We thank members of the Ansari lab and Prof. Parameswaran Ramanathan for helpful discussions. This work was supported by NIH grants CA133508 and HL099773, the H.&#8197;I. Romnes faculty fellowship, and the W.&#8197;M. Keck Medical Research Award to A.Z.A. G.S.E. was supported by a Peterson Fellowship from the Department of Biochemistry and Molecular Biosciences Training Grant NIH T32 GM07215. A.E. was supported by the Morgridge Graduate Fellowship and the Stem Cell and Regenerative Medicine Center Fellowship, and D.B. was supported by the NSEC grant from NSF.

1. Crosslinking in Live Cells
<ol>
	<li>Begin with ~2.5x107 H1 cells, or other cultured cells. 
	NOTE: This number of H1 cells corresponds to five 10-cm dishes (approximately 40% confluency).</li>
	<li>Grow cells in E8 media on dishes coated on a surface that supports pluripotent stem cells (see Materials List), and incubate them at 37 &#176;C in humidified atmosphere of 5% CO2. Harvest cells enzymatically (see Materials List). Note: Do not allow H1 cells to exceed 90% confluency; confluency induces spontaneous differentiation of H1 cells. To count cells, grow an extra 10-cm dish of cells, lift the cells enzymatically as described in sections 1.8-1.10, and count them with a hemocytometer.
	<ol>
		<li>Prepare E8 culture media as described in Chen et al.54&#160;</li>
	</ol>
	</li>
	<li>Prior to adding polyamide, remove the spent culture media with a Pasteur pipette attached to a vacuum trap. Add fresh media with a serological pipette and pipette dispenser (8 ml per 10-cm dish). 
	NOTE: Add the media to the side of the dish in order to avoid disrupting the cells. From this point onward protect the cells from light to avoid premature photo-crosslinking.</li>
	<li>Add polyamide with a pipette (8 &#181;l of 400 &#181;M polyamide in DMSO, 400 nM final concentration) directly to the culture media of each dish. Swirl the dish to disperse the polyamide evenly into the media. 
	NOTE: Concentration can be varied, but ensure that no cellular toxicity is observed for the treatment selected.</li>
	<li>Incubate the cells 24 hr 37 &#176;C in a humidified atmosphere of 5% CO2, and ensure they are protected from light. 
	NOTE: The incubation time can be varied to measure polyamide binding across time.</li>
	<li>Wash each 10-cm dish with 4 ml PBS (1.05 mM potassium phosphate monobasic, 155.17 mM sodium chloride, 2.97 mM sodium phosphate dibasic) using a serological pipette and pipette dispenser. Aspirate PBS and add 3 ml E8 culture media.</li>
	<li>With the lights dimmed, remove the lid of 5 culture dishes and place the cells on a flat surface outside of the hood. Place a glass filter over the 5 culture dishes to filter out light with &#955; &#60; 300 nm. Place the UV source on top of filter. Crosslink samples 30 min with 365 nm UV irradiation (2.4 mW/cm2). 
	NOTE: Crosslinking time should be determined empirically.</li>
	<li>Transfer culture dishes back to the hood. Aspirate the media with a Pasteur pipette attached to a vacuum trap. Wash each 10-cm dish with 4 ml PBS. Aspirate the PBS. Add 3 ml enzyme for cell dissociation (see Materials List) per 10-cm dish to dissociate the cells. Incubate 5 min at 37 &#176;C. 
	NOTE: Do not pre-warm the enzyme.</li>
	<li>Quench the enzyme with 3 ml E8 media per dish. Transfer dissociated cells into one 15-ml conical tube. 
	NOTE: Place cells on ice from this point onward.</li>
	<li>Centrifuge dissociated cells 5 min, 500 x g at 4 &#176;C. Aspirate supernatant to remove enzyme and media. 
	NOTE: Pause point. Cells can be flash-frozen in liquid nitrogen, stored at -80 &#176;C for subsequent processing at a later time.</li>
</ol>
2. Isolation of Chromatin
<ol>
	<li>Add 1.2 ml COSMIC buffer (20 mM Tris-HCl [pH 8.1], 2 mM EDTA, 150 mM NaCl, 1% Triton-X100, 0.1% SDS) and pipette up and down several times to resuspend the cell pellet for each sample in 1.2 ml COSMIC buffer.
	<ol>
		<li>Add 133 &#181;l each of 100 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM benzamidine, and 150 &#181;M pepstatin protease inhibitors fresh to a final concentration of 1 mM for PMSF and benzamidine and 1.5 &#181;M for pepstatin. Split solution of cell lysate into two amber 1.7 ml microcentrifuge tubes.</li>
	</ol>
	</li>
	<li>Sonicate with sonicator 35 min (10 sec on, 10 sec off, 60% power) to fragment the genome to between 100 and 500 bp.
	<ol>
		<li>Keep the level of chromatin solution in the microcentrifuge tube parallel to the level of water in the reservoir. Confirm this level by visual inspection.&#160; 
		NOTE: Confirm that DNA was sheared with a 1.5% agarose gel.55</li>
		<li>Optimize the sonication time empirically. Use a minimal amount of ice in the reservoir to chill the samples, and ensure the ice is not interfering between the samples and the cup horn.</li>
	</ol>
	</li>
