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://www.jove.com/files/ftp_upload/53510/53510fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td style="width:180px;">any source</td>
			<td style="width:172px;"></td>
			<td style="width:75px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benzamidine</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dynabeads MyOne Streptavidin C1</td>
			<td>Life Technologies</td>
			<td>65001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>Other sources can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">StemPro&#174; Accutase&#174; Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">pH paper</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">amber microcentrifuge tubes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microcentrifuge tubes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pyrex filter</td>
			<td>any source</td>
			<td></td>
			<td>Pyrex baking dishes are suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR master mix</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl (6 N)</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10-cm tissue culture dishes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Serological pipettes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pasteur pipettes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette tips</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15-mL conical tubes</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">centrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microcentrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nutator</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic separation rack</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Humidified CO2 incubator</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

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 Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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 ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

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 ...