The authors would like to thank members of the Green lab for helpful discussions. This work was supported in part by NIH grants R03AG052018 and R01GM124342 to E.M.G.

1. Pre-bind Antibody to Magnetic Beads
<ol>
	<li>Mix the protein A/G magnetic beads well and transfer 20 &#181;L of magnetic beads per one immunoprecipitation (IP) sample to a 1.5 mL tube. 
	NOTE: When pipetting beads, use wide-bore, low-retention tips.</li>
	<li>Place the tube with beads into a magnetic stand and allow beads to collect on side of the tube. Remove the supernatant.</li>
	<li>Wash the beads 3 times with 1 mL of cold Tris-buffered saline (TBS) (50 mM Tris-HCl pH 7.5, 150 mM NaCl).</li>
	<li>Add 200 &#181;L of TBS to beads and appropriate amount of antibody. Rotate at 8 rpm overnight at 4 &#176;C. 
	NOTE: For many histone PTM antibodies, 1-3 &#181;g of antibody per IP is sufficient. However, titration experiments are recommended to determine appropriate concentrations. Recommended concentrations for well-characterized, commercial histone PTM antibodies are often accurate and can be found in published reports or product literature.</li>
	<li>Place the beads in a magnetic stand and remove the supernatant. Wash them with 1 mL of TBS.</li>
	<li>Resuspend the beads in 20 &#181;L of TBS per IP sample plus an additional 5 &#181;L (in case of pipetting error) of TBS. 
	NOTE: The beads can be stored short-term at 4 &#176;C until use.</li>
</ol>
2. Grow Yeast Cells
<ol>
	<li>Inoculate 10 mL of YPD (10% yeast extract, 20% peptone and 20% dextrose) with a single colony of the appropriate strain and grow it at 30 &#176;C overnight in a shaking incubator at 220 rpm.</li>
	<li>Measure the optical density at 600 nm (OD600) of the yeast culture using a spectrophotometer. Dilute the culture to an OD600 = 0.2 in 100 mL YPD in a new flask.</li>
	<li>Grow the cells at 30 &#176;C in a shaking incubator at 220 rpm until they reach mid-log phase (OD600 = 0.6-0.8).</li>
</ol>
3. In Vivo Crosslinking of Proteins to DNA
<ol>
	<li>When cultures reach mid-log phase, add 2.7 mL of 37% formaldehyde directly to the medium, for a final concentration of 1% formaldehyde. Transfer the flask to a shaker at 25 &#176;C (or room temperature) and shake it at 50 rpm for 15 min.</li>
	<li>Add 5 mL of 2.5 M glycine to the medium and continue shaking at 50 rpm at room temperature for 5 min to quench the formaldehyde.</li>
	<li>Transfer the cells to centrifuge bottles and spin the cells at 5800 x g for 5 min at 4 &#176;C. Decant supernatant.
	<ol>
		<li>Wash the cells by resuspending them in 45 mL of cold TBS and transfer the suspension into a 50 mL conical tube. Spin the tube at 2800 x g for 3 min at 4 &#176;C. Remove the supernatant.</li>
		<li>Wash the cells in 10 mL of cold chIP Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% nonidet octyl phenoxypolyethoxylethanol (NP-40), stored at 4 &#176;C).</li>
		<li>Spin the cells at 2800 x g for 3 min at 4 &#176;C. Remove the supernatant. 
		NOTE: Cells can be frozen with liquid nitrogen and stored at -80 &#176;C until further processing.</li>
	</ol>
	</li>
</ol>
4. Make Yeast Lysates
<ol>
	<li>Resuspend the cells in 1 mL of cold ChIP Lysis Buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1:1,000 dilution of the yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 mL screw cap tube containing 200 &#181;L of glass beads.</li>
	<li>Transfer the cells to a bead beating apparatus for high speed agitation at 4 &#176;C and bead beat 6 times for 30 s each. Keep the cells on ice for at least 1 min between each bead beating.</li>
	<li>Transfer the lysate to a 1.5 mL tube using gel loading tips. Centrifuge the lysate at 15,500 x g for 20 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 250 &#181;L of MNase Digestion Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 21 mM CaCl2, 1% NP-40, stored at 4 &#176;C) by gently pipetting up and down.</li>
	<li>Add 2.5 &#181;L of MNase to the reactions. Mix them gently by inverting 4-6 times and immediately incubate it in a 37 &#176;C water bath for 20 min. 
	NOTE: Digest conditions should be optimized when a new stock of MNase is used. See step 10 for the protocol.</li>
	<li>Immediately place the tubes on ice to stop the reaction and add 5 &#181;L of 0.5 M EDTA for a final concentration of 10 mM EDTA. Mix them gently by inversion.</li>
	<li>Centrifuge the reaction at 15,500 x g for 15 min at 4 &#176;C and transfer the supernatant to a new 1.5 mL tube. 
	NOTE: Soluble (digested) chromatin will be in the supernatant. The pellet contains anything undigested and insoluble cell debris.</li>
</ol>
5. Immunoprecipitate (IP) Modified Histones
<ol>
	<li>Determine the protein concentration of each MNase-released chromatin fraction using a Bradford assay. Add 1 mL of Bradford reagent to each of 8 disposable cuvettes plus 1 additional cuvette for each protein sample to be tested.
