Name: genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae
Uploaded: 2017-06-03T15:00:00
Description: Watch this Scientific Journal Video about Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae at JoVE.com

We thank Moustafa Saleh and Jay Tourigny for critical reading of the manuscript and Steven Hahn and Steven Henikoff for mentorship and support during the development of ChEC-seq and its application to the Mediator complex. S.G. is supported by NIH grants R01GM053451 and R01GM075114 and G.E.Z. is supported by Indiana University startup funds.

1. Generation of Yeast Strains
<ol>
	<li>Generate a yeast strain bearing the factor of interest tagged with MNase.
	<ol>
		<li>PCR amplify the MNase tagging cassette from the desired vector (Table 1) using the specified reaction mixture (Table 2) and cycling conditions (Table 3). Mix 5 &#181;L of the PCR reaction and 1 &#181;L of 6X DNA loading dye. Run each PCR aliquot on a 0.8% agarose gel at 120 V for 40 min. The expected product size is ~2.3 kb.</l...

<ol>
	<li>Zentner, G. E., Kasinathan, S., Xin, B., Rohs, R., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChEC-seq+kinetics+discriminates+transcription+factor+binding+sites+by+DNA+sequence+and+shape+in+vivo.">ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.</a> Nat Commun. 6, 8733 (2015).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Dynamics+of+Enhanceosome+Assembly+in+Embryonic+Stem+Cells.">Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells.</a> Cell. 156 (6), 1274-1285 (2014).</li><li>Teytelman, L., Thurtle, D. M., Rine, J., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+expressed+loci+are+vulnerable+to+misleading+ChIP+localization+of+multiple+unrelated+proteins.">Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins.</a> Proc Natl Acad Sci USA. 110 (46), 18602-18607 (2013).</li><li>Park, D., Lee, Y., Bhupindersingh, G., Iyer, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+Misinterpretable+ChIP-seq+Bias+in+Yeast.">Widespread Misinterpretable ChIP-seq Bias in Yeast.</a> PLoS ONE. 8 (12), 83506 (2013).</li><li>Jain, D., Baldi, S., Zabel, A., Straub, T., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+promoters+give+rise+to+false+positive+'Phantom+Peaks'+in+ChIP-seq+experiments.">Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments.</a> Nucleic Acids Res. , (2015).</li><li>Krebs, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+transcription+factor+binding+map+accuracy+utilizing+knockout-mouse+models.">Optimization of transcription factor binding map accuracy utilizing knockout-mouse models.</a> Nucleic Acids Res. 42 (21), 13051-13060 (2014).</li><li>Paul, E., Zhu, Z. I., Landsman, D., Morse, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Association+of+Mediator+and+RNA+Polymerase+II+in+Wild-Type+and+Mediator+Mutant+Yeast.">Genome-Wide Association of Mediator and RNA Polymerase II in Wild-Type and Mediator Mutant Yeast.</a> Mol Cell Biol. 35 (1), 331-342 (2015).</li><li>Worsley Hunt, R., Wasserman, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+transcription+factors+motifs+are+a+systemic+component+of+ChIP-seq+datasets.">Non-targeted transcription factors motifs are a systemic component of ChIP-seq datasets.</a> Genome Biol. 15 (7), 412 (2014).</li><li>Vega, V. B., Cheung, E., Palanisamy, N., Sung, W. -. 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E., Ahmad, K., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+mapping+of+transcription+factor+binding+sites+on+native+chromatin.">High-resolution mapping of transcription factor binding sites on native chromatin.</a> Nat Methods. 11 (2), 203-209 (2014).</li><li>Skene, P. J., Hernandez, A. E., Groudine, M., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleosomal+barrier+to+promoter+escape+by+RNA+polymerase+II+is+overcome+by+the+chromatin+remodeler+Chd1.">The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin remodeler Chd1.</a> eLife. 3, 02042 (2014).</li><li>Aughey, G. N., Southall, T. 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We have shown that ChEC can map diverse classes of yeast proteins on chromatin, and anticipate that it will be broadly applicable to different families of TFs and other chromatin-binding factors in yeast. ChEC-seq is advantageous in that it does not require crosslinking, chromatin solubilization, or antibodies. Thus, ChEC avoids artifacts potentially present in X-ChIP-seq, such as the hyper-ChIPable artifact3,4, and native ChIP, such as false negatives due to inc...

