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.</li>
		<li>Transform the amplified tagging cassette into the strain of choice using standard lithium acetate transformation22 and perform selection.</li>
		<li>Verify correct integration of the tagging cassette with colony PCR.
		<ol>
			<li>Prepare the specified reaction mixture (Table 4) for the number of colonies to be screened. Keep on ice until needed.</li>
			<li>Pick a portion of each colony to be tested into the bottom of a PCR tube using a sterile toothpick or pipet tip.</li>
			<li>Microwave the picked colonies at high power for 1 min.</li>
			<li>Add 50 &#181;L of PCR mix (Table 4) to each microwaved colony and pipet up and down to mix. Perform PCR using the specified cycling conditions (Table 5).</li>
			<li>Mix the 50 &#181;L of PCR reaction and 10 &#181;L of 6X DNA loading dye directly in each PCR tube. Run 10 &#181;L of each sample on a 1% agarose gel at 120 V for 40 min. The expected product size is ~ 700 bp. 
			NOTE: If desired, verify appropriate expression of the MNase fusion protein via western blotting. A ~ 20.6 kilodalton shift in the molecular weight of the tagged protein is expected.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Generate a free MNase strain by homology-based cloning of the promoter of the gene of interest into pGZ136 (Table 1) and transformation and selection as in step 1.1.2.
	<ol>
		<li>Alternatively, assemble the promoter of the gene of interest and 3xFLAG-MNase-SV40 NLS into a plasmid vector, transform and select as in step 1.1.2, and grow the strain in the appropriate selective condition for maintenance of the plasmid.</li>
	</ol>
	</li>
</ol>
2. ChEC
<ol>
	<li>In the afternoon/evening of the day prior to the ChEC experiment, inoculate 3 mL yeast-peptone-dextrose (YPD) or appropriate selective medium with a single colony of the strain bearing the MNase-tagged factor. Incubate this culture overnight (180 RPM shaking, 30 &#176;C).</li>
	<li>In the morning of the day of the ChEC experiment, dilute the overnight culture into 50 mL of medium to an optical density at 600 nm (OD600) of 0.2 - 0.3. Incubate this culture (180 RPM shaking, 30 &#176;C) until it reaches an OD600 of 0.5-0.7. As the culture approaches the appropriate OD600, set heat block or water bath to 30 &#176;C and start thawing Buffer A additives (Table 6).</li>
	<li>Prepare 5 mL of Buffer A plus additives (Table 6) per culture. Keep buffer A at RT during the procedure.</li>
	<li>Prepare microfuge tubes containing 90 &#181;L of stop solution (Table 7) and 10 &#181;L of 10% SDS for each time point to be collected. For each new factor analyzed, take samples at 0 s, 30 s, 1 min, 2.5 min, 5 min, and 10 min.</li>
	<li>Decant culture into a 50 mL conical tube and centrifuge at 1,500 x g and room temperature (RT) for 1 min.</li>
	<li>Thoroughly resuspend cells in 1 mL of Buffer A and transfer the resuspended cells to a microfuge tube. Pellet resuspended cells in a microfuge at 1,500 x g and RT for 30 s.</li>
	<li>Aspirate supernatant and thoroughly resuspend cells in 1 mL of Buffer A by pipetting up and down. Pellet resuspended cells in a microfuge at 1,500 x g and RT for 30 s. Repeat this step once.</li>
	<li>Thoroughly resuspend cells in 570 &#181;L of Buffer A. Add 30 &#181;L of 2% digitonin (0.1% final concentration) to facilitate permeabilization of the cells, and invert to mix.</li>
	<li>Permeabilize cells in heat block or water bath at 30 &#176;C for 5 min.</li>
	<li>Pipet the reaction up and down to ensure an even distribution of cells. Remove a 100 &#181;L aliquot of permeabilized cells as a negative control to a microfuge tube containing stop solution and SDS (step 2.4) and vortex briefly to mix.</li>
	<li>Add 1.1 &#181;L of 1 M CaCl2 (2 mM final concentration) to permeabilized cells to activate MNase and invert several times quickly or vortex briefly to mix. Immediately return the mixed reaction at 30 &#176;C and start a timer.</li>
	<li>At each time point to be collected, pipet the reaction up and down to ensure an even distribution of cells, then remove a 100 &#181;L aliquot of permeabilized cells to a microfuge tube containing stop solution and SDS and vortex briefly to mix. For each new factor analyzed, take 0 s (no CaCl2&#160;added), 30 s, 1 min, 2.5 min, 5 min, and 10 min time points. 
	NOTE: ChEC experiments with factors that rapidly cleave DNA at 30&#176;C may be performed at lower temperatures to slow MNase cleavage kinetics.</li>
	<li>Once all time points have been collected, add 4 &#181;L of 20 mg/mL proteinase K to each sample, vortex briefly to mix, and incubate at 55 &#176;C for 30 min.</li>
	<li>Add 200 &#181;L of 25:24:1 phenol:chloroform:isoamyl alcohol to each sample, vortex vigorously to mix, and centrifuge at maximum speed and RT in a microfuge for 5 min.</li>
	<li>Remove each aqueous phase (~ 150 &#181;L) to a new tube. Add 30 &#181;g of glycogen and 500 &#181;L of 100% ethanol. Vortex vigorously to mix and precipitate on dry ice for 10 min or until solution is viscous.</li>
	<li>Pellet precipitated DNA at maximum speed and 4 &#176;C in a microfuge for 10 min.</li>
	<li>Decant supernatants and wash pellets with 1 mL of RT 70% ethanol.</li>
	<li>Decant ethanol, removing the remainder by inverting and gently tapping the tubes on a paper towel. Briefly spin samples using a benchtop centrifuge and use a pipet to remove residual ethanol, taking care not to disturb the pellet. Air-dry pellets for at RT for 5 min.</li>
	<li>While pellets are drying, make a master mix to resuspend dried pellets in 29 &#181;L of Tris, pH 8.0 + 1 &#181;L of 10 mg/mL RNase A each. Digest RNA at 37 &#176;C for 20 min.</li>
