作者要感谢 Tilman Borggrefe (Giessen 大学) 的友好分享超声波设备。来自大联盟的工作得到德意志 Forschungsgemeinschaft (SCHM 1417/8-3)、SFB/TRR81、SFB1021、SFB1213、卓越集群心肺系统 (ECCPS、激动 147/2)、德意志 Krebshilfe (111447) 和 IMPRS HLR 计划赠款的支持。最大的普朗克社会。

1. 天 1: 细胞文化
<ol><li>将要分析的单元格培养为所需的量。细胞培养条件和培养基取决于所调查的细胞类型。 注意: 原则上, 任何真核细胞都可以通过自由放任的方法对染色质的可达性进行分析。在这个实验中, 4 x 106 HEK-293T 细胞在 DMEM 培养基中的一个10厘米的盘子中播种, 补充了 10% (v/v) 的血清和 1% (w/v) 青霉素/链霉素, 并孵化在 5% CO2和37°c 24 小时, 直到细胞达到 70-80% 汇合 (~ 8 x 10每道菜的6单元格)。</li>
</ol>2. 2 天: 甲醛交联和细胞收获
注意: 甲醛是剧毒的, 必须总是在油烟机下使用。穿防护服 (手套和实验室大衣)。适当丢弃废物。
<ol><li>将甲醛直接添加到通风罩中的细胞培养基中, 以达到最终浓度为 1% (v/v) (270 µL 37% (v/v) 甲醛到10毫升的细胞培养培养基)。在室温下孵化10分钟 (20-25 °c), 并手动将板材每2分钟进行一次。 注: 交联时间可根据细胞类型进行调整。对于大多数细胞系, 10 分钟的交联是足够的。对于组织, 可能需要更长的孵化, 以允许所有细胞的固定。</li>
	<li>加入甘氨酸到最后浓度为0.125 米, 以淬火甲醛 (添加500µL 2.5 米甘氨酸的股票到10毫升培养基)。室温孵育5分钟, 每2分钟一次。</li>
	<li>用冰冷 PBS 清洗细胞 3x (8 克/升氯化钠, 0.2 克/升氯化钾, 1.44 克/升 Na2HPO4 · 2H2O, 0.24 g/升, 2 PO4, 用 HCl 或氢氧化钠调整 pH 值为7.4。通过加入10毫升的 PBS, 将培养基吸入并直接在细胞培养皿或烧瓶中冲洗。重复此步骤两次是非常小心的情况下, 松散黏附的细胞, 如 HEK-293T。在盘子的一侧小心地添加 PBS, 以避免细胞脱离。</li>
	<li>第三次冲洗后, 并用重悬在1毫升的冰冷 PBS 中的细胞, 并转移到1.5 毫升的反应管在冰上。必要时使用细胞刮刀分离细胞。 注: 当从有限的材料开始, 使用低 DNA 结合管, 以避免样本损失在过程中。</li>
	<li>离心机5分钟在 300 x g 在4°c 在一个冷却的桌上离心机。小心地吸掉上清。</li>
</ol>3. 细胞裂解和染色质破碎
<ol><li>并用重悬细胞颗粒在1毫升自由裂解缓冲 (1% (w/v), 10 毫米 EDTA, 50 毫米三盐酸 pH 值 8.1, 10 µg/毫升 leupeptin, 10 µg/毫升抑肽酶, 2 毫米 PMSF), 并并用重悬细胞通过仔细吹打几次。在冰上孵化20分钟。如果需要, 请在本步骤-80 °c 存储样品。</li>
	<li>油脂实验交联 DNA 剪切它的平均大小为 200-300 bp。必须为每个样本源和使用的超声波设备确定 sonicator 的特定设置。在任何情况下, 确保 DNA 的有效剪切。在步骤5.14 中描述了 DNA 剪切的优化步骤。在超声波期间冷却样品, 避免加热样品。</li>
	<li>离心样品15分钟和 1.3万 x g 在4°c。</li>
	<li>将上清液转移到新的反应管上。将样品分成100µL 整除数。如果需要, 将样品存放在摄氏-80 摄氏度。 注意: 此时可以中断协议。</li>
</ol>4. 控制 DNA 的脱交联
<ol><li>从步骤3.4 中提取100µL 整除, 以逆转交联, 并用作总 DNA 的参考。添加10µL 的 RNase (10 毫克/毫升) 和孵化1小时在37摄氏度。</li>
	<li>添加10µL 蛋白酶 K (20 毫克/毫升)。使用可编程热块孵化4小时在37摄氏度, 然后6小时65°c, 以切割蛋白质和逆转 crosslinks。最后, 将样品保持在4摄氏度, 直到协议恢复, 然后再进行步骤5。 注意: 其他自由放任协议使用未交联作为参考的细胞的染色质样本, 从而取消了对脱交联11的需要。这些样品没有交联的事实可能导致超声波效率或 dna 稳定性的差异, 因此使用与测试样本相同的程序的总 DNA 参考样本更准确。</li>
