We thank Dr. Bas van Steensel for providing the DamID mammalian expression vectors. We thank Yale Center for Genome Analysis and the Genomics Core in Yale Stem Cell Center for advice on preparing NGS libraries and implementing high throughput DNA sequencing. This work was supported by the startup funding from Yale School of Medicine, a Scientist Development Grant from American Heart Association (12SDG11630031) and a Seed Grant from Connecticut Innovations, Inc. (13-SCA-YALE-15).

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The authors have nothing to disclose.

Whether Dam-tagged proteins retain the functions of endogenous proteins should be examined before a DamID-seq experiment. The subcellular localization of Dam-tagged nuclear envelope proteins should always be determined and compared with that of the endogenous proteins. For studying transcription factors, it is suggested to examine whether the Dam-fusion protein can rescue the functions of the endogenous protein in regulating gene expression. This functional test can be performed in organisms in which knockout mutants of ...

DNA-Methyltransferase-Adenin-Identifikation (DAMiD) 1,2 ist eine Methode, um Protein-DNA-Wechselwirkungen in vivo zu detektieren und ist ein alternativer Ansatz zur Chromatin-Immunopräzipitation (ChIP) 3. Es verwendet eine relativ geringe Menge an Zellen und keine chemische Vernetzung des Proteins mit der DNA oder einem hochspezifischen Antikörpers erforderlich. Letzteres ist insbesondere dann hilfreich, wenn das Zielprotein ist lose oder indirekt mit DNA verbunden. DAMiD wurde erfolgreich verwendet, um die Bindungsstellen aus einer Vielzahl von Proteinen, einschließlich Kernhüllenproteine ​​4-10, Chromatin-assoziierte Proteine ​​11-13, Chromatin modifizierender Enzyme 14, Transkriptionsfaktoren und Co-Faktoren und 15-18 RNAi Maschinen 19 abzubilden. Das Verfahren ist in mehreren Organismen, einschließlich S. erhoben cerevisiae 13, S. pombe 7, C. elegans 9,17, D. melanogaster 5,11,18,20, A. thaliana 21,22 sowie Maus und humanen Zelllinien 6,8,10,23,24. Die Entwicklung des DAMiD Assay wurde auf den spezifischen Nachweis von Adenin-methylierten DNA-Fragmente in eukaryontischen Zellen, die endogene Adenin-Methylierung 2 fehlt basiert. Ein exprimiertes Fusionsprotein, bestehend aus der DNA-bindenden Proteins von Interesse und E. coli DNA-Adenin-methyltransferase (Dam), können die Adeninbase in GATC-Sequenzen, die in räumlicher Nähe befinden (am deutlichsten in 1 kb und bis zu etwa 5 kb) an die Bindungsstellen des Proteins in das Genom 2 methylieren. Die modifizierten DNA-Fragmenten, spezifisch amplifiziert und hybridisiert werden, um Mikroarrays, um die genomische Bindungsstellen des Proteins von Interesse 1,25,26 zu erfassen. Diese ursprüngliche DAMiD Verfahren wurde durch die Verfügbarkeit von Mikroarrays und der Dichte des vorbestimmten Sonden beschränkt. Daher haben wir in integrierten Hochdurchsatz-Sequenzierungum DAMiD 10 und bezeichnet das Verfahren, wie DAMiD-seq. Die große Anzahl von kurzen liest aus DAMiD-seq generiert ermöglicht eine präzise Lokalisierung von Protein-DNA-Interaktionen Genom-weiten. Wir fanden, dass DAMiD-seq vorgesehen eine höhere Auflösung und einen breiteren Dynamikbereich als DAMiD von Mikroarray für die Untersuchung genom Kernlamina (NL) 10 Verbände. Dieses verbesserte Verfahren ermöglicht Sondieren NL Assoziationen innerhalb Genstrukturen 10 und erleichtert den Vergleich mit anderen Hochdurchsatz-Daten, wie Chip-seq und RNA-seq. Die hier beschriebene DAMiD-seq-Protokoll wurde ursprünglich für Mapping-Genom-NL Verbände 10 entwickelt. Wir erzielten ein Fusionsprotein angebunden Maus oder Mensch Lamin B1 bis E. coli DNA-Adenin-Methyltransferase und getestet, das Protokoll in 3T3 embryonalen Maus-Fibroblasten, Myoblasten C2C12 Maus 10 und IMR90 menschlichen fötalen Lungenfibroblasten (Daten nicht veröffentlicht). In diesem Protokoll, beginnen wir mit constructing Vektoren und ausdrücken Dam-gebundenen Fusionsproteine ​​durch lentivirale Infektion in Säugetierzellen 24. Als nächstes beschreiben wir die ausführliche Protokolle zum Verstärken Adenin-methylierten DNA-Fragmente und Sequenzierung vorbereitet Bibliotheken, die in anderen Organismen anwendbar sein sollte.

