这项工作得到了 v 学者奖 (V2014-004) 和 v 学者加奖 (D2016-030) 从 v 的癌症研究基金会和美国癌症协会机构研究补助金 (ACS 表意文字补助金 58-006-53) 的支持, 以普渡大学癌症中心研究.e.g.p. 获得了普渡大学药物化学和分子药理学系创研究生捐赠奖的支持。

1. 准备工作 <ol> <li> 准备100毫升低渗溶液缓冲器 A: 0.3 米蔗糖, 60 毫米氯化钾, 60 毫米三 pH 8.0, 2 毫米 EDTA, 0.5% NP-40。存储在4和 #186; C. 
	注意: 某些细胞系, 如 HEK293T, 需要较不严格的溶条件。如果原子核溶解容易, 使用修改的缓冲 A:25 毫米 HEPES pH 7.6, 25 毫米氯化钾, 5 毫米氯化镁 2 , 0.05 毫米 EDTA, 0.1% NP-40, 和10% 甘油. </li>
	<li> 准备一个250毫升的 2x mRIPA 溶液的库存解决方案: 100 mM pH 8.0, 2% NP-40, 和0.5% 钠胆酸. </li>
	<li> 准备100毫升的5米氯化钠的库存解决方案. </li>
	<li> 使用 2x mRIPA 和5米 nacl 溶液, 为六盐浓度的每一个都准备50毫升的 1x mRIPA 溶液: 0 毫米, 100 毫米, 200 毫米, 300 毫米, 400 毫米, 和500毫米氯化钠。在开始实验之前, 冷却所有的冰上的解决方案. </li>
	<li> 在所需条件下增大单元格线. 
	注: 在确定击倒蛋白质的效果时, 必须将 SSE 与野生细胞进行比较。这些 SSE 必须并排执行。举例来说, 使用化学抑制剂或刺激, 请参阅 3-5 节. </li>
</ol> 2。基本顺序盐萃取 <ol> <li> 每种情况下收获800万细胞, 用5毫升冰冷 PBS 洗涤两次. 
	注意: 有同等数量的细胞具有同等的蛋白质浓度是至关重要的。细胞的确切数量可能需要针对单个细胞系或特定的蛋白质进行优化。重要的是不要有太多, 因为增加的蛋白质浓度将阻止观察的洗脱曲线, 这是依赖于平衡结合。然而, 太少, #160; 细胞可能没有足够的蛋白质来检测曲线, 染色质颗粒更难隔离, 并可以在分数之间丢失. </li>
	<li> 重新悬浮在1毫升的缓冲液 + 蛋白酶抑制剂, 转移到1.5 毫升微离心管, 并旋转顶部超过底部在4和 #186; C 为 10 min. </li>
	<li> 通过离心 6000 x g 分离原子核 5 min 在4和 #186; C. 
	注: 在这一步之后, 核信封仍应完好无损。如果核包完好无损, 颗粒将 re-suspend 充分;然而, 当信封是裂解和染色质释放, 颗粒不会 re-suspend。如果在缓冲孵化后核包络不完整, 请使用修改后的缓冲区 a. </li>
	<li> 添加200和 #181; L mRIPA 0 毫米氯化钠 + 蛋白酶抑制剂, 每核颗粒前 re-suspending 颗粒。质每样移15次, 用1毫升吸管。球团应该 re-suspend 容易, 然而, 原子核将溶解, 因为它是 pipetted 和样品将变得厚实和黏, 并且困难的吸管。
	<ol> <li> 为了将球团拉入笔尖, 在离心管的底部插入吸管尖端的末端。一旦 DNA 从细胞核中释放出来, 颗粒就不会完全溶解, 但移会破坏它. </li>
	</ol> </li> <li> 当所有样本均已匀浆时, 在冰上孵育所有样品3分钟. 
	注意: 孵化时间可能需要根据感兴趣的蛋白质进行优化. </li>
	<li> 在 6500 x g 处离心3分钟, 在4和 #186 分离染色质颗粒; C. </li>
	<li> 将上清液转移到清洁的1.5 毫升离心管。这将是0毫米的分数。这0毫米 mRIPA 溶液可以作为一个洗涤步骤来溶解细胞核并去除任何与染色质无关的松散蛋白. </li>