</ol>5. 3 天: 苯酚: 氯仿纯化 DNA
<ol><li>从步骤3.4 中取不交联的整除。添加10µL 的 RNase (10 毫克/毫升) 和孵化1小时在37摄氏度。</li>
	<li>从步骤4.2 中取非交联整除表单步骤5.1 和脱交联样品, 并进行并行处理。添加 H2O 以达到最后的卷300µL。</li>
	<li>在通风罩和漩涡中加入300µL 的 phenol:chloroform:isoamyl 醇 (25:24:1)。 注意: 苯酚具有腐蚀性和毒性, 必须始终使用在通风罩下。穿戴防护服 (手套和实验室大衣), 确保反应管紧密封闭, 适当处理废弃物。</li>
	<li>离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>小心地将上水相的280µL 转移到一个新的反应管上。确保不要从也含有蛋白质的界面中取任何碎片。将剩余的较低阶段丢弃为有机溶剂废料。</li>
	<li>重复步骤5.3 至 5.5, 小心地将上水相的270µL 转移到新的反应管上。</li>
	<li>在通风罩和涡流中加入270µL 氯仿。 注: 此步骤用于清除样品中残留的苯酚。</li>
	<li>离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>小心地将上水相的250µL 转移到新的反应管上, 注意不要从界面中携带任何碎片。将剩余的样品作为有机溶剂废料丢弃。</li>
	<li>添加25µL 5 米氯化钠和250µL 异丙醇。添加5µL 的糖原 (20 毫克/毫升) 作为 DNA 载体。在室温下, 通过反转管和孵育20分钟来混合。离心机为10分钟在 1.3万 x g 在4°c 沉淀脱氧核糖核酸。</li>
	<li>用400µL 70% (v/v) 乙醇清洗 DNA 颗粒。离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>仔细吸上清液, 让颗粒在室温下干燥10分钟。并用重悬在100µL 的缓冲 (10 毫米三 pH 8, 1 毫米 EDTA)。</li>
	<li>在65摄氏度的4小时内孵化无交联的游离 dna 样本, 以去除 dna 间的 crosslinks。把样品存放在摄氏-20 摄氏度。</li>
	<li>使用分光光度计量化 DNA 的数量, 并运行整除检查 2% (w/v) 琼脂糖凝胶的碎片大小。根据琼脂糖凝胶电泳 DNA 尺寸分析结果, 调整剪切时间和强度, 以获得 200-300 bp 的平均尺寸。</li>
</ol>6. 定量实时 pcr (qRT pcr) 测定游离与总 DNA
<ol><li>核 dna (以下简称自由 dna) 与总 dna 的比值由 qRT PCR17、18决定。样品也可以通过基因芯片杂交或深度测序进行定量分析, 以进行全组测定。</li>
	<li>为要测试的区域设计 qPCR 底漆, 并进行正负控制。 注意: 适当的阳性对照是在主动转录的管家基因 (如 ACTB、GAPDH、TPI1 或 TUBA1A (编码β肌动蛋白、GAPDH、TPI 或α蛋白) 的启动子中的引物。适当的阴性对照发现在染色质地区或基因沙漠。其他合适的负控制可以设计在与动态调节的轨迹相邻的基因组区域, 以控制本地副本数差异 (表 1)。</li>
	<li>将 DNA 样品稀释为最终浓度为10的µL 和 TE 缓冲器, 并将稀释系数计入最终计算 (见步骤 6.5)。</li>
	<li>在 triplicates 中建立一个96井板的 qPCR 反应, 每个井的反应组合如下: 模板 DNA:20 ng (2 µL), 前底漆 200 nM (0.4 µL 5 µM 股票), 反向底漆 200 nm (0.4 µL 股票µM), 一个商业准备使用含有绿色染料的反应混合物 (5 µL 从2x 股票), 和 H2O 到10µL。</li>
	<li>在实时 qPCR 设备中运行, 使用绝对量化模式, 并确定不同引物对样品的 Ct。在考虑到步骤6.3 中的稀释因子的情况下, 计算每种底漆的游离 dna 与总 dna 的比值, 方法如下: <img alt="Equation 1" src="/files/ftp_upload/57272/57272eq1.jpg"> 在表 2中给出了代表性的结果和一个示例计算。</li>
	<li>为了弥补样本之间的差异, 正常化为正控制回收率: <img alt="Equation 2" src="/files/ftp_upload/57272/57272eq2.jpg">
</li>
</ol>