<table><tbody><tr><td>ViraPower Lentiviral Expression Systems</td><td>Life Technologies</td><td>K4950-00, K4960-00, K4970-00, K4975-00, K4980-00, K4985-00, K4990-00, K367-20, K370-20, and K371-20</td><td></td></tr><tr><td>Gateway BP Clonase II Enzyme Mix</td><td>Life Technologies</td><td>11789-020</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme Mix</td><td>Life Technologies</td><td>11791-020</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit (250)</td><td>QIAGEN</td><td>69506 or 69504&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Gateway pDONR 201</td><td>Life Technologies</td><td>11798-014</td><td></td></tr><tr><td>293T cells</td><td>American Type Culture Collection</td><td>CRL-11268</td><td></td></tr><tr><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/25300054&quot;&gt;Trypsin-EDTA (0.05%), phenol red&lt;/a&gt;</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Tris</td><td></td><td></td><td>brand not critical</td></tr><tr><td>EDTA</td><td></td><td></td><td>brand not critical</td></tr><tr><td>200 Proof EtOH</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Isopropanol</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Sodium Acetate</td><td></td><td></td><td>brand not critical</td></tr><tr><td>DpnI</td><td>New England Biolabs</td><td>R0176</td><td>supplied with buffer</td></tr><tr><td>DamID adaptors &quot;AdRt&quot; and &quot;AdRb&quot;</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 24; no phosphorylation of the 5&#039; or 3&#039; end to prevent self-ligation.</td></tr><tr><td>T4 DNA Ligase</td><td>Roche Life Science</td><td>10481220001</td><td>supplied with buffer</td></tr><tr><td>DpnII</td><td>New England Biolabs</td><td>R0543</td><td>supplied with buffer</td></tr><tr><td>DamID PCR primer &quot;AdR_PCR&quot;</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 24</td></tr><tr><td>Deoxynucleotide (dNTP) Solution Set</td><td>New England Biolabs</td><td>N0446</td><td>100 mM each of dATP, dCTP, dGTP and dTTP</td></tr><tr><td>Advantage 2&amp;nbsp; Polymerase Mix</td><td>Clontech</td><td>639201</td><td>supplied with buffer</td></tr><tr><td>1Kb Plus DNA Ladder</td><td>Life Technologies</td><td>10787018</td><td>1.0 &amp;micro;g/&amp;micro;l</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28104 or 28106</td><td></td></tr><tr><td>MinElute PCR Purification Kit</td><td>QIAGEN</td><td>28004 or 28006</td><td>for an elution volume of less than 30 &amp;micro;l</td></tr><tr><td>SPRI beads / Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63880</td><td>apply extra mixing and more elution time if less than 40 &amp;micro;l elution buffer is used</td></tr><tr><td>Buffer EB</td><td>QIAGEN</td><td>19086</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England Biolabs</td><td>M0348</td><td>supplied with buffer</td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>New England Biolabs</td><td>B0202</td><td></td></tr><tr><td>T4 DNA Polymerase</td><td>New England Biolabs</td><td>M0203</td><td></td></tr><tr><td>DNA Polymerase I, Large (Klenow) Fragment</td><td>New England Biolabs</td><td>M0210</td><td></td></tr><tr><td>T4 Polynuleotide Kinase</td><td>New England Biolabs</td><td>M0201</td><td></td></tr><tr><td>Klenow Fragment (3&amp;rsquo; &amp;#8594; 5&amp;rsquo; exo-)</td><td>New England Biolabs</td><td>M0212</td><td>supplied with buffer</td></tr><tr><td>sequencing adaptors</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 28</td></tr><tr><td>Quick Ligation Kit</td><td>New England Biolabs</td><td>M2200</td><td>used in 3.4.3; supplied with Quick Ligation Reaction Buffer and Quick T4 DNA Ligase</td></tr><tr><td>sequencing primer 1 and 2</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 28</td></tr><tr><td>KAPA HiFi PCR Kit</td><td>Kapa Biosystems</td><td>KK2101 or KK2102</td><td>supplied with KAPA HiFi DNA Polymerase, 5x KAPA HiFi Fidelity Buffer and 10 mM dNTP mix</td></tr><tr><td>agarose</td><td>Sigma Aldrich</td><td>A4679</td><td></td></tr><tr><td>ethidium bromide</td><td>Sigma Aldrich</td><td>E1510-10ML</td><td>10 mg/ml</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704 or 28706</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-Rad Laboratories</td><td>1725121 or 1725122</td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td>brand not critical</td></tr><tr><td>0.45 &amp;#956;m PVDF Filter</td><td></td><td></td><td>brand not critical</td></tr><tr><td>25 ml Seringe</td><td></td><td></td><td>brand not critical</td></tr><tr><td>10 cm Tissue Culture Plates</td><td></td><td></td><td>brand not critical</td></tr><tr><td>6-well Tissue Culture Plates</td><td></td><td></td><td>brand not critical</td></tr><tr><td>S1000 Thermal Cycler</td><td>Bio-Rad Laboratories</td><td></td><td></td></tr><tr><td>C1000 Touch Thermal Cycler</td><td>Bio-Rad Laboratories</td><td></td><td>for qPCR</td></tr><tr><td>Vortex Mixer</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Dry Block Heater or Thermomixer</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Gel electrophoresis system with power supply</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Magnet stand</td><td></td><td></td><td>for purification of DNA with SPRI beads; should hold 1.5-2 ml tubes; brand not critical</td></tr><tr><td>UV transilluminator</td><td></td><td></td><td>brand not critical</td></tr><tr><td>E-gel electrophoresis system</td><td>Life Technologies</td><td>G6400, G6500, G6512ST</td><td></td></tr></tbody></table>

