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
Summary
通过结合本地和交联染色质免疫沉淀高分辨率质谱，ChroP方法使解剖的组蛋白修饰，变体和非histonic协同作用的蛋白质在功能上不同的染色质域的复合蛋白质结构。
Abstract
染色质是一种高动态核蛋白复合物由DNA和蛋白质，其控制各个DNA依赖性过程。在特定区域的染色质结构和功能是由组蛋白的翻译后修饰（hPTMs）和变体，染色质结合蛋白，包括转录因子，和DNA甲基化的局部富集调节。染色组合物中的不同的功能区的蛋白质组分析的方法已经被缺乏有效的协议，以丰富这些域在适当的纯度和量为后续的深入分析物的质谱（MS）到目前为止妨碍。我们在这里介绍了新设计的染色质蛋白组学策略，命名ChroP（CHRO晨报P roteomics），由此制备型染色质免疫沉淀是用来隔离不同的染色质区域，其特征在hPTMs方面，变体和共同相关联的非histonic蛋白质，是通过MS进行分析。我们说明他重新ChroP的转录沉默的异染色质区域的富集和分析，其标志是三甲基化的组蛋白H3赖氨酸9的存在成立。所取得的结果证明ChroP在彻底表征异蛋白质组，并证明它作为理解如何染色质的独特的蛋白质决定簇相互作用和协同效应，建立基因座的特定结构和功能结构的有力的分析策略的潜力。
Introduction
染色质是涉及为所有的DNA介导的过程主模板一个高度动态的核蛋白复合物。核小体是染色质的基本重复单元和由含各规范组蛋白H2A，H2B，H3和H4，在其周围147 bp的DNA被包裹1,2两分子的蛋白质八聚体核心的。所有核心组蛋白的结构为球状结构域和一个灵活的N端“尾巴”突出了核小体之外。一种用于调节染色质结构和动力学的主要机制是基于共价键的翻译后修饰（翻译后修饰），其主要发生在N-末端组蛋白3,4。组蛋白修饰可以通过改变高阶染色质结构，通过改变之间的组蛋白-DNA或核小体之间的接触，并从而控制DNA结合蛋白（ 顺机制）的可访问功能，或通过作为dockin克位点的调节蛋白，无论是作为单个单元，或者嵌入在多聚体复合物。这样调节蛋白可以发挥它们的功能以不同的方式：通过直接调节基因表达（ 即 TAF蛋白），或通过改变核小体定位（ 即染色质重塑复合物），或者通过修改其他组蛋白残基（ 即蛋白质具有甲基转移或乙酰基转移酶活性）（ 反式机制）5。观察到在某些具体的染色质基因座不同的PTM模式群集导致了阐述这一假设在不同地点的不同修饰可协同作用以生成分子编码介导相关的DNA的功能状态。在“组蛋白密码假说”已经获得了很大的共识在多年，但它的实验验证已受困于技术限制6,7。
质谱（MS）为基础的蛋白质组学已成为一个功能强大的工具来映射组蛋白修饰模式和表征染色质结合蛋白8。 MS检测的变形作为肽的实验和理论质量之间的特定Δmass。在个人组蛋白的水平，微软提供了一个公正和全面的方法来映射翻译后修饰，允许其中9-14新的修改和揭示interplays的检测。
在最近几年，已经开发了多种策略来剖析染色质的蛋白质组合物，包括完整的有丝分裂染色体15，可溶性HPTM结合蛋白16-18的标识和特定染色质区域的分离和分析的表征（ 即端粒）19,20。然而，组蛋白翻译后修饰，变体和染色质相关蛋白之间的位点特异性的协同作用的研究仍然是不完整。在这里，我们描述了一种新的方法，命名为ChroP（染色体晨报P roteomics）21，我们已经开发了能够有效表征功能不同的染色质域。这种方法适应染色质免疫沉淀技术（ChIP），在表观遗传学研究中使用一套行之有效的方案，为充实样品的有效质谱为基础的蛋白质组学分析。我们已经开发出两种不同的协议，这取决于所用的输入和由MS处理的问题染色质的类型;具体为：1）芯片消化MNase不固定本地染色质是用来净化单核和解剖合作关联hPTMs（N-ChroP）; 2）交联染色质碎片通过超声处理芯片采用的是与SILAC为主interactomics策略来描述所有共同丰富的染色质结合蛋白（X-ChroP）的组合。我们举例说明这里的N-和X-ChroP的丰富和研究异，采用H3K9me3为诱饵进行免疫沉淀步骤的结合。使用ChroP的可扩展研究无论是不同的区域染色质上，或变化过渡到一个不同的功能状态，在同一区域内的染色质组成，从而铺平了道路表观遗传学中的各种应用。
Protocol
Representative Results
Discussion
