Name: sequential salt extractions for the analysis of bulk chromatin binding properties of chromatin modifying complexes
Uploaded: 2017-10-02T13:00:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes at JoVE.com

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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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&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>8360-04</td><td></td></tr><tr><td>Potassium Chloride, Granular 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>6858-04</td><td></td></tr><tr><td>Trizma(R) base,Primary Standard and Buffer, &gt;=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&amp;uml; (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, &gt;=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 &amp;micro;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&#039;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

8.3K 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

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

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

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 ...

科研

遗传学

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

医学

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

癌症研究

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 ...

生物化学

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

生物学

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

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

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

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

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

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

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

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

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此视频中的章节

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

在疾病的染色质蛋白质组的定量分析

验证组蛋白乙酰转移酶抑制剂的检测

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

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