Mapping the binding sites of transcription factors (TFs), chromatin remodelers, and other chromatin-associated regulatory factors is key to understanding all chromatin-based processes. While chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) approaches have been used to gain many important insights into genome biology, they have notable limitations. We recently introduced an alternative method, termed chromatin endogenous cleavage and high-throughput sequencing (ChEC-seq)1, to circumvent these drawbacks.
ChIP-seq is most often performed with an initial formaldehyde crosslinking step (X-ChIP-seq) to preserve protein-DNA interactions. However, a number of recent studies have indicated that X-ChIP-seq captures transient or nonspecific protein-DNA interactions2,3,4,5,6,7,8, giving rise to false positive binding sites. In addition, sonication, commonly used to fragment chromatin in X-ChIP-seq experiments, preferentially shears regions of open chromatin, leading to biased recovery of fragments from these regions9,10. Sonication also yields a heterogeneous mixture of fragment lengths, ultimately limiting binding site resolution, though the addition of an exonuclease digestion step can greatly improve resolution11,12. Native ChIP methods such as occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC)13 do not use crosslinking and fragment chromatin with micrococcal nuclease (MNase), alleviating potential biases associated with formaldehyde crosslinking and sonication. However, the solubility of many chromatin-bound proteins under the relatively mild conditions required for native chromatin extraction is poor, potentially leading to reduced dynamic range and/or false negatives14.
While various iterations of ChIP-seq are most commonly used for genome-wide mapping of protein-DNA interactions, several mapping techniques based on fusion of proteins of interest to various DNA-modifying enzymes have also been implemented. One such approach is DNA adenine methyltransferase identification (DamID)15, wherein a chromatin-binding protein of interest is genetically fused to Dam and this fusion is expressed in cells or animals, resulting in methylation of GATC sequences proximal to binding sites for the protein. DamID is advantageous in that it does not rely upon immunoprecipitation and so avoids crosslinking, antibodies, or chromatin solubilization. It is also performed in vivo. However, the resolution of DamID is limited to the kilobase scale and the methylating activity of the Dam fusion protein is constitutive. A second method based on enzymatic fusion is Calling Card-seq16, which employs fusion of a factor of interest to a transposase, directing site-specific integration of transposons. Like DamID, Calling Card-seq is not based on immunoprecipitation and thus has similar advantages, with the added benefit of increased resolution. However, Calling Card-seq may be limited by sequence biases of transposases and is also reliant on the presence of restriction sites close to transposon insertion sites.
A third enzymatic fusion method, developed in the Laemmli lab, is chromatin endogenous cleavage (ChEC)17. In ChEC, a fusion between a chromatin-associated protein and MNase is expressed in cells, and upon calcium addition to activate MNase, DNA is cleaved proximal to binding sites for the tagged factor (Figure 1). In conjunction with Southern blotting, ChEC has been used to characterize chromatin structure and protein binding at a number of individual loci in yeast17,18, and has been combined with low-resolution microarray analysis to probe the interaction of nuclear pore components with the yeast genome19. ChEC offers benefits similar to DamID and Calling Card-seq, and its resolution is nearly single-base pair when analyzed by primer extension19. ChEC is also controllable: robust DNA cleavage by MNase depends on the addition of millimolar calcium, ensuring that MNase is inactive at the low free calcium concentrations observed in live yeast cells20.
Previously, we postulated that combining ChEC with high-throughput sequencing (ChEC-seq) would provide high-resolution maps of TF binding sites. Indeed, ChEC-seq generated high-resolution maps of the budding yeast general regulatory factors (GRFs) Abf1, Rap1, and Reb1 across the genome1. We have also successfully applied ChEC-seq to the modular Mediator complex, a conserved, essential global transcriptional coactivator21, expanding the applicability of ChEC-seq to megadalton-size complexes that do not directly contact DNA and may be difficult to map by ChIP-based methods. ChEC-seq is a powerful method both for independent validation of ChIP-seq results and generation of new insights into the regulation of chromatin-resident processes. Here, we present a step-by-step protocol for the implementation of this method in budding yeast.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">dNTPs</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">N0447</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Q5 high-fidelity DNA polymerase</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">M0491L</td>
			<td>Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">TrackIt 1 Kb Plus DNA ladder</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10488085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Taq DNA polymerase</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">M0273L</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:307px;">cOmplete Mini EDTA-free protease inhibitor cocktail</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:119px;">11836170001</td>
			<td>It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit Mnase cleavage by chelation of Ca2+.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">PMSF</td>
			<td style="width:159px;">ACROS Organics</td>
			<td style="width:119px;">AC215740010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Digitonin, High Purity</td>
			<td style="width:159px;">EMD Millipore</td>
			<td style="width:119px;">300410-250MG</td>
			<td>Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Proteinase K, 20 mg/mL</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:119px;">25530049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">RNase A, 10 mg/mL</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EN0531</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Ampure XP beads</td>
			<td style="width:159px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td>Ampure-like beads can be generated using a published protocol (ref 24).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">MagneSphere magnetic rack</td>
			<td style="width:159px;">Promega</td>
			<td style="width:119px;">Z5342</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In the case of a successful ChEC experiment, analysis of DNA by agarose gel electrophoresis will reveal a calcium-dependent increase in DNA fragmentation over-time, as indicated by smearing and eventual complete digestion of genomic DNA. In some cases, a ladder of bands similar to that seen with a traditional MNase digestion is observed after extended digestion. This is the case for ChEC analysis of Reb1, a general regulatory factor that binds nucleosome-depleted regions (NDRs) (<strong c...

