulter</td><td>A63987</td><td>We recommend to use these paramagnetic beads. Aliquot and store at 4&amp;deg;C</td></tr><tr><td>ChIP library preparation kit</td><td>KapaBiosystems</td><td>KK8504</td><td>Or use the kit of your choice</td></tr><tr><td>DNA low bind microcentrifuge tubes</td><td>Eppendorf</td><td>Z666548-250EA</td><td>or equivalent</td></tr><tr><td>Dynabeads Protein G</td><td>Invitrogen</td><td>10004D</td><td>Use these superparamagnetic beads coupled to protein G in step 4.3.1.; Bring to RT before use</td></tr><tr><td>Glycine</td><td>gereral lab supplier</td><td></td><td>Prepare a 2M stock solution</td></tr><tr><td>Glycogen</td><td>Roche</td><td>10-901-393-001</td><td></td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td>Use this kit (or equivalent) to purify chromatin in step 4.2.4.</td></tr><tr><td>Phosphatase Inhibitor (PhosStop)</td><td>Roche</td><td>4906837001</td><td>Add freshly to the buffer and keep on ice</td></tr><tr><td>Power SYBRgreen Master mix</td><td>Thermo Fisher</td><td>4367659</td><td></td></tr><tr><td>Protease Inhibitor (cOmplete, EDTA-free)</td><td>Roche</td><td>11873580001</td><td>Add freshly to the buffer and keep on ice</td></tr><tr><td>Proteinase K</td><td>Invitrogen</td><td>25530049</td><td></td></tr><tr><td>Qubit dsDNA HS Assay kit</td><td>Invitrogen</td><td>Q32851</td><td>Use this kit for quantifying DNA quantity in step 4.6.4. on a Qubit Fluorometer</td></tr><tr><td>Rnase, DNase free</td><td>Roche</td><td>11-119915001</td><td></td></tr><tr><td>Salmon sperm (sonicated to around 100bp)</td><td>Sigma</td><td>D1626</td><td></td></tr><tr><td>TE pH 8.0</td><td>gereral lab supplier</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies (ChIP grade if possible)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-RNA Pol II [8WG16]</td><td>abcam</td><td>ab817</td><td></td></tr><tr><td>anti-Histon H3K36me3</td><td>abcam</td><td>ab9050</td><td></td></tr><tr><td>or antibody of interest</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Binding/Blocking buffer</td><td></td><td>Store at RT</td><td>PBS with 0.5 % BSA and 0.5 % Tween 20</td></tr><tr><td>Cell-Lysis buffer</td><td></td><td>Store at RT</td><td>5 mM Pipes [pH 8.0], 85 mM KCl, and 0.5 % NP40</td></tr><tr><td>ChIP IP buffer</td><td></td><td>Store at RT</td><td>0.01 % SDS; 1. 1% Triton X-100;1.2&amp;nbsp;mM EDTA; 16.7&amp;nbsp;mM Tris-HCl, pH 8.1; 16.7&amp;nbsp;mM NaCl</td></tr><tr><td>Elution buffer</td><td></td><td>Store at RT up to 6 months</td><td>10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 300 mM NaCl and 0.5 % SDS</td></tr><tr><td>Nuclei-Lysis buffer</td><td></td><td>Store at RT</td><td>50 mM Tris [pH 8.0], 10 mM EDTA, and 1 % SDS</td></tr><tr><td>Wash buffer I</td><td></td><td>Store at RT</td><td>0.1 % SDS; 1 % Triton X-100; 2 mM EDTA; 20mM Tris-HCL pH 8.1; 150 mM NaCl</td></tr><tr><td>Wash buffer II</td><td></td><td>Store at RT</td><td>0.1 % SDS; 1 % Triton X-100; 2&amp;nbsp;mM EDTA; 20&amp;nbsp;mM Tris-HCL pH 8.1; 500&amp;nbsp;mM NaCl</td></tr><tr><td>Wash buffer III</td><td></td><td>Store at RT</td><td>0.25&amp;nbsp;M LiCl; 1% NP-40; 1&amp;nbsp;mM EDTA; 10&amp;nbsp;mM Tris-HCl, pH 8.1</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2100 Bioanalyzer instrument</td><td>Agilent</td><td>G2939BA</td><td>use this instrument for electrophoretical analysis</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Bioruptor TBX microtubes 1.5 ml</td><td>Diagenode</td><td>C30010010</td><td></td></tr><tr><td>or tubes special for your sonication device</td><td></td><td></td><td></td></tr><tr><td>Bioruptor sonication device or sonication device of your choice</td><td></td><td></td><td>Sonication of T cells with Bioruptor: 20 - 25 cycles (30 s on, 30 s off at high in two 1.5 ml bioruptor microtubes with 500 &amp;micro;l each tube)</td></tr><tr><td>Magnetic stand for tubes</td><td></td><td></td><td></td></tr><tr><td>Thermomixer</td><td></td><td></td><td></td></tr><tr><td>Agarose gel electrophoresis</td><td></td><td></td><td></td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Scientific</td><td></td><td>Use this Fluorometer for quantifying low amounts of RNA/DNA</td></tr></tbody></table>