	<li> 添加200和 #181; L mRIPA 100 毫米氯化钠 + 蛋白酶抑制剂, 每一个染色质颗粒前 re-suspending 颗粒。通过移15倍的颗粒来分解染色质颗粒. 
	注意: 关键是要与 pipetted 中的球团的次数一致. </li>
	<li> 在冰上孵育3分钟。这一孵化步骤允许所有样本达到平衡状态, 这是特别重要的, 当有多个样本. </li>
	<li> 离心机在 6500 x g, 3 分钟在4和 #186; C 和转移上清到一个干净的1.5 毫升管. </li>
	<li> 对剩余的盐浓度重复 2.6-2.10. 
	注: 400 毫米氯化钠后, mRIPA 颗粒应清晰, 7月29日, 不会停留在底部的管。颗粒可以放在离心管的盖子上, 而上清分离. </li>
	<li> 添加70和 #181; 将4x 蛋白质加载到每一个分数中, 并加载30和 #181; 每一个分数的 l 对 SDS 丙烯酰胺凝胶进行西部印迹分析. 
	注: 要确定蛋白质的结合轮廓, 关键是要装载等量的裂解物, 而不是载入等量的蛋白质浓度. </li>
	<li> 通过转移到膜上并使用有兴趣的蛋白质的主要抗体来执行标准的免疫印迹. </li>
	<li> 从染色质定量蛋白质洗, 用成像仪在红外荧光 IRDye 二次抗体和图像印迹中孵育印迹。虽然其他的开发方法可以使用, 我们建议荧光或红外成像, 因为它是更定量的性质. </li>
	<li> 使用 ImageJ 或类似软件计算每个盐浓度的蛋白质洗强度。通过绘制对盐浓度的波段强度, 可以确定你感兴趣的蛋白质的洗脱模式. </li>
</ol> 3。在 #34; 读取器和 #34; 抑制剂 <ol> <li> 获取两组单元格 (400万) 并将原子核隔离为标准的 SSE. </li>
	<li> 在200和 #181 中重新挂起两个组; mRIPA 0 毫米氯化钠, 孵育3分钟。这将允许去除细胞核中的任何游离蛋白. </li>
	<li> 添加200和 #181; 每粒 mRIPA 0 毫米氯化钠。将抑制剂 (2 和 #181; 1 毫米 (+) JQ1) 添加到一个样品和一甲基亚砜到对照组. </li>
	<li> 用移15次搅动颗粒, 在冰上孵育5分钟. </li>
	<li> 离心机在 6500 x g, 3 分钟在4和 #186; C 和转移上清到一个干净的1.5 毫升管. </li>
	<li> 重复 3.3-4 对所有盐浓度和执行一个标准的西方印迹. </li>
</ol> 4。序贯盐萃取在 #34; 作家和 #34; 抑制剂 <ol> <li> 使用抑制剂 (10 和 #181; M) 或亚甲基亚砜治疗3小时的细胞 </li>
	<li> 为每种治疗收集400万单元格. </li>
	<li> 为示例执行一个标准的 SSE, 并将抑制剂添加到所有缓冲区. </li>
</ol> 5。DNA 损伤后的序贯盐提取 <ol> <li> 使用1和 #181 处理单元格; M. 1 h. </li>
	<li> 更改单元格上的媒体并允许它们恢复3小时. </li>
	<li> 收集800万单元格并执行基本的 SSE </li>
</ol> 6。非顺序盐提取 <ol> <li> 收集1200万单元格并使用 PBS 进行清洗. </li>
	<li> 拆分单元格, 使每个微离心管有200万细胞. </li>
	<li> 在500和 #181 中重新挂起; 缓冲 A L, 孵育10分钟. </li>
	<li> 用离心法将原子核分离 6000 x g. </li>
	<li> 在200和 #181 中重新悬浮颗粒; 每个 mRIPA 缓冲液 + 氯化钠. </li>
	<li> 质每样移15次, 用1毫升吸管针尖. </li>
	<li> 在冰上孵育3分钟 </li>
	<li> 通过离心 6500 x g 分离染色质, 并将上清液转移至清洁管。添加70和 #181; l 加载染料和运行30和 #181; 我在一个 SDS 页的凝胶中进行印迹分析. </li>
</ol>