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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 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 ...

The overall goal of this procedure is to generate genome-wide maps of protein-DNA interactions in situ with high resolution and low background, without the need for formaldehyde crosslinking, chromatin solubilization, or antibodies. This method can help answer key questions in the transcription field, such as where various regulatory factors associate with chromatin, relative to gene-regulatory regions. The main advantage of this technique is that is provides high resolution genomic binding maps with negligible background, and avoids limitations associated with standard ChEC-seq protocols.Demonstrating the procedure will be Sebastian Grunberg, my colleague from the Fred Hutchinson Cancer Research Center. In the afternoon or evening of the day prior to the experiment, start a pre-culture of the experimental use strain. Inoculate three milliliters of used peptone dextrose, or an appropriate selective medium with a single colony of the strain, bearing the MNase tagged factor.Incubate this culture overnight in a 30 degrees Celsius shaker incubator. On the following morning, dilute the overnight culture into 50 milliliters of medium to an optical density at 600 nanometers of 0.2 to 0.3. Incubate the culture in the shaker incubator until it reaches an optical density at 600 nanometers of 0.5 to 0.7.Prepare the needed amount of Buffer A plus additives, and keep it room temperature during the procedure. Prepare microfuge tubes for sample collection. For each new factor analyzed, collect samples without calcium addition, and add 30 seconds, one minute, 2.5 minutes, 5 minutes, and 10 minutes after calcium addition.To each tube add 90 microliters of stop solution, and 10 microliters of 10%SDS. When the culture has reached an optical density at 600 nanometers of 0.5 to 0.7, decant the culture into a 50 milliliter conical tube, and centrifuge at 1, 500 times g for one minute at room temperature. Remove the supernatant, thoroughly resuspend the cells in one milliliter of Buffer A, and transfer the resuspended cells to a microfuge tube.Pellet the resuspended cells in a microfuge at 1, 500 times g for 30 seconds at room temperature. Aspirate the supernatant, and thoroughly resuspend the cells in one milliliter of Buffer A by pipetting up and down. Pellet the resuspended cells at 1, 500 times g for 30 seconds at room temperature.Remove the supernatant, resuspend the cells in one milliliter of Buffer A, and centrifuge again. After removing the supernatant, thoroughly resuspend the cells in 570 microliters of Buffer A.Add 30 microliters of 2%digitonin to facilitate permeabilization of the cells, and invert to mix. Permeabilize the cells in a heat block at 30 degrees Celsius for five minutes.Pipette the reaction up and down to ensure an even distribution of cells. As a negative control, transfer 100 microliter aliquot of permeabilized cells to a microfuge tube containing stop solution and SDS, and vortex briefly to mix. Prior to beginning the time course it's critical to have the tubes of stop solution, the pipette, pipette-tips, and a timer next to the heat blocks so that the early time points can be collected efficiently.To start the time course, add 1.1 microliters of one-molar calcium chloride to the permeabilized cells to activate MNase, and invert several times quickly to mix. Immediately return the mixed reaction to 30 degrees Celsius and start a timer. At each time point to be collected, pipette the reaction up and down to ensure an even distribution of cells, and then remove a 100 microliter aliquot of permeabilized cells to a microfuge tube containing stop solution and SDS.Vortex briefly to mix. Once all time points have been collected, add four microliters of 20 milligrams per milliliter proteinase K to each sample. Vortex briefly to mix, and incubate at 55 degrees Celsius for 30 minutes.Add 200 microliters of phenol:chloroform:isoamyl alcohol to each sample, vortex vigorously to mix, and centrifuge at maximum speed for five minutes at room temperature. Remove the aqueous phase to a new tube. Add 30 micrograms of glycogen, and 500 milliliters of 100%ethanol.Vortex vigorously to mix, and precipitate on dry ice for 10 minutes, or until the solution is viscous. Pellet the precipitated DNA by centrifuging at maximum speed, and four degrees Celsius for 10 minutes. Decant the supernatant and wash each pellet with one milliliter of room temperature 70%ethanol.Decant the ethanol and remove the remainder by inverting, and gently tapping the tubes on a paper towel. Briefly spin the samples using a bench-top centrifuge, then use a pipette to remove residual ehtanol, taking care not to disturb the pellet. Air-dry the pellets at room temperature for five minutes.While the DNA pellets are drying, make a master mix of Tris buffer and RNase A for resuspending the pellets once they are dried, and vortex to mix. Add 30 microliters of the RNase A solution to each dried DNA pellet, vortex to mix, and incubate at 37 degrees Celsius for 20 minutes to digest RNa. After 20 minutes mix one microliter of 6X DNA loading dye with five microliters of each sample, and run on a 1.5%agarose gel at 120 volts for 40 minutes.Begin the size selection procedure by adding to each sample, 75 microliters of SPRI beads in a polyethylene glycol solution. Pipette up and down 10 times to mix the beads and DNA mixture, and then incubate at room temperature for five minutes. During the SPRI bead incubation, prepare microfuge tubes containing 96 microliters of 10 millimolar Tris pH 8.0, and four microliters of five molar sodium chloride for each sample.Place the tubes containing the beads and DNA mixture in a magnetic rack for two minutes to collect beads on the side of the tube, then transfer each supernatant to a new tube containing Tris and sodium chloride. Add 200 microliters of phenol:chloroform:isoamyl alcohol to each sample, vortex to mix, and centrifuge at maximum speed at room temperature for five minutes. Remove each aqueous phase to a new tube, add 30 micrograms of glycogen, and 500 microliters of 100%ethanol.Precipitate DNA on dry-ice for 10 minutes, or until the solution is viscous. Pellet the precipitated DNA by centrifuging at maximum speed and four degrees Celsius for 10 minutes. Decant the supernatant and wash each pellet with one milliliter of 70%ethanol.Decant the ethanol, removing the remainder by inverting and gently tapping the tubes on a paper towel. Briefly spin the samples using a bench-top centrifuge, and use a pipette to remove residual ethanol, taking care not to disturb the pellet. Air-dry the pellets at room temperature for five minutes.Resuspend each dried pellet in 25 microliters of Tris pH 8. Determine the concentration of each sample using a high-sensitivity assay. Agarose gel electrophoresis of DNA from a chromatin endogenous cleavage analysis of Reb1, a general regulatory factor that binds nucleosome-depleted regions, reveals a calcium-dependent increase in DNA fragmentation over time as indicated by smearing, and eventual complete digestion of genomic DNA.Visualization of fragment ends from a Reb1 chromatin endogenous cleavage sequencing experiment reveals robust enrichment of Reb1 over background, derived from the free MNase strain after 30 seconds of calcium treatment. Similarly, substantial DNA cleavage was observed by a fusion of the mediator subunit Med8 with MNase, but not free MNase-driven by the Med8 promoter one minute after calcium addition. To compare Reb1 chromatin endogenous cleavage sequencing data to chromatin immunoprecipitation and high throughput sequencing data, 1, 992 Reb1 peaks determined by the organic method were motif centered, and the average fragment end count at each base position in a 100 base pair window around the motif midpoint was determined.A striking asymmetry and cleavage was observed with the majority of Reb1-MNase released fragment ends mapping to the upstream side of the motif. Once mastered, this technique can be done in one full working day, if it performed properly. When attempting this procedure it's important to remember to limit the number of samples assayed in parallel, to ensure accurate pipetting and handling times.After its development, this technique paved the way for researchers in the field of transcription to, for the first time, unambiguously characterize the genomic association of a non-DNA binding, megadalton-size protein complex in yeast. After watching this video, you should have a good idea of how to perform the ChEC-Seq method for genome-wide mapping of protein-binding sites in the budding yeast genome.

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