damid-seq: genome-wide mapping of protein-dna interactions by high throughput sequencing of adenine-methylated dna fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

Dam-V5-LmnB1 Fusionsprotein wurde überprüft, um mit dem endogenen Lamin B-Protein durch Immunfluoreszenzfärbung (Abbildung 1) co-lokalisiert werden. Die erfolgreiche PCR-Amplifikation von Adenin-methylierten DNA-Fragmente ist ein wichtiger Schritt für DAMiD-seq. Die Versuchsproben sollten einen Abstrich von 0,2 zu verstärken - 2 kb, während der Negativkontrollen (ohne DpnI ohne Ligase oder ohne PCR-Vorla...

Watch this Scientific Journal Video about DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments at JoVE.com

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

Wir beschreiben hier einen Test durch Kopplung DNA-Adenin-Methyltransferase-Identifikation (DAMiD), um Hochdurchsatz-Sequenzierung (DAMiD-seq). Dieses verbesserte Verfahren bietet eine höhere Auflösung und einen größeren Dynamikbereich und erlaubt die Analyse DAMiD-seq Daten in Verbindung mit anderen Hochdurchsatz-Sequenzierung Daten wie ChIP-seq, RNA-seq usw.

DAMiD-seq: Genomweite Mapping von Protein-DNA-Wechselwirkungen von Hochdurchsatz-Sequenzierung von Adenin-methylierten DNA-Fragmenten

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

damid-seq-genome-wide-mapping-protein-dna-interactions-high

Research

JoVE Journal

Biology

19.4K Aufrufe.  Yale University School of Medicine.  Wir beschreiben hier einen Test durch Kopplung DNA-Adenin-Methyltransferase-Identifikation (DAMiD), um Hochdurchsatz-Sequenzierung (DAMiD-seq). Dieses verbesserte Verfahren bietet eine höhere Auflösung und einen größeren Dynamikbereich und erlaubt die Analyse DAMiD-seq Daten in Verbindung mit anderen Hochdurchsatz-Sequenzierung Daten wie ChIP-seq, RNA-seq usw. 

Serie: DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

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 ...

Forschung

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 ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

Informatischen Analyse von Sequenzdaten von Batch-Hefe-2-Hybrid-Bildschirme

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

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 ...

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here.

Kartierung von 3D-Genominteraktionen von Säugetieren mit Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

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

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iClip - Transkriptom-weiten Mapping von Protein-RNA Wechselwirkungen mit Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Biologie

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 ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Probing hoher Dichte Funktionale Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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).

DAMiD-seq: Genomweite Mapping von Protein-DNA-Wechselwirkungen von Hochdurchsatz-Sequenzierung von Adenin-methylierten DNA-Fragmenten

Zusammenfassung

Weitere Videos entdecken

Genom-weite Abbildung von Protein-DNA-Wechselwirkungen mit ChEC-seq in