We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22, 1813-1831 (2012).</li><li>Serandour, A. A., Brown, G. D., Cohen, J. D., Carroll, J. 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The authors have nothing to disclose.

<html>我々は、クロマチンが近い塩基対の解像度で公平な、ゲノムワイドな方法で相互作用タンパク質の正確な結合位置を決定するために、機能ゲノムプロトコルを提示します。免疫沈降物は、磁性樹脂のままに近い塩基対のマッピング分解能を達成するための最も重要なステップは、チップに富むDNAのエキソヌクレアーゼ処理です。表向きは、タンパク質複合体は、潜在的に任意のサブユニ?...

クロマチン免疫沈降（チップ）が選択的に生きている細胞内の指定されたタンパク質と相互作用するDNA断片を濃縮することにより、遺伝子調節のメカニズムを研究するための強力な方法です。技術は、ハイスループットシークエンシング（チップ配列番号1）をオリゴヌクレオチドマイクロアレイ（チップオンチップ）のハイブリダイゼーションを単一遺伝子座（標準のChIP-定量PCR）の検出から、改善、チップ富化DNAフラグメントの検出方法が発展してきました。 ChIP-seqが幅広い応用、クロマチン異質と非特異的なDNA相互作用を見たが、偽陽性と不正確なマッピングにつながるデータ品質を妨げてきました。これらの制限を回避するために、博士はフランク・ピューは、チップエキソ方法2を開発しました。 ChIPのエキソの顕著な特徴は、それが効果的に位置転写因子結合をフットプリンティング、エキソヌクレアーゼ5 &#39;から3&#39;を組み込むことです。その結果、チップエキソ方法は、より高い解像度、detectioの大きなダイナミックレンジを達成しますnおよびより低いバックグラウンドノイズ。 ChIP-エキソは、より技術的に困難なのChIP-seqのよりマスターにではあるが研究は、多様な生物学的システム3-8を使用して、ユニークな超高解像度の洞察を得ることを目指しとして、それが広く採用されています。実際に、チップエキソ正常細菌、酵母、マウス、ラット、およびヒトの細胞系に適用されています。原理の証明としてチップエキソは当初、酵母転写因子2の一握りのための正確な結合モチーフを同定しました。技術はまた、転写開始前複合体の組織化を研究するために、種々のヒストン9,10のsubnucleosomal構造を解読するために、酵母中で使用しました。最近では、我々は人間のプロモーターで隣接TFIIBとポルII結合事象を解決するためのChIP-エキソを活用し、かつ広範な発散転写は11と錯体を形成する別個の開始から生じることを示しました。 ここで紹介するワークフローを最適化およびsされます哺乳類のChIP-エキソ（ 図1）のためにtreamlined。まず、生きている一次または組織培養細胞は、共有架橋を介してインビボでタンパク質-DNA相互作用を維持するためにホルムアルデヒドで処理されます。細胞を溶解し、クロマチンは〜100に剪断された - 500塩基対のサイズの断片。チップはその後、選択的に目的のタンパク質に架橋DNA断片のために豊かにします。この時点でのChIP-seqのライブラリーは、典型的には、本質的に、数百塩基対の平均断片サイズに検出分解能を制限する、調製されます。しかし、のChIP-エキソはラムダエキソヌクレアーゼでタンパク質-DNA架橋部位の左右の5 &#39;DNA境界をトリミングすることによって、この制限を克服します。以下に詳述するようにシークエンシングライブラリーは、その後、エキソヌクレアーゼ消化DNAから構成されています。得られたネストされた5 &#39;境界は、タンパク質-DNA相互作用（ 図1、ステップ14）とのインビボでのフットプリントを表し、ハイスループットシークエンシングによって検出されます。 AltキーChIP-エキソ方法論は、チップ-seqのよりも複雑であるハフ、ほとんどの工程間の遷移はサンプルのロスや実験的なばらつきを最小限に抑えるシンプルなビーズの洗浄が必要です。重要なことに、チップエキソは、チップのseq、またのChIP-エキソと成功するはずのChIP-seqのと成功している任意のサンプルの洗練されたバージョンがあるため。 ChIP-seqのとは根本的に別個のデータ構造でのChIP-エキソ結果とタンパク質-DNA相互作用のin vivoフットプリント。共通のChIP-seqの発信者が最も正確なピークのコールを取得するために、ChIPのエキソデータに適用することができるが、我々は、具体的に念頭に置いてのChIP-エキソデータのユニークな構造で設計されたバイオインフォマティクスツールをお勧めします。これらはGenetrack、GEM、MACE、Peakzilla、およびExoProfiler 12-15が含まれます。