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作者没有什么可透露的。

自由放任是一种稳健和廉价的方法, 允许识别无核区域。它基于的原理, DNA 包裹在核可以很容易地交联到组蛋白。苯酚: 氯仿提取是用来将非交联和无蛋白质的 dna 片段从蛋白质束缚 dna 中分离出来, 这是在低酚相和在一定程度上在界面 (图 1) 中发现的。因此, 无核区域在水相中游离 DNA 的分数中占了比例。许多出版物描述了自由放任方法23、24、25、26的详细协议, 可以通过协商来补充此处描述的过程。
与其他用于确定染色质结构的方法相似, 自由放任法也主要依赖于 DNA 的最佳剪切。如果片段太长, 任何无核的区域将被相邻的染色质绑定 DNA 掩盖。如果碎片太小, qPCR 或深度测序的检测就变得非常困难。因此, DNA 的超声波是一个关键参数, 必须加以控制和优化, 以获得大约 200-300 bp 长度的碎片, 这代表 1-2 核 (图 4)。
另一个可能影响最终结果的参数是样品的交联。虽然这里描述的固定程序对大多数细胞系是有效的, 研究员必须仔细评估这一步骤为特定的样本研究, 特别是当使用组织, 在那里可能需要更长的固定时间, 以允许甲醛到达所有组织样品中的细胞。
不像其他方法, 如 DNase I 或 micrococcal 核酸酶过敏, 芯片, 或 ATAC, 自由放任不依赖于酶活性或特定的抗体, 因此它不需要滴定或优化的反应条件。这使得自由放任是一个理想的方法来衡量染色质可及性的实验室很少经验的染色质领域。此外, 在必要时, 还需要进行试验, 以优化 DNA 剪切步骤和交联时间。
该方法最初是在酵母6中开发的, 但也已扩展到哺乳动物细胞。用自由放任法纯化的 DNA 可以用于深度测序, 允许对染色质状态进行全基因组的表征。这在许多研究中已经证明是有用的8,9,10,11,12,13,19,27,28。
DNase 实验结果表明, 与其他方法 (如14) 所获得的结果有很大的重叠, 尽管出现了一些差异。这是由于方法上的差异, 例如放宽或改造核仍然可能阻碍分裂由 DNase I, 而这些 dna 区域将不太容易产生 DNA/蛋白 crosslinks, 从而将确定为无核使用自由放任9的区域。此外, 非组蛋白的存在, 如 RNA 聚合酶或读者蛋白质的表观遗传标记可能导致 DNA/蛋白 crosslinks, 因此将被解释为蛋白质包装的区域在自由放任实验。这些限制是所有用于确定染色质状态的方法所固有的, 从而提高了使用不同方法的组合以获得全面的洞察力的必要性。

