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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The authors have nothing to disclose.

<html> Wir präsentieren eine funktionelle Genom Protokoll die genaue Bindungsstelle für Chromatin-interagierende Proteine ​​in einer unvoreingenommenen, genomweite Weise in der Nähe von Basenpaar-Auflösung zu bestimmen. Der wichtigste Schritt in der Nähe von Basenpaar Abbildungsauflösung zu erreichen, ist die Exonuclease-Behandlung des ChIP angereicherte DNA während der Immunopräzipitat auf dem magnetischen Harz bleibt. Vordergründig könnte Proteinkomplexe möglicherweise blockieren in vivo Fußabd...

Chromatinimmunpräzipitation (Chip-) ist eine leistungsfähige Methode Mechanismen der Genregulation zu untersuchen, indem selektiv für die DNA-Fragmente zu bereichern, die in lebenden Zellen mit einem bestimmten Protein in Wechselwirkung treten. Nachweisverfahren von ChIP angereicherte DNA - Fragmente haben sich weiterentwickelt , wie die Technologie verbessert, von der Erfassung eines einzelnen Locus (Standard ChIP-qPCR) zur Hybridisierung an Oligonukleotid - Mikroarrays (ChIP-Chip) auf Hochdurchsatz - Sequenzierung (ChIP-seq) 1. Obwohl ChIP-seq weit verbreitete Anwendung, Chromatin Heterogenität und unspezifischer DNA-Wechselwirkungen Datenqualität gesehen haben, was zu falsch-positive erschwert und ungenau Mapping. Um diese Einschränkungen zu umgehen, entwickelte Dr. Frank Pugh dem Chip-exo - Methode 2. Das hervorstechendste Merkmal von ChIP-exo ist, dass es eine 5 &#39;zu 3&#39; Exonuklease enthält, effektiv Transkriptionsfaktor-Bindungsstellen Footprinting. Im Ergebnis erreicht der Chip-exo-Methodik höhere Auflösung, größeren Dynamikbereich von detection und unteren Hintergrundgeräusche. Obwohl ChIP-exo technisch schwer zu meistern ist als ChIP-seq ist es weithin angenommen wird , wie Studien einzigartige , ultrahochauflösende Einblicke mit verschiedenen biologischen Systemen 3-8 gewinnen wollen. Tatsächlich ChIP-exo wurde auf Bakterien, Hefe, Maus, Ratte und menschlichen Zellsysteme erfolgreich eingesetzt. Als Beweis für das Prinzip wurde ChIP-exo ursprünglich verwendet , um die genaue Bindungsmotiv für eine Handvoll von Hefe - Transkriptions zu identifizieren 2 Faktoren ab . Die Technik wurde auch in Hefe verwendet , um die Organisation des Transkriptions Präinitiationskomplex zu studieren, und subnucleosomal Struktur verschiedener Histone 9,10 zu dechiffrieren. In jüngerer Zeit, Leveraged wir ChIP-exo benachbarten TFIIB und Pol II an menschlichen Promotoren Bindungsereignisse zu lösen, und zeigte , dass weit verbreitete divergente Transkription entsteht aus verschiedenen Initiations 11 - Komplexe. Der Workflow hier präsentiert wird optimiert und streamlined für Säugetier ChIP-exo (Abbildung 1). Erstens leben primäre oder Gewebekulturzellen mit Formaldehyd behandelt in vivo Protein-DNA - Wechselwirkungen durch eine kovalente Vernetzungs zu erhalten. Die Zellen werden lysiert und Chromatin abgeschert ~ 100-500 Basenpaar Größe Fragmente. ChIP dann reichert selektiv für DNA an das Protein von Interesse vernetzt Fragmente. An diesem Punkt ChIP-seq-Bibliotheken werden typischerweise hergestellt, die von Natur aus begrenzt die Detektionsauflösung auf die durchschnittliche Fragmentgröße von wenigen hundert Basenpaaren. Jedoch ChIP-exo überwindet diese Einschränkung durch die linken und rechten 5&#39;-DNA Grenzen der Protein-DNA-Vernetzungsstelle mit Lambda-Exonuklease Trimmen. Sequencing Bibliotheken werden dann aus Exonuklease verdaut DNA konstruiert, wie unten beschrieben. Die resultierenden verschachtelten 5 &#39;Grenzen stellen eine in vivo Footprint des Protein-DNA - Interaktion (Abbildung 1, Schritt 14) und werden durch Hochdurchsatz - Sequenzierung nachgewiesen. Although der Chip-exo-Methodik mehr beteiligt als ChIP-seq ist, Übergänge zwischen den meisten Schritten erfordert einfache Perle Waschen, die Probenverlust und experimentelle Variabilität minimiert. Wichtig ist, dass da ChIP-exo ist eine verfeinerte Version von ChIP-seq, jede Probe, die mit ChIP-seq sollte auch erfolgreich sein, mit ChIP-exo erfolgreich ist. Die in - vivo - Fußabdrucks von Protein-DNA - Wechselwirkungen mit ChIP-exo Ergebnisse in einer grundlegend unterschiedliche Datenstruktur von ChIP-seq. Obwohl häufig vorkommend ChIP-seq Anrufern ChIP-exo-Daten angewendet werden kann, die präzisesten Spitzen Anrufe zu erhalten, empfehlen wir die Bioinformatik Tools, die speziell mit der einzigartigen Struktur von ChIP-exo-Daten im Auge behalten. Dazu gehören Genetrack, GEM, MACE, Peakzilla und ExoProfiler 12-15.