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作者没有相互竞争的金融利益。

蛋白质和染色质相互作用通过盐萃取的表征是一种常用的方法, 已经使用了几十年14,15;然而, 它没有得到系统的优化, 以揭示它的充分效用。我们演示了这种顺序方法提供了一种快速和廉价的方法来区分染色质结合的变化时, 蛋白质或环境的改变。SSE 具有高度的适应性和优化, 重要的是, 它在技术上是简单的, 不需要专门的设备。在开始之前需要优化的关?...

真核细胞中的 DNA 调控是一个复杂而复杂的系统, 它受到一系列蛋白质的严密控制, 它们能够协调对细胞内和细胞外刺激的反应。dna 被包裹在组蛋白 octamers 形成核, 它可以松散地沿 dna 分布或压缩成紧密的线圈1。这种结构安排的 DNA 和组蛋白被称为染色质, 这是由一个网络的蛋白质, 读取, 写入, 并抹去后的翻译修改 (PTM) 组组的2。一些组蛋白 PTMs, 如乙酰化, 改变了它们所沉积的氨基酸的电荷, 改变了蛋白质组与 DNA2之间的相互作用。组蛋白 PTMs 也用于招募转录调节剂, 染色质 remodelers, dna 损伤修复机械, 和 dna 复制机械, 以特定的区域的染色体3。
大多数研究染色质相互作用的方法要么探测小规模的相互作用, 要么涉及大规模的全基因组分析。体外结合研究通常利用单个重组域与组蛋白多肽或 DNA 进行分析, 如电泳迁移率测定 (EMSA)、等温滴定量热法、荧光极化和肽 pulldowns。因为这些分析通常专注于单个蛋白质领域, 它们有助于理解一小部分的拼图, 但不允许我们理解多域蛋白质的合作性质, 更不用说它们在 multi-protein 中的作用了。配合.另一层复杂的是由大多数哺乳动物染色质修饰复合物的异质成分添加。这种蛋白质的异质性, 结合染色质景观的动态性质, 使它具有挑战性, 以重述在体内染色质蛋白的结合相互作用的染色质体外。
在体内方法已取得重大进展;然而, 他们往往是昂贵的, 费时, 技术上的挑战。染色质沉淀, 其次是测序 (芯片序列) 是非常有用的, 以确定蛋白质和组蛋白修饰在整个基因的本地化, 但它需要大量的优化4。蛋白质通常与染色质交联以保持相互作用;然而, 这可以产生人工交互作用, 并可能导致表位掩蔽5。此外, immunoprecipitations (ip) 要求高度特异的抗体, 并广泛优化 DNA 剪切和 IP 条件的芯片 qPCR 使用已知的绑定站点, 这通常是不可用的先验。在优化了芯片条件后, 样品的处理成本很高, 需要数周到数月才能进行序列分析。虽然这种方法对于鉴别基因组中染色质结合蛋白的定位是非常宝贵的, 但成本和时间的承诺使得它无法使用这种方法来产生关于小的变化可能影响全局绑定属性的假设。.
在本文中, 我们描述了如何使用顺序盐萃取 (SSE) 检测可以用来检查染色质结合蛋白的全球结合剖面, 并区分如何改变蛋白质, 复杂, 或全球 PTM 剖面可以改变相互作用。虽然盐提取物是一个普遍和广泛使用的技术, 我们展示了如何这种顺序方法是高度重现性和多才多艺。SSE 允许我们描述一个复杂的单个子单位, 甚至一个单一的域是如何促成复合体对大块染色质的整体亲和性的。SSE 也可以用来确定蛋白质的结合是否受染色质景观的变化的影响, 为组蛋白标记靶向提供了有趣的假说, 可以使用芯片序列和其他基因研究进行确认。
我们最初改编自吴 et al., 检查 Polybromo1 (PBRM1) 在 PBAF 染色质 remodeler6,7的绑定中的功能。利用这一技术, 我们确定了 PBRM1 在 PBAF 染色质重塑复合体中染色质结合的作用, 然后确定了六个体 bromodomains 对此函数的相对贡献7。
在这里, 我们描述如何优化这种方法来探索不同细胞类型的染色质结合, 以评估类似的染色质修饰复合物的相对结合亲和性, 以检查化学抑制剂的染色质蛋白质的位移, 并以确定染色质结合在染色质景观改变后的效果。

