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。

<ol>
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没有利益冲突的声明。

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?...

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。

<table><tbody><tr><td>DMEM&amp;nbsp;</td><td>Lonza</td><td>BE12-614F</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10270-106</td><td></td></tr><tr><td>SILAC DMEM</td><td>M-Medical</td><td>FA30E15086</td><td></td></tr><tr><td>Dialyzed FBS</td><td>Invitrogen</td><td>26400-044</td><td></td></tr><tr><td>Lysine 0 (12C6 14N2 L-lysine)</td><td>Sigma Aldrich</td><td>L8662</td><td></td></tr><tr><td>Arginine 0 (12C6 14N4 L-arginine)</td><td>Sigma Aldrich</td><td>A6969</td><td></td></tr><tr><td>Lysine 8 (13C6 15N2 L-lysine)</td><td>Sigma Aldrich</td><td>68041</td><td></td></tr><tr><td>Arginine 10 (13C6 15N4 L-arginine)</td><td>Sigma Aldrich</td><td>608033</td><td></td></tr><tr><td>Micrococcal Nuclease</td><td>Roche</td><td>10 107 921 001</td><td></td></tr><tr><td>Complete, EDTA-free Protease Inhibitor Cocktail Tablets</td><td>Roche</td><td>04 693 132 001</td><td></td></tr><tr><td>Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length</td><td>Spectrumlabs</td><td>132720</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>Anti-Histone H3 tri-methylated K9-ChIP grade</td><td>Abcam</td><td>ab8898</td><td></td></tr><tr><td>Histone H3 peptide tri-methyl K9&amp;nbsp;</td><td>Abcam</td><td>ab1773</td><td></td></tr><tr><td>Dynabeads Protein G</td><td>Invitrogen</td><td>100.04D</td><td></td></tr><tr><td>NuPAGE Novex 4-12%&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Bis-Tris Gel&amp;nbsp;</td><td>Invitrogen</td><td>NP0335BOX</td><td></td></tr><tr><td>Colloidal Blue Staining Kit</td><td>Invitrogen</td><td>LC6025</td><td></td></tr><tr><td>LDS Sample Buffer</td><td>Invitrogen</td><td>NP0007</td><td></td></tr><tr><td>Formaldheyde</td><td>Sigma Aldrich</td><td>F8775</td><td></td></tr><tr><td>Aceti anhydride-d6</td><td>Sigma Aldrich</td><td>175641-1G</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich</td><td>94318-50ML-F</td><td></td></tr><tr><td>Iodoacetamide &amp;ge;99% (HPLC), crystalline</td><td>Sigma Aldrich</td><td>I6125</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma Aldrich</td><td>43815</td><td></td></tr><tr><td>Sequencing Grade Modified Trypsin, Frozen 100 &amp;mu;g (5 &amp;times; 20 &amp;mu;g)</td><td>Promega</td><td>V5113</td><td></td></tr><tr><td>Nanospray OD 360&amp;mu;m x ID 75&amp;mu;m, tips ID 8 &amp;mu;m uncoated Pk 5</td><td>Microcolumn Srl</td><td>FS360-75-8-N-5-C15</td><td></td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 3 &amp;micro;m&amp;nbsp;&amp;nbsp; 15% C</td><td>Dr. Maisch GmbH</td><td>r13.aq.</td><td></td></tr><tr><td>Carbon extraction disk, 47 mm</td><td>Agilent Technologies</td><td>12145040</td><td></td></tr><tr><td>Cation extraction disk</td><td>Agilent Technologies</td><td>66889-U</td><td></td></tr></tbody></table>

the chrop approach combines chip and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin

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。

染色质免疫沉淀法是用于分析沿基因组中的蛋白或组蛋白修饰的定位一个强大的技术。在蛋白质组学当量，芯片随后基于MS的蛋白质组学识别定性和定量的hPTMs，组蛋白变体与染色质结合蛋白是与感兴趣的修饰或蛋白免疫沉淀一起，作为"诱饵"。在N-ChroP方法中，在图1A中，原生芯片，其中染色质消化MNase（ 图1B）所述，作为输入到从散装染色质独特的功能域净化。消化染色?...

Watch this Scientific Journal Video about The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin at JoVE.com

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

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。

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 ...

the-chrop-approach-combines-chip-mass-spectrometry-to-dissect-locus

Research

JoVE Journal

Biology

18.1K 次观看.  European Institute of Oncology. 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。 

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

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

肿瘤组织中高通量芯片测序的染色质状态全基因组图谱集成平台

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

科研

癌症研究

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 ...

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

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

生物学

组蛋白组学分析和翻译后修饰表征

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

作者聚焦： 通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

使用液相色谱法、捕获离子淌度谱法和飞行时间质谱法进行组蛋白修饰筛选

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

化学

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. ...

神经科学

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

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

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