real-time analysis of transcription factor binding, transcription, translation, and turnover to display global events during cellular activation

激活后, 细胞迅速改变其功能程序, 从而, 他们的基因表达谱。基因表达的巨大变化, 例如, 在细胞分化, 形态发生, 功能刺激 (如激活免疫细胞), 或在接触到药物和其他因素的地方环境。根据刺激和细胞类型, 这些变化迅速发生, 并在任何可能的基因调控水平。将应答细胞的所有分子过程显示给某种刺激/药物是分子生物学中最困难的任务之一。在这里, 我们描述了一个协议, 可以同时分析多层次的基因调控。我们比较, 特别是转录因子结合 (染色质-免疫沉淀排序 (芯片序列)), de从头转录 (4-thiouridine 序列 (4 su)), mRNA 处理和营业额以及翻译 (核糖体分析)。通过结合这些方法, 可以显示一个详细的和全基因组范围的行动过程。
在分析快速适应或改变系统时, 特别推荐测序新转录 RNA, 因为这描述了4sU 暴露期间所有基因的转录活动 (不管它们是 downregulated 的还是不确定的)。总 rna 序列和核糖体分析的组合使用另外允许计算 rna 营业额和翻译率。对高通量测序结果的生物信息学分析, 可以为数据的分析和解释提供多种方法。生成的数据还可以跟踪联合转录和替代拼接, 只是提到一些可能的结果。
这里描述的组合方法可以适用于不同的模型有机体或细胞类型, 包括主要细胞。此外, 我们为每个使用的方法提供详细的协议, 包括质量控制, 并讨论潜在的问题和隐患。