<table>
	<tbody>
		<tr>
			<td>Sodium Chloride, Crystal 2.5 Kg AR (ACS)</td>
			<td>Avantor/Macron</td>
			<td>7581-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, Crystal 500 G AR&#168; (ACS)</td>
			<td>Avantor/Macron</td>
			<td>8360-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride, Granular 500 G AR&#168; (ACS)</td>
			<td>Avantor/Macron</td>
			<td>6858-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma(R) base,Primary Standard and Buffer, &#62;=99.9% (titration), crystalline</td>
			<td>Sigma-Aldrich</td>
			<td>T1503-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (Ethylenedinitrilo) Tetraacetic Acid, Disodium Salt, Dihydrate 125 G AR (ACS)</td>
			<td>Avantor/Macron</td>
			<td>4931-02</td>
			<td></td>
		</tr>
		<tr>
			<td>NONIDET P-40 SUBSTRATE, 100mL UN3082</td>
			<td>Amresco</td>
			<td>E109-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride, 6-Hydrate, Crystal 500 G AR&#168; (ACS)</td>
			<td>Avantor/Macron</td>
			<td>5958-04</td>
			<td></td>
		</tr>
		<tr>
			<td>GLYCEROL, 1L</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>DEOXYCHOLIC ACID SODIUM SALT, 100g</td>
			<td>Amresco</td>
			<td>0613-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research plus pipette</td>
			<td>Fisher Scientific</td>
			<td>13-690-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424 R refrigerated microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary mouse IgG HRP-linked</td>
			<td>Cell Signaling</td>
			<td>7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary rabbit IgG HRP-linked</td>
			<td>Cell Signaling</td>
			<td>7074</td>
			<td></td>
		</tr>
		<tr>
			<td>BMI-1 antibody</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBRM1 antibody</td>
			<td>Bethyl</td>
			<td>A301-591A</td>
			<td></td>
		</tr>
		<tr>
			<td>ARID1a antibody</td>
			<td>Santa Cruz</td>
			<td>sc-32761</td>
			<td></td>
		</tr>
		<tr>
			<td>BRD4 antibody</td>
			<td>Bethyl</td>
			<td>A301-9852a</td>
			<td></td>
		</tr>
		<tr>
			<td>(+) JQ1</td>
			<td>Cayman Chemical Company</td>
			<td>11187</td>
			<td></td>
		</tr>
		<tr>
			<td>SAHA</td>
			<td>Cayman Chemical Company</td>
			<td>10009929</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin</td>
			<td>AK Scientific</td>
			<td>25316-40-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Macron</td>
			<td>4948-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Cell</td>
			<td>BioRad</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Gel Tank</td>
			<td>ThermoFisher</td>
			<td>A25977</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine (BSA)</td>
			<td>Amresco</td>
			<td>0332-100g</td>
			<td>Used in as a 5% solution in PBS-T as blocking solution for Western blots</td>
		</tr>
		<tr>
			<td>Bolt MOPS SDS Running Buffer (20x)</td>
			<td>Life Technologies</td>
			<td>B0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bolt LDS Sample Buffer (4x)</td>
			<td>Life Technologies</td>
			<td>B0007</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa</td>
			<td>ThermoFisher</td>
			<td>26619</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Alcohol, Anhydrous</td>
			<td>Macron</td>
			<td>3041-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin kEDTA, 1X</td>
			<td>Cellgro</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>SODIUM AZIDE, 250g UN1687</td>
			<td>Amresco</td>
			<td>0639-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>SODIUM DODECYL SULFATE (SDS)500g UN1325</td>
			<td>Amresco</td>
			<td>0227-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine,for electrophoresis, &#62;=99%</td>
			<td>Sigma-Aldrich</td>
			<td>G8898-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>BigTop Microcentrifuge Tubes, Polypropylene</td>
			<td>VWR</td>
			<td>20170-333</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Pipet Tips</td>
			<td>VWR</td>
			<td>83007-382</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Bis-Tris Precast Gels 4-12%</td>
			<td>Invitrogen</td>
			<td>NW04125BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin Hemisulfate, 5 MG</td>
			<td>RPI Research Products</td>
			<td>22035-0.005</td>
			<td>Used for Protease inhibitor solution.</td>
		</tr>
		<tr>
			<td>APROTININ, 10 MG</td>
			<td>RPI Research Products</td>
			<td>20550-0.01</td>
			<td>Used for Protease inhibitor solution.</td>
		</tr>
		<tr>
			<td>PEPSTATIN A, 5 MG</td>
			<td>RPI Research Products</td>
			<td>30100-0.005</td>
			<td>Used for Protease inhibitor solution.</td>
		</tr>
		<tr>
			<td>OVCA429 cells</td>
			<td>Gift from Karen Cowden Dahl. Ph.D.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A media</td>
			<td>Corning Mediatech</td>
			<td>10-050-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning Mediatech</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM), Powder</td>
			<td>Corning Mediatech</td>
			<td>50-011-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA (100X) non-essential amino acid</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>FB-11: Fetal Bovine Serum, U.S. Source</td>
			<td>Omega Scientific</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Power Source</td>
			<td>VWR</td>
			<td>13-690-032</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequential salt extractions for the analysis of bulk chromatin binding properties of chromatin modifying complexes