	<li>Mix 1 &#181;L of 6X DNA loading dye with 5 &#181;L of each sample and run on a 1.5% agarose gel at 120 V for 40 min.</li>
</ol>
3. Size Selection
NOTE: The goal of size selection is to remove multi-kilobase fragments of genomic DNA from the sample to be sequenced and enrich fragments of ~150 bp (approximately the size of nucleosomal DNA) or smaller. In sequencing data, fragments &#60;400 bp are enriched, with a notable peak around the size of nucleosomal DNA (~150 bp) and a peak or broad distribution of subnucleosomal fragments.
<ol>
	<li>To each sample, add 75 &#181;L of solid-phase reversible immobilization (SPRI) beads in a polyethylene glycol (PEG) solution23 and pipet up and down 10 times to mix. Incubate the bead:DNA mixture at RT for 5 min.</li>
	<li>During the SPRI bead incubation, prepare microfuge tubes containing 96 &#181;L of 10 mM Tris, pH 8.0 and 4 &#181;L of 5 M NaCl for each sample.</li>
	<li>Place tubes in a magnetic rack to collect beads on the side of the tube. Allow the beads to collect on the side of the tube for 2 min.</li>
	<li>Transfer each supernatant to a new tube containing Tris and NaCl (step 3.2).</li>
	<li>Add 200 &#181;L of 25:24:1 phenol:chloroform:isoamyl alcohol to each sample, vortex to mix, and centrifuge at maximum speed and RT in a microfuge for 5 min.</li>
	<li>Remove each aqueous phase to a new tube. Add 30 &#181;g of glycogen and 500 &#181;L of 100% ethanol. Precipitate DNA on dry ice for 10 min or until solution is viscous.</li>
	<li>Pellet precipitated DNA at maximum speed and 4 &#176;C in a microfuge for 10 min.</li>
	<li>Decant supernatants and wash pellets with 1 mL of 70% ethanol.</li>
	<li>Decant ethanol, removing the remainder by inverting and gently tapping the tubes on a paper towel. Briefly spin samples using a benchtop centrifuge and use a pipet to remove residual ethanol, taking care not to disturb the pellet. Air-dry pellets for at RT for 5 min.</li>
	<li>Resuspend dried pellets in 25 &#181;L of Tris, pH 8.0. Determine sample concentration using a high-sensitivity assay. For TF ChEC experiments, 10-100 ng DNA is routinely recovered after size selection.</li>
	<li>Prepare sequencing libraries. Library preparation protocols that do not rely upon size selection, as previously described24, are desirable to allow sequencing of a large range of fragment sizes (25-500 bp).</li>
	<li>Sequence libraries in paired-end mode. A minimum of 25 bases of sequence on each end is needed for high quality read mapping. Between 1 and 2 million reads provides sufficient coverage for a ChEC sample. 
	NOTE: Analysis of ChEC-seq data is a complex procedure and beyond the scope of this article. Detailed information on data analysis can be found in previous publications1,21, and the custom scripts used for analysis are available on GitHub (https://github.com/zentnerlab/chec-seq). HOMER25 (http://homer.salk.edu) can also be used for command-line analysis of ChEC-seq data.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>dNTPs</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>Q5 high-fidelity DNA polymerase</td><td>NEB</td><td>M0491L</td><td>Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.</td></tr><tr><td>TrackIt 1 Kb Plus DNA ladder</td><td>ThermoFisher Scientific</td><td>10488085</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>NEB</td><td>M0273L</td><td></td></tr><tr><td>cOmplete Mini EDTA-free protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>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 Ca&lt;sup&gt;2+&lt;/sup&gt;.</td></tr><tr><td>PMSF</td><td>ACROS Organics</td><td>AC215740010</td><td></td></tr><tr><td>Digitonin, High Purity</td><td>EMD Millipore</td><td>300410-250MG</td><td>Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.</td></tr><tr><td>Proteinase K, 20 mg/mL</td><td>Invitrogen</td><td>25530049</td><td></td></tr><tr><td>RNase A, 10 mg/mL</td><td>ThermoFisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Ampure XP beads</td><td>Beckman Coulter</td><td>A63880</td><td>Ampure-like beads can be generated using a published protocol (ref 24).</td></tr><tr><td>MagneSphere magnetic rack</td><td>Promega</td><td>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 ...

genome-wide-mapping-protein-dna-interactions-with-chec-seq

Universidad San Sebastian

Research

JoVE Journal

Genetics

11.0K Views.  Fred Hutchinson Cancer Research Center. 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.

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

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

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemistry

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biology

Locus-Specific Chromatin Isolation from Yeast Cells

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

Author Spotlight: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

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

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Genome-wide 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 ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

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

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Chapters in this video

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

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

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

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

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae