Name: damid-seq: genome-wide mapping of protein-dna interactions by high throughput sequencing of adenine-methylated dna fragments
Uploaded: 2016-01-27T18:00:00.000+00:00
Duration: 9 min 14 s
Description: 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

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).

<ol>
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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 adenine methyltransferase identification (DamID) 1,2 is a method to detect protein-DNA interactions in vivo and is an alternative approach to chromatin immunoprecipitation (ChIP) 3. It uses a relatively low amount of cells and does not require chemical cross-linking of protein with DNA or a highly specific antibody. The latter is particularly helpful when the target protein is loosely or indirectly associated with DNA. DamID has been successfully used to map the binding sites of a variety of proteins including nuclear envelope proteins 4-10, chromatin associated proteins 11-13, chromatin modifying enzymes 14, transcription factors and co-factors15-18 and RNAi machineries 19. The method is applicable in multiple organisms including S. cerevisiae 13, S. pombe 7, C. elegans 9,17, D. melanogaster 5,11,18,20, A. thaliana 21,22 as well as mouse and human cell lines 6,8,10,23,24.
The development of the DamID assay was based on the specific detection of adenine-methylated DNA fragments in eukaryotic cells that lack endogenous adenine methylation 2. An expressed fusion protein, consisting of the DNA-binding protein of interest and E. coli DNA adenine methyltransferase (Dam), can methylate the adenine base in GATC sequences that are in spatial proximity (most significantly within 1 kb and up to roughly 5 kb) to the binding sites of the protein in the genome 2. The modified DNA fragments can be specifically amplified and hybridized to microarrays to detect the genomic binding sites of the protein of interest 1,25,26. This original DamID method was limited by the availability of microarrays and the density of predetermined probes. We have therefore integrated high throughput sequencing into DamID 10 and designated the method as DamID-seq. The large number of short reads generated from DamID-seq enables precise localization of protein-DNA interactions genome-wide. We found that DamID-seq provided a higher resolution and a wider dynamic range than DamID by microarray for studying genome-nuclear lamina (NL) associations 10. This improved method allows probing NL associations within gene structures 10 and facilitates comparisons with other high throughput sequencing data, such as ChIP-seq and RNA-seq.
The DamID-seq protocol described here was initially developed for mapping genome-NL associations 10. We generated a fusion protein by tethering mouse or human Lamin B1 to E. coli DNA adenine methyltransferase and tested the protocol in 3T3 mouse embryonic fibroblasts, C2C12 mouse myoblasts 10 and IMR90 human fetal lung fibroblasts (data not published). In this protocol, we start with constructing vectors and expressing Dam-tethered fusion proteins by lentiviral infection in mammalian cells 24. Next, we describe the detailed protocols of amplifying adenine-methylated DNA fragments and preparing sequencing libraries that should be applicable in other organisms.

<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.

The Dam-V5-LmnB1 fusion protein was verified to be co-localized with the endogenous Lamin B protein by immunofluorescence staining (Figure 1).
The successful PCR amplification of adenine-methylated DNA fragments is a key step for DamID-seq. The experimental samples should amplify a smear of 0.2 - 2 kb while the negative controls (without DpnI, without ligase or without PCR template) should result in no-or clearl...

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

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.3K Views.  Yale University School of Medicine. We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

Video: 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.

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

Genetics

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.

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.

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.

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

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Biochemistry

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.

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

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.

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

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.

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

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.

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

Summary

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Chapters in this video

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

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

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

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

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

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

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

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

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

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