4sU 标记: 验证最佳4sU 标记条件 (凋亡、核应力、胞浆应力)、时间和浓度:高水平的4sU 可以抑制 rRNA 的生产和加工, 诱导细胞质以及核应力30。因此, 需要对细胞的4诱导应力和细胞凋亡进行检测。建议采用西方印迹分析方法可视化 p53 积累, 表明核应力, 增加 phospho-EIF2a 水平, 显示胞浆应力和荧光活化细胞分类 () 对细胞凋亡的分析。高浓度和长时间接触4sU 或药物, 如 thapsigargin 或亚砷酸可用于诱发细胞压力。为诱导凋亡或细胞死亡, 细胞以 BH3I-1 (500 ng/µL) 治疗, 或在95摄氏度 (热休克) 中孵化5分钟。蛋白 V/7-AAD 染色用于测定凋亡 (蛋白 V) 和死亡 (7) 细胞。标记体外生成的主 Th1 细胞为0.5 小时, 500 µM 4sU (最终浓度) 或 1 h, 200 µM 4sU 既不诱发细胞压力或凋亡的迹象 (图 1), 但导致足够的4sU 合并。
RNA 标记时间也可以缩短 (≤5分钟), 这导致短寿命的内含子序列的增加比更长的标记时间。为了可视化共转录拼接率, 4 su 标记时间不应超过30分钟。有关4sUlabeling 的详细信息, 请参阅 Rädle et。19
质量控制:rna 完整性在处理 rna 时是非常重要的。通过 electrophoretical 分析检查4个 biotinylation 标记 rna 的 rna 质量是最方便的 (参见材料表)。考虑从步骤2.2.2 中验证分离的 rna, 特别是在使用它进行总 RNA 序列排序时。rna 完整性编号 (≥8) 应被确认, 以确保 rna 完整性, 以便进一步处理 (图 3)。
Electrophoretical 分析也可以用来验证新转录的 RNA。要知道, 新转录的 rna 含有明显较不成熟的 rRNAs 与总 RNA 相比, 典型的 rRNA 带是不太突出。
核糖体分析: cdna 文库的 PCR 扩增: cdna 放大 (步骤 4.9.1) 是确保良好测序结果的关键步骤。通过 electrophoretical 分析对放大后的库进行分析。一个好的放大库示例显示了大约 140-160 bp (图 4A) 的峰值。应避免使用过多的适配器脂肪酸 (图 4B), 并应根据制造商的协议 (PCR 产品的页面纯化) 进一步纯化这些样品。过多的模板或太多的 PCR 周期会导致过度放大, 其特征是高于预期的分子量带、涂抹的 PCR 产品和适配器二聚体产品 (图 4C)。对于大多数样本 1-5 µL 的发送 cDNA 和 9 PCR 周期的扩增, 通常会产生足够数量的正确的 PCR 产品。
芯片: 染色质剪切:每种类型的细胞都需要调整最佳的剪切条件。预先确定剪切条件 (例如、周期数、高或低功耗)。使用相同数量的细胞和相同体积的测试目的, 因为较低的细胞密度增加剪切效率。尽量避免过度或不受剪切的染色质。大的染色质碎片可以通过堵塞来显著地影响芯片的结果, 过度剪切可以破坏兴趣蛋白上的表位, 导致抗体的结合效率降低。在本实验中, 当剪切染色质的主要分数约为 1000 bp 或稍低 (图 5A) 时, 取得了最佳效果。
通过 qPCR 验证芯片:在启动芯片之前, 最好测试使用的抗体是否适合芯片 (如果可能的话, 使用芯片级抗体) 的芯片 qPCR。在启动库准备之前, 验证芯片是否按 qPCR 排序 (请参见步骤 5.6.5)。设计引物, 绑定到已知的兴趣蛋白质的目标站点。如果一个基因中的确切靶点是未知的, 几个引物对可以用来扫描基因和相关的调控元素。对于 Th1 单元格的 RNAPII 芯片Ifng, 该在刺激时转录上调, 肌动蛋白引物可用作正向控制。Sox9和胰岛素作为一个负控制, 因为这些基因不是在 Th1 细胞中表达的 (图 5B)。记住不要使用外显子-跨越引物, 这通常用于 qPCR 的 mRNA。IgG 控制也可用于证明所用抗体的特异性。Immunoprecipitated DNA 可以用合适的荧光计来测量 (参见材料表)。免疫球蛋白控制的 nonspecifically 绑定 dna 的数量应该大大降低, 与 DNA 的数量绑定的抗体的利益。
复制: 生物学意义的证明:强烈建议对从同一池中开始的所有方法执行动力学实验, 以确保单元格对所有未经处理和治疗的样本具有相同的标识 (图 2)。然而, 建议采取小整除数的主要时间点为每个方法比较样本与生物复制 (例如, 通过 qPCR, 资产管制分析).这允许粗略估计, 如果两个复制的治疗是可重现的, 你可以进行排序。复制的验证应通过 bioinformatical 分析进行。结果的重现性可以用散布图 (图 6) 来评估复制和可视化之间的 FPKM 值之间的相关性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56752/56752fig1.jpg"> 图 1: 验证无扰动细胞生理学的最佳 4 su 标记条件 (从 Davari et al.11) (A) 通过资产管制分析法检测细胞凋亡:体外生成的 Th0 细胞以不同浓度的 4sU (括号内表示) 分别治疗0.5 小时、1小时和2小时。BH3I-1 治疗诱导蛋白 V 的凋亡, 而热休克 (5 分钟95摄氏度) 被用来诱导细胞死亡, 由7。(B) 对4sU 治疗和活化 T 细胞 p53 的西方印迹分析: 样品被标记为200µM 4sU, 显示激活时间, 但标记为500µM 4sU 的 0.5 h 时间点除外。(C) 在具有相同标记条件 (B) 的激活 Th1 细胞中 phospho-EIF2a 和总 EIF2a 的西方印迹分析。Thapsigargin 被用来作为一个积极的控制。<a href="http://cloudflare.jove.com/files/ftp_upload/56752/56752fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56752/56752fig2.jpg"> 图 2: 用于跟踪全基因组变化的动力学设置示意图 该方案说明了将4的 su 序列、总 RNA 序列、核糖体剖面和芯片序结合在细胞治疗上研究全基因组变化的设置。池单元格, 并预留所需的单元格数以进行未经处理的控制。为每个方法和时间点处理剩余的单元格和拆分。标签未经处理的/治疗的细胞为4苏序列与4sU 如所述。每个方法的时间点和样本取决于所检查的特定生物学问题。为每个时间点和方法抽取样本, 并遵循协议的专用部分。<a href="http://cloudflare.jove.com/files/ftp_upload/56752/56752fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56752/56752fig3.jpg"> 图 3: 4 个 su 标记 RNA 的质量控制 Bioanalyzer 对活化 Th1 细胞的总 rna 和生物素化 rna 进行了分析。18S rRNA 和 28S rRNA 显示, 并且 rna 完整性数字 (玲) 由仪器给确定 rna 的完整性。应≥8, 以确保 RNA 的完整性。<a href="http://cloudflare.jove.com/files/ftp_upload/56752/56752fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56752/56752fig4.jpg"> 图 4: 核糖体分析库的 Bioanalyzer 配置文件 (A) 一个好的库: 该示例显示了预期大小范围 (140-160 bp) 的峰值, 不需要进一步纯化。(B) 本示例显示了相对于所需产品 (140-160 bp) 的过多适配器二聚体放大产品 (120 bp)。这个图书馆需要进一步的净化。(C) 过度放大的样本: 高于预期的分子量峰值和涂抹的 PCR amplicons 是可见的 (由箭头表示)。<a href="http://cloudflare.jove.com/files/ftp_upload/56752/56752fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56752/56752fig5.jpg"> 图 5: qPCR 剪切和验证芯片后的最佳染色质尺寸 (A) 琼脂糖凝胶图片显示被剪切的染色质的最佳片段大小从三个样品被剪切了25个周期在 sonicator 和纯化如在协议之前描述。(B) 总 RNAPII 芯片 (反 RNA II、8WG16、ab817) 的 Q PCR 结果表示为输入的百分比。Ifng和肌动蛋白引物用作阳性, 而Sox9和胰岛素为负控制 (两种基因均未在活化 Th1 细胞中表达)。<a href="http://cloudflare.jove.com/files/ftp_upload/56752/56752fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56752/56752fig6.jpg"> 图 6: 生物复制的比较 (来自 Davari et ) 的数字。11) 在激活 Th1 细胞刺激后, 新转录 (4sU) RNA 4 h 复制之间的表达值 (FPKM) 的代表性散点图比较。绿线表示相等的 FPKM 值和秩相关在每个情节被表明。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>标签持续时间 (分钟)</td>
			<td>推荐的4sU 浓度 (µM)</td>
		</tr>
<tr>
<td>120</td>
			<td>100-200</td>
		</tr>
<tr>
<td>60</td>
			<td>200-500</td>
		</tr>
<tr>
<td>15-30</td>
			<td>500-1000</td>
		</tr>
<tr>
<td>< 10</td>
			<td>500-2000</td>
		</tr>
</tbody></table>表 1: 推荐的4sU 浓度 (从 Rädle, et al.19) 推荐的4sU 浓度的范围是指不同的标签时间。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>方法</td>
			<td>单元格编号</td>
			<td>RNA 数量</td>
		</tr>
<tr>
<td>4 su 标记</td>
			<td>≥2 x 107
</td>
			<td>≥60µg</td>
		</tr>
<tr>
<td>核糖体分析</td>
			<td>≥2 x107
</td>
			<td></td>
		</tr>
<tr>
<td>芯片序列</td>
			<td>≥2 x 107 -3 x 107
</td>
			<td></td>
		</tr>
</tbody></table>表 2: 所需的初级 T 细胞数量 每个方法所需的主要 T 单元格的最小数量。使用其他单元格类型时, 金额可以减少。

Watch this Scientific Journal Video about Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation at JoVE.com

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

该协议描述了芯片序列的组合使用, 4 的 su 序列, 总 RNA 序列, 和核糖体分析的细胞系和主细胞。它能够跟踪转录因子绑定、从头转录、RNA 处理、营业额和翻译的变化, 并在激活和/或快速变化的细胞中显示事件的总体过程。

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

real-time-analysis-transcription-factor-binding-transcription

Research

JoVE Journal

Genetics

13.5K 次观看.  Helmholtz Zentrum München. 该协议描述了芯片序列的组合使用, 4 的 su 序列, 总 RNA 序列, 和核糖体分析的细胞系和主细胞。它能够跟踪转录因子绑定、从头转录、RNA 处理、营业额和翻译的变化, 并在激活和/或快速变化的细胞中显示事件的总体过程。

视频: Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

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 ...

科研

遗传学

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

免疫与感染

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

癌症研究

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

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

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

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 ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

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

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

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

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

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

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA双链断裂时转录活性的活细胞成像

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

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