我们最近已描述ChroP，定量策略染色质中的蛋白质组分的大规模表征。 ChroP结合的表观遗传领域使用两种互补的方法，芯片和质谱，从自己的优势中获利，克服各自的局限性。沉淀耦合到深度测序技术（ChIP-SEQ）允许的组蛋白修饰在核小体少35的分辨率的全基因组的映射。虽然便于其敏感性，基于抗体的测定是在自己有能力区分类似的修改和解剖的组蛋白密码36组合方面的限制。另?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究最初发表在分子细胞蛋白质组学。索尔多米和染色质功能域Bonaldi T的蛋白质组学研究揭示新型增效作用之间鲜明的异染色质成分的MCP。 2013; 12：64-80。 ©美国社会生物化学与分子生物学。我们感谢罗伯塔Noberini（技术和IEO的意大利学院，意大利）的手稿的批判性阅读。结核病工作是由来自乔瓦尼Armenise哈佛基金会职业发展计划，意大利癌症研究协会和健康的意大利外交部资助。 MS工作是由一个FIRC奖学金支持。
Materials
| DMEM | Lonza | BE12-614F | |
| FBS | Invitrogen | 10270-106 | |
| SILAC DMEM | M-Medical | FA30E15086 | |
| Dialyzed FBS | Invitrogen | 26400-044 | |
| Lysine 0 (12C6 14N2 L-lysine) | Sigma Aldrich | L8662 | |
| Arginine 0 (12C6 14N4 L-arginine) | Sigma Aldrich | A6969 | |
| Lysine 8 (13C6 15N2 L-lysine) | Sigma Aldrich | 68041 | |
| Arginine 10 (13C6 15N4 L-arginine) | Sigma Aldrich | 608033 | |
| Micrococcal Nuclease | Roche | 10 107 921 001 | |
| Complete, EDTA-free Protease Inhibitor Cocktail Tablets | Roche | 04 693 132 001 | |
| Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length | Spectrumlabs | 132720 | |
| QIAquick PCR purification kit | QIAGEN | 28104 | |
| Anti-Histone H3 tri-methylated K9-ChIP grade | Abcam | ab8898 | |
| Histone H3 peptide tri-methyl K9 | Abcam | ab1773 | |
| Dynabeads Protein G | Invitrogen | 100.04D | |
| NuPAGE Novex 4-12% Bis-Tris Gel | Invitrogen | NP0335BOX | |
| Colloidal Blue Staining Kit | Invitrogen | LC6025 | |
| LDS Sample Buffer | Invitrogen | NP0007 | |
| Formaldheyde | Sigma Aldrich | F8775 | |
| Aceti anhydride-d6 | Sigma Aldrich | 175641-1G | |
| Formic Acid | Sigma Aldrich | 94318-50ML-F | |
| Iodoacetamide ≥99% (HPLC), crystalline | Sigma Aldrich | I6125 | |
| DL-Dithiothreitol | Sigma Aldrich | 43815 | |
| Sequencing Grade Modified Trypsin, Frozen 100ug (5 × 20μg) | Promega | V5113 | |
| Nanospray OD 360μm x ID 75μm, tips ID 8μm uncoated Pk 5 | Microcolumn Srl | FS360-75-8-N-5-C15 | |
| ReproSil-Pur 120 C18-AQ, 3 µm 15 % C | Dr. Maisch GmbH | r13.aq. | |
| C18 extraction disk, 47 mm | Agilent Technologies | 12145004 | |
| Carbon extraction disk, 47 mm | Agilent Technologies | 12145040 | |
| Cation extraction disk | Agilent Technologies | 66889-U |
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