<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

Chromatinimmunpräzipitation (Chip-) ist ein unverzichtbares Werkzeug in den Bereichen Epigenetik und Genregulation, die spezifische Protein-DNA-Wechselwirkungen isoliert. ChIP gekoppelt Hochdurchsatz-Sequenzierung (ChIP-seq) wird allgemein die genomische Lage von Proteinen zu bestimmen, die mit Chromatin in Wechselwirkung treten. Allerdings ChIP-seq wird durch eine relativ geringe Abbildungsauflösung von mehreren hundert Basenpaaren und hohen Hintergrundsignal behindert. Der Chip-exo-Methode ist eine verfeinerte Version von ChIP-seq, die auf sowohl Auflösung und Rauschen wesentlich verbessert. Der Hauptunterschied der ChIP-exo-Methodik ist die Einarbeitung von lambda-Exonuclease-Verdauung in der Bibliothek Vorbereitungsarbeitsablauf, um effektiv die linke Standfläche und rechts 5 &#39;DNA-Grenzen des Protein-DNA-Vernetzungsstelle. Der Chip-exo-Bibliotheken werden dann einer Hochdurchsatz-Sequenzierung unterzogen. Die resultierenden Daten können genutzt werden, um eine einzigartige und extrem hochauflösende Einblicke in die funktionelle Organisation of das Genom. Hier beschreiben wir die ChIP-exo-Methode, die wir optimiert und rationalisiert für Säugetier-Systeme der nächsten Generation Sequenzierung-durch-Synthese-Plattform.

Die folgenden Abbildungen zeigen repräsentative Ergebnisse aus dem Chip-exo-Protokoll hier vorgestellt. Im Gegensatz zu herkömmlichen ChIP-seq Methodologien mit wenigen enzymatischen Schritten, ChIP-exo erfordert elf sequentiell abhängigen enzymatischen Reaktionen (Abbildung 1). Somit muss darauf geachtet werden bei jedem Schritt unternommen werden, um sicherzustellen, dass jede Reaktionskomponente seiner jeweiligen Reaktions Mastermix zugesetzt wird. Wir empfehlen, e...

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

Die ChIP-exo-Methode: Identifizierung von Protein-DNA-Wechselwirkungen mit nahezu Basenpaar-Präzision | Biologie | Video

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.

Der Chip-exo-Methode: Ermittlung von Protein-DNA-Wechselwirkungen mit Near-Basenpaar Precision

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

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16.5K Aufrufe.  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.

Serie: 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.

Genom-weite Abbildung von Protein-DNA-Wechselwirkungen mit ChEC-seq in<em&gt; Saccharomyces cerevisiae</em

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

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Genetik

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.

Anpassung der Hybridisierung erfassen der Chromatin-assoziierte Proteine für Proteomics für Säugerzellen

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.

Hochauflösende Kartierung von Protein-DNA-Wechselwirkungen in Mausstammzell-abgeleiteten Neuronen mit Chromatin-Immunpräzipitation-Exonuklease (ChIP-Exo)

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.

CD-Spektroskopie zur Untersuchung von DNA-Protein-Wechselwirkungen

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

Biochemie

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.

Generierung von High Quality Chromatin Immunpräzipitation DNA Vorlage für Hochdurchsatz-Sequenzierung (ChIP-seq)

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

Biologie

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.

Identifizierung von Protein-Interaktionspartner in Säugetierzellen mit SILAC-Immunpräzipitation Quantitative Proteomics

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.

DNA-Affinitäts-gereinigten Chip (DAP-Chip)-Methode, um Gene für bakterielle Ziele fest, Zwei-Komponenten-Systeme Regulatory

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).

Genomweite Kartierung der Drug-DNA-Wechselwirkungen in Zellen mit COSMIC (Vernetzung von kleinen Molekülen, um Chromatin isolieren)

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.

In Situ-Überwachung von transient gebildeten molekularen Chaperon-Baugruppen in Bakterien, Hefe und menschlichen Zellen

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.

Visualisierung und Quantifizierung endonukleasebasierter ortsspezifischer DNA-Schäden

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