真核细胞的基因组 DNA 被紧密地包装成核, 需要被去除或改造以允许其他蛋白质与 DNA 相互作用。基因的转录活化通常会导致其 nucleosomal 结构更加开放, 特别是在 +1 核1中, 以促进 RNA 聚合酶的转录。此外, 诸如促进剂和促进剂等调控区域在其染色质可及性上进行动态变化, 以允许与染色质修饰符或转录因子的相互作用2。已经制定了几种方法来确定这些核的区域。这些内容包括对核酸的敏感度, 如 DNase I3或 micrococcal 核酸酶 4, 它只能在核不紧密环绕的情况下访问 DNA.其他鉴定开放染色质或游离 DNA 的方法包括 transposase 介导的 retrotransposable 元素 (transposase 可接触染色质、ATAC)5或染色质免疫沉淀 (芯片) 的整合, 使用抗组蛋白抗体。自由放任方法不是基于酶的能力达到 DNA 或抗体对某一特定蛋白质的结合, 而是依靠苯酚净化无核区域: 氯仿提取6,7。在这种方法中, DNA 是共价键与染色质蛋白的交联剂甲醛, 然后被剪切到小块超声波。紧密包装的染色质区域将有丰富的 dna/蛋白 crosslinks, 而没有或很少核的 dna 区域将很少或没有交联的 dna/蛋白复合物。苯酚: 氯仿萃取允许纯化水相中的无交联游离 dna, 而与蛋白质交联的 dna 则在有机酚相或水有机相间的界面中与蛋白质一起捕获。这种游离 dna 的量化与总 DNA 样本的引用相比, 可以识别染色质自由区。该方法可用于描述个别基因组区域, 如下所述, 但也适用于鉴定全基因组的染色质可达性时, 结合到深测序, 如所述的不同模式8,9,10,11,12,13. 由 ATAC 和其他方法 (如序列) 获得的结果在很大程度上是重叠的14。自由放任法是一种非常健壮、廉价、易于执行的方法来识别无核区域。与其他方法不同, 它不需要进行试验以确定核酸 (DNase 序列、MNase 序列) 或 transposases (ATAC 序列) 所需的适当消化水平, 并在最近的一次审查15中详细讨论了这一点。唯一要调整的参数是超声波条件, 在某些情况下是固定时间。本文描述了一个直接的协议, 它在图 1中显示, 不需要任何特殊设备 (sonicator)。该协议可以在相当短的时间内进行, 并使自由放任一个简单和可靠的方法来测试的染色质可达性的多种不同的细胞类型, 从肺细胞11到从小鼠肝脏得到的主要细胞16。

<table><tbody><tr><td>Phenol:chloroform:isoamyl alcohol 25:24:1</td><td>Carl Roth</td><td>A156.1</td><td></td></tr><tr><td>Glycogen, 20 mg/ml</td><td>Thermo Fisher</td><td>R0561</td><td></td></tr><tr><td>ABsolute QPCR Mix, SYBR-Green, Rox</td><td>Thermo Fisher</td><td>AB1162B</td><td></td></tr><tr><td>Proteinase K, 20 mg/ml</td><td>Thermo Fisher</td><td>EO0491</td><td></td></tr><tr><td>RNase A, 10 mg/ml</td><td>Thermo Fisher</td><td>EN0531</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L8511</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1603</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma</td><td>78830</td><td></td></tr><tr><td>milliTUBE 1 ml AFA Fiber</td><td>Covaris</td><td>520130</td><td></td></tr><tr><td>Holder milliTUBE 1ml</td><td>Covaris</td><td>500371</td><td></td></tr><tr><td>Covaris S220 Focused-ultrasonicator</td><td>Covaris</td><td></td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>Applied Biosistems</td><td>4376600</td><td></td></tr></tbody></table>

formaldehyde-assisted isolation of regulatory elements to measure chromatin accessibility in mammalian cells

适当的基因表达对细胞外线索的反应, 即组织和谱系特异基因转录, 关键依赖于高定义的染色质组织状态。核的动态结构由多个机制控制, 并形成转录输出程序。因此, 重要的是要确定一个可靠的方式, 最好是独立于抗体, 这可能是一个潜在的混杂的实验变异来源的特异性染色质的可访问度。染色质可达性可以用各种方法来衡量, 包括甲醛辅助的调控元素 (自由放任) 的分离, 这使得在相对较少的细胞数中测定一般的染色质可达性。在这里, 我们描述了一个自由放任协议, 允许简单, 可靠, 快速识别的基因组区域与低蛋白质占用。在这种方法中, DNA 是共价键与染色质蛋白结合使用甲醛作为交联剂, 并剪切到小块。游离的 DNA 之后被丰富使用苯酚: 氯仿萃取。游离 DNA 的比值是由定量聚合酶链反应 (qPCR) 或 dna 测序 (dna 序列) 与代表总 DNA 的控制样本相比较确定的。具有较松散的染色质结构的区域在游离 DNA 样本中丰富, 从而允许鉴定低染色质压实的基因组区域。