<table><tbody><tr><td>37% formaldehyde, methanol free, 10 x 10 ml ampules</td><td>ThermoFisher Scientific</td><td>28908</td><td>Section 3</td></tr><tr><td>Complete Protease Inhibitor cocktail (CPI)</td><td>Roche Life Science</td><td>11873580001</td><td>Sections 4, 8</td></tr><tr><td>Bioruptor sonicator</td><td>Diagenode</td><td>UCD200</td><td>Section 5</td></tr><tr><td>15 ml polystyrene tubes</td><td>BD Falcon</td><td>352095</td><td>Section 5</td></tr><tr><td>MagSepharose Protein G Xtra beads</td><td>GE Healthcare</td><td>28-9670-66</td><td>Section 6</td></tr><tr><td>DynaMag-1.5 ml Side Magnetic Rack</td><td>Invitrogen</td><td>12321D</td><td>Sections 6-17, 22</td></tr><tr><td>Mini-Tube Rotator</td><td>Fisher Scientific</td><td>05-450-127</td><td>Sections 7, 22</td></tr><tr><td>T4 DNA Polymerase</td><td>New England BioLabs</td><td>M0203</td><td>Section 9</td></tr><tr><td>DNA Polymerase I, Klenow</td><td>New England BioLabs</td><td>M0210</td><td>Section 9</td></tr><tr><td>10x NEBuffer 2 (10x Reaction Buffer 2)</td><td>New England BioLabs</td><td>B7002</td><td>Sections 9-11, 13, 15, 20</td></tr><tr><td>Thermomixer C</td><td>Eppendorf</td><td>5382</td><td>Sections 9-16</td></tr><tr><td>ATP</td><td>Roche Life Science</td><td>010419979001</td><td>Sections 9, 11, 13</td></tr><tr><td>dNTPs</td><td>New England BioLabs</td><td>N0447</td><td>Sections 9, 12, 19, 23</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England BioLabs</td><td>M0201</td><td>Sections 9, 13</td></tr><tr><td>dATP</td><td>New England BioLabs</td><td>N0440</td><td>Sections 10, 20</td></tr><tr><td>Klenow 3&#039;-5&#039; Exo Minus</td><td>New England BioLabs</td><td>M0212</td><td>Sections 10, 20</td></tr><tr><td>T4 DNA ligase&amp;nbsp;</td><td>New England BioLabs</td><td>M0202</td><td>Sections 11, 21</td></tr><tr><td>&amp;Phi;-29 DNA Polymerase</td><td>New England BioLabs</td><td>M0269</td><td>Sections 12, 19</td></tr><tr><td>10x &amp;Phi;-29 Buffer</td><td>New England BioLabs</td><td>B0269</td><td>Sections 12, 19</td></tr><tr><td>Lambda Exonuclease</td><td>New England BioLabs</td><td>M0262</td><td>Section 14</td></tr><tr><td>10x Lambda Buffer</td><td>New England BioLabs</td><td>B0262</td><td>Section 14</td></tr><tr><td>RecJ&lt;sub&gt;f&lt;/sub&gt; Exonuclease</td><td>New England BioLabs</td><td>M0264</td><td>Section 15</td></tr><tr><td>Proteinase K</td><td>Roche Life Science</td><td>03115828001</td><td>Section 16</td></tr><tr><td>Glycogen</td><td>Roche Life Science</td><td>010901393001</td><td>Section 18</td></tr><tr><td>10x T4 Ligase Buffer</td><td>New England BioLabs</td><td>B0202</td><td>Section 21</td></tr><tr><td>AMPure XP (clean up) beads&amp;nbsp;</td><td>Beckman Coulter</td><td>A63881</td><td>Section 22</td></tr><tr><td>Q5 Hot Start DNA Polymerase&amp;nbsp;</td><td>New England BioLabs</td><td>M0493</td><td>Section 23</td></tr><tr><td>5x Q5 Buffer (5x PCR Buffer)</td><td>New England BioLabs</td><td>B9027</td><td>Section 23</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Section 25</td></tr><tr><td>Qubit Fluorometer</td><td>Invitrogen</td><td>Q33216</td><td>Section 26</td></tr><tr><td>Optical Clear Qubit tubes&amp;nbsp;</td><td>Invitrogen</td><td>Q32856</td><td>Section 26</td></tr><tr><td>Qubit dsDNA High Sensitivity Assay kit</td><td>Invitrogen</td><td>Q32851</td><td>Section 26</td></tr></tbody></table>

the chip-exo method: identifying protein-dna interactions with near base pair precision

クロマチン免疫沈降（チップ）エピジェネティクスと特定のタンパク質-DNA相互作用を分離する遺伝子調節の分野で不可欠なツールです。ハイスループットシークエンシング（チップ配列）に結合されたチップは、一般に、クロマチンと相互作用するタンパク質のゲノム位置を決定するために使用されます。しかしチップ-配列は数百塩基対の比較的低いマッピング分解能および高いバックグラウンドシグナルによって妨げられます。 ChIP-エキソ方法は、実質的に解像度とノイズの両方を改善するのChIP-seqのの洗練されたバージョンです。 ChIP-エキソ方法論の重要な違いは、効果的にタンパク質-DNA架橋部位の左右の5 &#39;DNAボーダーフットプリントするためのライブラリ作成ワークフローのラムダエキソヌクレアーゼ消化の組み込みです。 ChIPのエキソライブラリーは、高スループット配列決定に供されます。得られたデータは、機能的な組織O中にユニークな超高解像度の洞察を提供するために活用することができゲノムF。ここで、我々は、哺乳動物系と次世代シーケンシング・バイ・合成プラットフォーム向けに最適化し、合理化しているのChIP-エキソ方法について説明します。