由于染色质的复杂性质和大多数哺乳动物染色质修饰络合物的异质性, 对染色质靶向蛋白的结合性质的阐明具有很大的挑战性。为了克服这些障碍, 我们采用了一种顺序盐萃取 (SSE) 测定方法来评估染色质结合物的相对结合的亲和力。这种简单而直接的测定方法可用于非, 以评估两个相关复合物的结合亲和性的相对差异, 当亚基丢失或单个域被灭活时, 复杂度的亲和性变化, 以及染色质景观改变后的结合亲和力。通过在不断增加的盐量中依次 re-suspending 大块染色质, 我们能够从染色质中分析特定蛋白质的洗脱。使用这些轮廓, 我们能够确定染色质修饰复合物或染色质环境的改变如何影响结合作用。连接 SSE 与其他在体外和在体内化验, 我们可以确定的作用, 单个领域和蛋白质的功能, 在一个复杂的各种染色质环境。

本文从文献6中, 论证了常用的顺序盐萃取 (SSE) 方法的优点和应用。在图 1中, 我们通过检测 ARID1a 和 PBRM1 的洗脱模式, 比较了 SSE 在提取蛋白质并发中的重现性。我们一贯观察, ARID1a, 一个曝气生物滤子亚基, elutes 主要在200毫米氯化钠和 PBRM1, 一个独家 PBAF 亚基, elutes 主要在300毫米氯化钠。图 1a显示了三独立的 SSE...

Watch this Scientific Journal Video about Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes at JoVE.com

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

顺序盐提取染色质结合蛋白是一个有用的工具, 以确定结合性质的大蛋白复合物。该方法可用于评价单个亚基或域在蛋白质复合体与大块染色质的整体亲和性中的作用。

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 ...

sequential-salt-extractions-for-analysis-bulk-chromatin-binding

Research

JoVE Journal

Genetics

8.2K 次观看.  Purdue University. 顺序盐提取染色质结合蛋白是一个有用的工具, 以确定结合性质的大蛋白复合物。该方法可用于评价单个亚基或域在蛋白质复合体与大块染色质的整体亲和性中的作用。

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

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

科研

遗传学

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

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 ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

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 ...

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

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

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

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

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...