我们使用自由放任方法来测量 HEK293T 细胞中 MHC II 类基因的染色质可达性的差异, 如发布的19。MHC II 类编码基因表达在抗原呈现细胞 (apc), 无论是组成性或响应细胞因子信号 20.MHCII 基因的表达是由激活剂复合物驱动的, 它与特定的 DNA 元素结合在一起, 被称为 XY 盒, 位于这些基因的启动子和增强器中21,22。这反映在染色质水平, 正如我们对 MHC II 类基因 HLA-DPB1 和 HLA DMA 中选定区域的自由放任分析所揭示的那样。这些实验表明, 染色质是开放的区域, 包括 XY 盒, 而它更紧凑的基因体 (图 2)。
作为一个例子, 在这个特定的实验中, 我们稀释了总 DNA 样本10倍 (稀释因子 10), 而游离 DNA 样本没有稀释 (稀释因子 1) 的 qRT PCR。在表 2中列出了为不同区域测试的 Cts, 以及获取最终恢复比率和相对恢复比率的计算。利用 ACTB 启动子作为正向控制, 获得了相对回收率。请注意, 这些计算是用于生成图 2中所示结果的复制之一。
在不同的背景下, 在诱导基因表达模型中测试了染色质可及性。在这种情况下, 测定染色质压实的快速变化对促炎性刺激肿瘤坏死因子 (TNF) 的反应。HeLa 细胞未被治疗或刺激与 TNF 为1小时, 然后自由地测量在启动子区域的染色质压实, 控制 TNF 应答细胞因子白细胞介素 8 (IL8) 基因的表达。这些结果表明, 在 TNF 刺激细胞的 IL8 启动子中, 染色质的可达性明显增强 (图 3)。肿瘤坏死因子刺激后 IL-8 启动子的可达性甚至高于 ACTB 启动子中发现的明显的染色质重塑在这个调控区域。由于在本例中获得的回收率高于作为正控 (ACTB 启动子) 的区域所获得的采收率, 相对回收率高于一。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57272/57272fig1.jpg"> 图 1: 自由放任工作流.细胞通过添加甲醛 (A) 直接在细胞培养瓶或盘子中交联。在甲醛淬火后, 细胞被洗三次与冰冷 PBS (B), 并转移到一个反应管, 他们在悬浮溶解缓冲和微气泡剪切 DNA (C).提取的染色质的一个整除是通过加热65摄氏度去交联, 以获得总 DNA 参考 (D), 然后利用苯酚提取游离 dna: 交联的氯仿和脱交联的整除数 (E).最后, 对 dna 进行量化, 并计算出自由与总 dna 的比值 (F)。<a href="https://cloudflare.jove.com/files/ftp_upload/57272/57272fig1large.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/57272/57272fig2.jpg"> 图 2: HEK293T 细胞中 MHC II 类基因簇中染色质可达性的测定.HEK293T 细胞的分析。自由与总 DNA 的比值 (i. e, 相对回收率) 确定为 ACTB 基因的启动子作为阳性对照, 染色质区域. 12 作为阴性对照, 以及 MHC 的调控区和基因体第二类基因 HLA-DMA 和 HLA-DPB1。上部部分显示了引物的轨迹和位置的示意图表示。误差线表示在 triplicates 中测量的两个生物复制的标准偏差。从已发布的报表19中修改了该图形。<a href="https://cloudflare.jove.com/files/ftp_upload/57272/57272fig2large.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/57272/57272fig3.jpg"> 图 3: IL8 启动子中 DNA 可访问性的变化对 TNF 治疗的反应.用 TNF (20 ng/mL) 刺激 HeLa 细胞1小时, 并通过自由放任分析。ACTB (阳性对照) 的启动子 (染色质) 和 IL8 启动子的自由与总 DNA 的比值是确定的. 12 (负控制)。误差线表示从三生物复制中测量的重复值的标准误差。<a href="https://cloudflare.jove.com/files/ftp_upload/57272/57272fig3large.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/57272/57272fig4.jpg"> 图 4: 优化超声波条件.从293T 细胞提取的染色质是 sonified 使用一个重点 sonicator 与适当的管 (材料表)。染色质是微气泡为被表明的时间以重复三十年代以以下设置: 峰值功率 150, 义务因素 15, 周期每爆发 500, 跟随三十年代: 峰值功率 2.5, 责任因素 15, 周期每爆裂500。所产生的染色质通过苯酚纯化: 氯仿萃取并载入2% 琼脂糖凝胶。溴了一种溴化染色凝胶, 并在 bp 中标明了分子量标记的位置。在实验中, 超声波20分钟后获得了最佳染色质尺寸 (200-300 bp)。<a href="https://cloudflare.jove.com/files/ftp_upload/57272/57272fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆</td>
			<td>序列</td>
		</tr>
<tr>
<td>ACTB-启动子 F</td>
			<td>AAAGGCAACTTTCGGAACGG</td>
		</tr>
<tr>
<td>ACTB-启动子 R</td>
			<td>TTCCTCAATCTCGCTCTCGC</td>
		</tr>
<tr>
<td>人权中心12负控 F</td>
			<td>ATGGTTGCCACTGGGGATCT</td>
		</tr>
<tr>
<td>人权中心12负控制 R</td>
			<td>TGCCAAAGCCTAGGGGAAGA</td>
		</tr>
<tr>
<td>HLA-DMA-XY 框 F</td>
			<td>CATCAGTCACTGGGGAGACG</td>
		</tr>
<tr>
<td>HLA-DMA-XY 框 R</td>
			<td>GCTTCCCAGCCCAGTTACAT</td>
		</tr>
<tr>
<td>HLA-DMA-5'-F</td>
			<td>GGAGAGAACAATCTCCGCTTCA</td>
		</tr>
<tr>
<td>HLA-DMA-5'-R</td>
			<td>AGCTGCTATGTGTGGTTGGT</td>
		</tr>
<tr>
<td>HLA-DMA-3'-F</td>
			<td>TGGGGACCTAGTTAGGGAGC</td>
		</tr>
<tr>
<td>HLA-DMA-3'-R</td>
			<td>AGATCCATGGGAGGAGGCTT</td>
		</tr>
<tr>
<td>HLA-DPB1-XY-Box-F</td>
			<td>GTCCAATCCCAGGGTCACAG</td>
		</tr>
<tr>
<td>HLA-DPB1-XY-Box-R</td>
			<td>TGAAAAGAGCTGCAGTCAGGA</td>
		</tr>
<tr>
<td>HLA-DPB1-5'-F</td>
			<td>GCGTGTTCATGTCTGCATCC</td>
		</tr>
<tr>
<td>HLA-DPB1-5'-R</td>
			<td>TGATCCTCAGAGCCTGGACA</td>
		</tr>
<tr>
<td>HLA-DPB1-3'-F</td>
			<td>CCAGCCTAGGGTGAATGTTT</td>
		</tr>
<tr>
<td>HLA-DPB1-3'-R</td>
			<td>GCCTGGGTAGAAATCCGTCA</td>
		</tr>
<tr>
<td>IL8 启动子 F</td>
			<td>GTGATGACTCAGGTTTGCCCT</td>
		</tr>
<tr>
<td>IL8 启动子 R</td>
			<td>CTTATGGAGTGCTCCGGTGG</td>
		</tr>
</tbody></table>表 1: qPCR 底漆。
<img alt="Table 2" src="/files/ftp_upload/57272/57272table2.jpg"> 表 2: 自由放任相对恢复比率的示例计算.<a href="http://cloudflare.jove.com/files/ftp_upload/57272/Table_2.xlsx">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells at JoVE.com

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

核结构的可塑性受动态后生机制的控制, 包括非编码 rna、DNA 甲基化、核重新定位以及组蛋白组成和修饰。在这里, 我们描述了甲醛辅助的调控元素 (自由放任) 协议的隔离, 它允许以可重现、廉价和直截了当的方式确定染色质的可访问性。

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

formaldehyde-assisted-isolation-regulatory-elements-to-measure

Research

JoVE Journal

Genetics

11.2K 次观看.  University Hospital Virgen del Rocío. 核结构的可塑性受动态后生机制的控制, 包括非编码 rna、DNA 甲基化、核重新定位以及组蛋白组成和修饰。在这里, 我们描述了甲醛辅助的调控元素 (自由放任) 协议的隔离, 它允许以可重现、廉价和直截了当的方式确定染色质的可访问性。

视频: Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

科研

遗传学

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

改进的小鼠骨骼肌染色质沉淀的协议

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

生物化学

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

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 ...

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 ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

生物工程

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

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此视频中的章节

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

改进的小鼠骨骼肌染色质沉淀的协议

小鼠棕色脂肪组织的染色质免疫沉淀

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

酿酒酵母的单拷贝基因位点染色质纯化

酿酒酵母的单拷贝基因位点染色质纯化

酿酒酵母的单拷贝基因位点染色质纯化

用化学表观修饰因子重定向细胞机械抑制基因转录

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养