	<li>Centrifuge the sample 12,000 x g 10 min. Save the aqueous solution which contains soluble chromatin by transferring it to a new amber microcentrifuge tube with a pipette. Discard the pellet.</li>
	<li>Transfer 110 &#181;l (10%) of sample to a new microcentrifuge tube and label it Input DNA. Store at -80 &#176;C. Save the rest of the chromatin sample on ice for use in Step 3.3.</li>
</ol>
3. Capture of Ligand&#8211;DNA Crosslinks
<ol>
	<li>Use a pipette to dispense 60 &#181;l streptavidin-coated magnetic beads in a microcentrifuge tube. Add 1 ml COSMIC buffer and mix on a nutator 5 min at RT. Place magnetic beads on magnetic separation rack 2 min to capture beads. Remove COSMIC buffer with a pipette. 
	NOTE: Do not allow the beads to dry out. Proceed immediately to the next step.</li>
	<li>Add chromatin sample (~1 ml) to beads (60 &#181;l) and resuspend the beads. Incubate chromatin with streptavidin-coated magnetic beads at least 4 hr on a rotating, rocking mixer at 4 &#176;C. Incubate the samples O/N if desired.</li>
</ol>
4. Isolation of Affinity-purified DNA
<ol>
	<li>Wash beads with 7 min interval at RT with the following wash buffers to remove non-specific interactions. Use a magnetic separation rack to capture beads after each wash. Resuspend the beads after each change in washing buffer.</li>
	<li>Prepare wash buffers in distilled deionized water and filter (0.2 &#181;m) before use. Store wash Buffers 1 and 2 at 4 &#176;C for several months. Prepare Wash Buffer 3 fresh each day. Add and remove wash buffers from the sample with a pipette. For 2 and 4, add 1 and 3 (5 mM), respectively, in the washes. Samples can additionally be washed twice with COSMIC buffer (once 12 hr, once 4 hr) prior to the washes listed below.
	<ol>
		<li>Wash once with Wash Buffer 1 (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 3% SDS). Wash once with Wash Buffer 2 (10 mM Tris-Cl [pH 8.0], 250 mM LiCl, 1 mM EDTA, 0.5% NP40, 1% sodium deoxycholate). Wash twice with Wash Buffer 3 (4 M urea, 10 mM Tris-Cl [pH 7.5], 1 mM EDTA, 0.1% NP-40). Wash twice with Tris EDTA (TE) buffer (10 mM Tris-Cl [pH 8.0], 1 mM EDTA).</li>
	</ol>
	</li>
	<li>Resuspend the beads in 200 &#956;l TE buffer with a pipette. This sample of captured DNA is referred to as affinity-purified (AP) DNA.</li>
	<li>Supplement the Input and AP DNA with 10x Crosslink Reversal Buffer (100 mM Tris-Cl [pH 8.0], 1 M KOH, 4 mM EDTA) to 1x final concentration.56,57 Incubate 30 min at 90 &#176;C. 
	NOTE: 254 nm UV irradiation can also be used to reverse the psoralen crosslink,58 but cyclobutyl pyrimidine dimers can be formed from irradiation at this wavelength and interfere with downstream processing of DNA.</li>
	<li>For AP DNA, place the microcentrifuge tube containing the sample on magnetic separation rack 2 min at RT and isolating liquid (DNA) with a pipette. Transfer the AP DNA (which has been released from the beads) to a new amber microcentrifuge tube. 
	NOTE: The desired AP DNA is in the liquid, no longer attached to the beads.</li>
	<li>Neutralize Input and AP DNA with concentrated HCl to pH 7 by adding approximately 1 &#181;l 6 N HCl per 100 &#956;l sample with a pipette. Confirm the samples are neutralized by adding ~0.5 &#181;l onto pH paper with a pipette. WARNING: Concentrated HCl is a strong corrosive acid. Handle according to your institution&#8217;s guidelines for the appropriate personal protective equipment.</li>
	<li>Add RNase A (100 mg/ml) to 0.2 &#181;g/&#181;l final concentration to both Input and AP DNA. Incubate 1 hr at 37 &#176;C. Add Proteinase K (20 mg/ml) to 0.2 &#181;g/&#181;l final concentration to both Input and AP DNA, add. Incubate 1 hr at 55 &#176;C.</li>
	<li>Purify the DNA with a DNA column cleanup kit (see Materials List).55 Elute DNA in 58 &#956;l DNA-grade H2O. Store Input and AP samples at -20 or -80 &#176;C</li>
	<li>Analyze AP DNA by quantitative polymerase chain reaction (qPCR) with locus-specific primers, and/or by next-generation sequencing. For qPCR, use 2 &#181;l AP DNA per locus with the following parameters: 1 cycle of 95 &#176;C 10 min and 40 cycles of 95 &#176;C 20 sec, 54 &#176;C or 56 &#176;C 20 sec, 72 &#176;C 40 sec. 
	NOTE: The annealing temperature should be modified according to the melting temperature of the primer pairs used. Select an annealing temperature that minimizes the quantitation cycle with no nonspecific amplification. 
	NOTE: For analysis by next-generation sequencing, aim for at least 10 million mapped reads. The number of mapped reads can be increased by increasing the amount of AP DNA and by combining fewer samples into a run for sequencing. More reads improve the sensitivity. Standard packages for ChIP-seq analysis (e.g., HOMER,59 MACS,60 and SPP61) work with COSMIC-seq data. As with other methods that rely upon next-generation sequencing, including genome sequencing and ChIP-seq, repetitive regions create ambiguities in alignment and assembly and thus remain a technical challenge.</li>
</ol>

A.Z.A. is the sole proprietor of Vista Motif, LLC and WINStep Forward.

One of the primary challenges with conventional ChIP is the identification of suitable antibodies. ChIP depends heavily upon the quality of the antibody, and most commercial antibodies are unacceptable for ChIP. In fact, the Encyclopedia of DNA Elements (ENCODE) consortium found only 20% of commercial antibodies to be suitable for ChIP assays.50 With COSMIC, antibodies are replaced by streptavidin. Because polyamides are functionalized with biotin, streptavidin is used in place of an antibody to capture polyam...

The information to make each cell in the human body is encoded in DNA. The selective use of that information governs the fate of a cell. Transcription factors (TFs) are proteins that bind to specific DNA sequences to express a particular subset of the genes in the genome, and the malfunction of TFs is linked to the onset of a wide array of diseases, including developmental defects, cancer, and diabetes.1,2 We have been interested in developing molecules that can selectively bind to the genome and modulate gene regulatory networks.
Polyamides composed of N-methylpyrrole and N-methylimidazole are rationally-designed molecules that can target DNA with specificities and affinities that rival natural transcription factors.3-6 These molecules bind to specific sequences in the minor groove of DNA.4,5,7-11 Polyamides have been employed to both repress and activate the expression of specific genes.4,12-19 They also have interesting antiviral20-24 and anticancer12,13,25-30 properties. One attractive feature of polyamides is their ability to access DNA sequences that are methylated31,32 and wrapped around histone proteins9,10,33.
To measure the comprehensive binding specificities of DNA-binding molecules, our lab created the cognate site identifier (CSI) method.34-39 The predicted occurrence of binding sites based on in vitro specificities (genomescapes) can be displayed on the genome, because the in vitro binding intensities are directly proportional to association constants (Ka).34,35,37 These genomescapes provide insight into polyamide occupancy across the genome, but measuring polyamide binding in live cells has been a challenge. DNA is tightly packaged in the nucleus, which could influence the accessibility of binding sites. The accessibility of these chromatinized DNA sequences to polyamides remains a mystery.
Recently, many methods to study interactions between small molecules and nucleic acids have emerged.40-48 The chemical affinity capture and massively-parallel DNA sequencing (chem-seq) is one such technique. Chem-seq uses formaldehyde to crosslink small molecules to a genomic target of interest and a biotinylated derivative of a small molecule of interest to capture the ligand&#8211;target interaction.48,49
Formaldehyde crosslinking leads to indirect interactions that can produce false positives.50 We developed a new method, the crosslinking of small molecules to isolate chromatin (COSMIC),51 with a photocrosslinker to eliminate these so-called &#8220;phantom&#8221; peaks.50 To begin, we designed and synthesized trifunctional derivatives of polyamides. These molecules contained a DNA-binding polyamide, a photocrosslinker (psoralen), and an affinity handle (biotin, Figure 1). With trifunctional polyamides, we can covalently capture polyamide&#8211;DNA interactions with 365 nm UV irradiation, a wavelength that does not damage DNA or induce non-psoralen-based crosslinking.51 Next, we fragment the genome and purify the captured DNA under stringent, semi-denaturing conditions to decrease DNA that is non-covalently bound. Thus, we view COSMIC as a method related to chem-seq, but with a more direct readout of DNA targeting. Importantly, the weak (Ka 103-104 M-1) affinity of psoralen for DNA does not detectably impact polyamide specificity.51,52 The enriched DNA fragments can be analyzed by either quantitative polymerase chain reaction51 (COSMIC-qPCR) or by next-generation sequencing53 (COSMIC-seq). These data enable an unbiased, genome-guided design of ligands that interact with their desired genomic loci and minimize off-target effects.
<img alt="Figure 1" src="/files/ftp_upload/53510/53510fig1.jpg"/> 
Figure 1. Bioactive polyamides and COSMIC scheme. (A)&#8197;Hairpin polyamides 1&#8211;2 target the DNA sequence 5&#8242;-WACGTW-3&#8242;. Linear polyamides 3&#8211;4 target 5&#8242;-AAGAAGAAG-3&#8242;. Rings of N-methylimidazole are bolded for clarity. Open and filled circles represent N-methylpyrrole and N-methylimidazole, respectively. Square represents 3-chlorothiophene, and diamonds represent &#946;-alanine. Psoralen and biotin are denoted by P and B, respectively. (B)&#8197;COSMIC scheme. Cells are treated with trifunctional derivatives of polyamides. After crosslinking with 365 nm UV irradiation, cells are lysed and genomic DNA is sheared. Streptavidin-coated magnetic beads are added to capture polyamide&#8211;DNA adducts. The DNA is released and can be analyzed by quantitative PCR (qPCR) or by next-generation sequencing (NGS). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53510/53510fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>any source</td><td></td><td></td></tr><tr><td>Benzamidine</td><td>any source</td><td></td><td></td></tr><tr><td>Pepstatin</td><td>any source</td><td></td><td></td></tr><tr><td>Proteinase K</td><td>any source</td><td></td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin C1</td><td>Life Technologies</td><td>65001</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td>Other sources can be used</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>Life Technologies</td><td>A1110501</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>We have tried other manufacturers of DNA columns with success.</td></tr><tr><td>TruSeq ChIP Sample Prep Kit</td><td>Illumina</td><td>IP-202-1012</td><td>This Kit can be used to prepare COSMIC DNA for next-generation sequencing</td></tr><tr><td>Matrigel Basement Membrane Matrix</td><td>BD Biosciences</td><td>356231</td><td>Used to coat plates in order to grow H1 ESCs</td></tr><tr><td>pH paper</td><td>any source</td><td></td><td></td></tr><tr><td>amber microcentrifuge tubes</td><td>any source</td><td></td><td></td></tr><tr><td>microcentrifuge tubes</td><td>any source</td><td></td><td></td></tr><tr><td>pyrex filter</td><td>any source</td><td></td><td>Pyrex baking dishes are suitable</td></tr><tr><td>qPCR master mix</td><td>any source</td><td></td><td></td></tr><tr><td>RNase</td><td>any source</td><td></td><td></td></tr><tr><td>HCl (6 N)</td><td>any source</td><td></td><td></td></tr><tr><td>10-cm tissue culture dishes</td><td>any source</td><td></td><td></td></tr><tr><td>Serological pipettes</td><td>any source</td><td></td><td></td></tr><tr><td>Pasteur pipettes</td><td>any source</td><td></td><td></td></tr><tr><td>Pipette tips</td><td>any source</td><td></td><td></td></tr><tr><td>15-ml conical tubes</td><td>any source</td><td></td><td></td></tr><tr><td>centrifuge</td><td>any source</td><td></td><td></td></tr><tr><td>microcentrifuge</td><td>any source</td><td></td><td></td></tr><tr><td>nutator</td><td>any source</td><td></td><td></td></tr><tr><td>Magnetic separation rack</td><td>any source</td><td></td><td></td></tr><tr><td>UV source</td><td>CalSun</td><td>B001BH0A1A</td><td>Other UV sources can be used, but crosslinking time must be optimized empirically</td></tr><tr><td>Misonix Sonicator</td><td>Qsonica</td><td>S4000 with 431C1 cup horn</td><td>Other sonicators can be used, but sonication conditions must be optimized empirically</td></tr><tr><td>Humidified CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>any source</td><td></td><td></td></tr><tr><td>Biological safety cabinet with vacuum outlet</td><td>any source</td><td></td><td></td></tr></tbody></table>

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.

To account for non-uniform genome fragmentation and other variables, the purified DNA should always be normalized against a reference of Input DNA. Primers specific to a locus of interest can be used. It is helpful to also analyze a locus where the molecule is not expected to bind, as a negative control. We see a &#62;100-fold increase in polyamide occupancy upon irradiation with 365-nm light (Figure 2).
Enriched DNA can also be analyzed by next-generation sequencing. DNA is p...

Watch this Scientific Journal Video about Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin at JoVE.com

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).

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 ...

genome-wide-mapping-drug-dna-interactions-cells-with-cosmic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.2K Views.  University of Wisconsin–Madison. 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).

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

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 ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

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 ...

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

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.

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

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

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.

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 ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, ...

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

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Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

Associated Chromosome Trap for Identifying Long-range DNA Interactions

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

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C