次の図は、ここで紹介するのChIP-エキソプロトコルから代表的な結果を示しています。いくつかの酵素工程と、伝統的なのChIP-seqの方法論とは対照的に、ChIPのエキソは11順次依存酵素反応（ 図1）が必要です。このように、注意が各反応成分が各反応マスターミックスに添加されることを確実にするために各ステップで注意しなければなりません。私たち?...

Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

ChIP-exo メソッド: ほぼ塩基対の精度でタンパク質と DNA の相互作用を特定 |生物学 |ビデオ

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

ChIP-エキソ方法：近く塩基対の精度でタンパク質 -  DNA相互作用を同定します

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

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16.6K ビュー.  Vanderbilt University. Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

ビデオ: The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

ChEC-seqとのタンパク質 - DNA相互作用のゲノムワイドマッピング<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

遺伝学

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

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

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

交配の適応は哺乳類細胞プロテオミクスのクロマチン関連タンパク質の捕捉します。

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

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

クロマチン免疫沈降-エキソヌクレアーゼ(ChIP-Exo)を用いたマウス幹細胞由来ニューロンにおけるタンパク質-DNA相互作用の高分解能マッピング

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

DNA-タンパク質相互作用を研究するためのCD分光法

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

生化学

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.

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

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

生物学

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC免疫沈降定量的プロテオミクスを用いて哺乳動物細胞におけるタンパク質相互作用パートナーの同定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

細菌のための2つの成分​​規制システムを遺伝子標的を決定するために、DNA親和性精製チップ（DAP-チップ）方法

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

COSMICと細胞の薬剤-DNA相互作用のゲノムワイドなマッピング（小分子の架橋は、クロマチンを分離するために）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

細菌、酵母、ヒト細胞における一過性形成分子シャペロンアセンブリのモニタリング

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

エンドヌクレアーゼベースの部位特異的DNA損傷の可視化と定量化

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

ChIP-エキソ方法：近く塩基対の精度でタンパク質 - DNA相互作用を同定します

要約

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この動画の章

ChEC-seqとのタンパク質 - DNA相互作用のゲノムワイドマッピング

交配の適応は哺乳類細胞プロテオミクスのクロマチン関連タンパク質の捕捉します。

クロマチン免疫沈降-エキソヌクレアーゼ(ChIP-Exo)を用いたマウス幹細胞由来ニューロンにおけるタンパク質-DNA相互作用の高分解能マッピング

DNA-タンパク質相互作用を研究するためのCD分光法

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

SILAC免疫沈降定量的プロテオミクスを用いて哺乳動物細胞におけるタンパク質相互作用パートナーの同定

細菌のための2つの成分規制システムを遺伝子標的を決定するために、DNA親和性精製チップ（DAP-チップ）方法

COSMICと細胞の薬剤-DNA相互作用のゲノムワイドなマッピング（小分子の架橋は、クロマチンを分離するために）

細菌、酵母、ヒト細胞における一過性形成分子シャペロンアセンブリのモニタリング

エンドヌクレアーゼベースの部位特異的DNA損傷の可視化と定量化

ChIP-エキソ方法：近く塩基対の精度でタンパク質 - DNA相互作用を同定します

要約

さらに動画を探す

この動画の章

ChEC-seqとのタンパク質 - DNA相互作用のゲノムワイドマッピング

交配の適応は哺乳類細胞プロテオミクスのクロマチン関連タンパク質の捕捉します。

クロマチン免疫沈降-エキソヌクレアーゼ(ChIP-Exo)を用いたマウス幹細胞由来ニューロンにおけるタンパク質-DNA相互作用の高分解能マッピング

DNA-タンパク質相互作用を研究するためのCD分光法

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

SILAC免疫沈降定量的プロテオミクスを用いて哺乳動物細胞におけるタンパク質相互作用パートナーの同定

細菌のための2つの成分​​規制システムを遺伝子標的を決定するために、DNA親和性精製チップ（DAP-チップ）方法

COSMICと細胞の薬剤-DNA相互作用のゲノムワイドなマッピング（小分子の架橋は、クロマチンを分離するために）

細菌、酵母、ヒト細胞における一過性形成分子シャペロンアセンブリのモニタリング

エンドヌクレアーゼベースの部位特異的DNA損傷の可視化と定量化

細菌のための2つの成分規制システムを遺伝子標的を決定するために、DNA親和性精製チップ（DAP-チップ）方法