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 polyamide&#8211;DNA crosslinks. The interaction between biotin and streptavidin is one of the strongest known in nature and one thousand to one million times stronger than the interaction of an antibody with its ligand.62 We harness this robust interaction, and the intrinsic stability of streptavidin, by subjecting captured polyamide&#8211;DNA crosslinks to harsh washes that would denature an antibody. These washes reduce the background signal of COSMIC. Furthermore, biotinylated proteins such as histones unfold and dissociate from DNA under these harsh conditions.63,64
The addition of the biotin moiety increases the size of the molecule, which could be reduced by substitution with a &#8216;clickable&#8217; handle such as an alkyne.43-46 This handle could be used to introduce the biotin moiety after the cells have been lysed. Taken together, COSMIC avoids one of the major sources of unreliability that plagues many ChIP experiments.
Although formaldehyde will crosslink protein&#8211;DNA interactions, formaldehyde also crosslinks protein&#8211;protein interactions.65,66 Thus, formaldehyde can lead to the misidentification of DNA-binding events that are indirect or transient.65,66 This issue is especially prominent in highly transcribed regions.67 COSMIC uses a photocrosslinker (psoralen) instead of formaldehyde to avoid the issues of formaldehyde-based crosslinking. Because a light-sensitive crosslinker is used, the sample must be shielded from light to prevent premature crosslinking.
We have performed COSMIC in multiple conditions and cell types. The cell type, concentration of polyamide, and duration of treatment can all be varied, but it is important to confirm that polyamide treatment does not induce cellular toxicity. It is also critical to empirically determine a sonication time that produces sheared DNA with a size between 100 and 500 bp. If the COSMIC signal is low, optimize the number of cells, incubation time, concentration of polyamide, and UV crosslinking time.
The bioinformatic methods to analyze ChIP-seq data are compatible with COSMIC-seq data. Similar to ChIP, COSMIC requires normalization to a reference sample of fragmented DNA that has not been enriched with an antibody or streptavidin. This sample, normally called input DNA, is designed to control for many possible sources of bias including sonication efficiency, PCR bias, sequencing bias, and mappability of DNA fragments. We have found success with the ENCODE guidelines to identify peaks in our COSMIC-seq datasets.50,68
Some of the most successful therapeutic agents are molecules that bind to DNA and interfere with an array of genomic transactions.43,69,70 COSMIC can be readily applied to small molecules other than polyamides. The crosslinker can be UV active, as we have chosen, or it could also be DNA-reactive.69,70 In summary, COSMIC reveals the binding sites of polyamides and other ligands throughout the genome and uncovers new strategies to selectively target DNA.

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://www-jove-com.remotexs.ntu.edu.sg/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 prepared for sequencing in the same manner as ChIP DNA is prepared (e.g., with a commercial sample prep kit, see Materials List). Even with next-generation sequencing, we still use qPCR to confirm enrichment of DNA at loci where we expect the polyamide to be bound. Once sequenced, raw DNA reads are aligned to the genome and density traces are prepared with the same software designed to analyze ChIP-seq data (Figure 3). Based on our analysis of polyamide distributions in cells, we found that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. We developed an algorithm to score the entire genome for binding with our in vitro CSI data (Figure 4A). This scoring method revealed that different genomic loci with similar predicted binding scores exhibit the diverse clustering of multiple sites of varying affinities (Figure 4B).
<img alt="Figure 2" src="/files/ftp_upload/53510/53510fig2.jpg" /> 
Figure 2. Results of COSMIC-qPCR. Effect of 365 nm UV irradiation on the fraction of AP/input from HEK293 cells. AP, affinity-purified. Results are plotted on log scale and represent mean &#177; s.e.m.
<img alt="Figure 3" src="/files/ftp_upload/53510/53510fig3.jpg"/> 
Figure 3. Results of COSMIC-seq in H1 cells. COSMIC-seq tag density track for linear polyamide 4 designed to target AAG repeats. Tag density was normalized to 107 tags and displayed with the Integrated Genome Viewer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53510/53510fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53510/53510fig4.jpg" /> 
Figure 4. Model of polyamide binding.&#160;(A) Violin plots of predicted scores for 2 and 4 binding across the entire genome. Representative genomescapes for 4 are also shown. With the bioinformatic scoring method employed, genomic loci can sum to the same predicted score in different ways. (B) Loci with multiple low- and medium-affinity sequences show similar polyamide occupancy to loci with few high-affinity sequences. Reprinted with permission.51 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53510/53510fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K Views.  University of Wisconsin–Madison.  Het identificeren van de directe doelstellingen van genoom-gerichte moleculen blijft een grote uitdaging. Om te begrijpen hoe DNA-bindende moleculen die aangrijpen op de genoom, ontwikkelden we een methode die is gebaseerd op de verknoping van kleine moleculen aan chromatine isoleren (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

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of both synthetic lethal interactors leads to loss of cell viability or cell death. The best studied synthetic lethal interaction is between BRCA1/2 and PARP1, with PARP1 inhibitors being used in clinical practice to treat patients with BRCA1/2 mutated tumors. Large genetic screens in model organisms but also in haploid human cell lines have led to the identification of numerous additional synthetic lethal interaction pairs, all being potential targets of interest in the development of novel tumor therapies. One approach is to therapeutically target genes with a synthetic lethal interactor that is mutated or significantly downregulated in the tumor of interest. A second approach is to formulate drug combinations addressing synthetic lethal interactions. In this article, we outline a data integration workflow to evaluate and identify drug combinations targeting synthetic lethal interactions. We make use of available datasets on synthetic lethal interaction pairs, homology mapping resources, drug-target links from dedicated databases, as well as information on drugs being investigated in clinical trials in the disease area of interest. We further highlight key findings of two recent studies of our group on drug combination assessment in the context of ovarian and breast cancer.

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

Cancer Research

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 ...

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, ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Biochemistry

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, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Bioengineering

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 ...

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

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