	<ol>
		<li>Prepare a stock solution of 2 mg/mL bovine serum albumin (BSA).</li>
		<li>Add the appropriate volume to achieve the following concentrations of BSA in 1 mL of Bradford reagent: 0 &#956;g/mL, 0.5 &#956;g/mL, 1 &#956;g/mL, 2 &#956;g/mL, 4 &#956;g/mL, 6 &#956;g/mL, 8 &#956;g/mL and 10 &#956;g/mL. Add 2 &#956;L of the MNase-released chromatin fraction to the additional cuvettes.</li>
		<li>Cover the top of each cuvette with a small piece of parafilm and invert the cuvettes 4-6 times to mix the solution well. Incubate the cuvettes at room temperature for 15 min.</li>
		<li>Using a visible light spectrophotometer, measure the absorbance at 595 nm for the standard curve and experimental samples. 
		Note: Use the cuvette without BSA (0 &#956;g/mL) to blank the spectrophotometer prior to taking the first measurement.</li>
		<li>Plot a standard curve using the absorbance values for the different concentrations of BSA on software associated with the spectrophotometer or spreadsheet software. Based on the standard curve and the dilution factor used (1:500), use the absorbance values to calculate the protein concentration for each of the chromatin fraction samples.</li>
	</ol>
	</li>
	<li>For each IP, add 50 &#181;g of total protein from the MNase-released chromatin fraction to ChIP Lysis Buffer to reach a final volume of 1 mL. 
	NOTE: If setting up more than one IP per lysate, make a master mix of the lysate and ChIP Lysis Buffer and aliquot 1 mL to individual tubes for the IP.</li>
	<li>Remove 100 &#181;L from the IP mix to process as the input sample. Freeze the input sample at -20 &#176;C until step 7.1. 
	NOTE: Any remaining chromatin sample can also be frozen at -20 &#176;C and used for a subsequent IP if desired.</li>
	<li>Add 20 &#181;L of magnetic Protein A/G beads pre-bound with antibody to each IP sample. Rotate the sample at 8 rpm for 3 h (standard) or overnight (if preferred) at 4 &#176;C.</li>
</ol>
6. Wash IPs and Elute Histone-DNA Complexes
<ol>
	<li>Place the tubes in a magnetic stand and let beads collect on the side of the tube. Remove the supernatant using a pipette. Wash the beads successively with 1 mL of each of the buffers below.
	<ol>
		<li>Perform each wash by rotating at 8 rpm for 5 min at 4 &#176;C, placing beads in a magnetic stand, removing supernatant, and adding next buffer: 2x - ChIP Lysis Buffer, 2x - High Salt Wash Buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% NP-40, stored at 4 &#176;C), 1x - LiCl/detergent Wash Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, stored at 4 &#176;C), 1x - TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA, stored at 4 &#176;C), and 1x - TE .</li>
		<li>With last TE wash, transfer beads to a new tube using 0.5 mL of TE to transfer most of the beads, then another 0.5 mL of TE to wash the tube and transfer remaining beads.</li>
	</ol>
	</li>
	<li>Add 250 &#181;L of freshly made ChIP Elution Buffer (1% SDS, 1 mM NaHCO3). Vortex the solution briefly. Rotate the samples at 8 rpm for 15 min at room temperature.</li>
	<li>Place tube in a magnetic stand and collect the beads. Carefully transfer the supernatant to a new tube and avoid the beads. 
	NOTE: The eluate contains immunoprecipitated proteins and associated DNA.</li>
	<li>Add another 250 &#181;L of ChIP Elution Buffer to beads. Rotate the samples at 8 rpm for 15 min at room temperature.</li>
	<li>Carefully transfer the supernatant to the tube containing the first elution. If necessary, remove a smaller volume (240 &#181;L) for all IPs to avoid disturbing the beads.</li>
</ol>
7. Reverse Protein-DNA Crosslinks
<ol>
	<li>Thaw the input samples and add 400 &#181;L of ChIP Elution Buffer to each input.</li>
	<li>To both input and IP samples, add 20 &#181;L of 5 M NaCl, 5 &#181;L of 20 mg/mL glycogen, and 12.5 &#181;L of 20 mg/mL Proteinase K. Mix well by inverting or flicking tubes. Incubate the samples in a 65 &#176;C water bath overnight.</li>
</ol>
8. Purify and Concentrate DNA
<ol>
	<li>Add 10 &#181;L of 10mg/mL RNase A to the input and IP samples. Incubate the samples in a 37 &#176;C water bath for 30 min.</li>
	<li>Add 600 &#181;L of phenol:chlorofrom:isoamyl alcohol (25:24:1 PCI) to the aqueous solution and mix them well. Spin them at 15,500 x g for 5 min at room temperature.
	<ol>
		<li>Remove the aqueous layer to a new tube. Add 600 &#181;L of PCI and mix them well. Spin the tube at 15,500 x g for 5 min at room temperature. Transfer the aqueous layer to a new tube. 
		CAUTION: Work in the fume hood and wear appropriate personal protective equipment when working with PCI. Dispose liquid and solid waste as instructed by institutional guidelines.</li>
	</ol>
	</li>
	<li>Add 0.1x volume of 3M sodium acetate and 1 mL of cold 100% ethanol to precipitate the DNA. Invert the tube 4-6 times to mix the solution well. Incubate at -20 &#176;C overnight.
	<ol>
		<li>Spin the tube at 15,500 x g for 20 min at 4 &#176;C. Carefully remove the supernatant with a pipette.</li>
		<li>Wash the pellet in 1 mL of cold 70% ethanol. Spin it at 15,500 x g for 10 min at 4 &#176;C. Carefully remove the supernatant with a pipette.</li>
		<li>Air-dry the pellets in the hood for approximately 20 min (until completely dry). Resuspend IP samples in 50 &#181;L of nuclease-free water and resuspend input samples in 100 &#181;L of nuclease-free water. Store the DNA samples at -20 &#176;C until performing qPCR.</li>
	</ol>
	</li>
</ol>
9. Quantitative PCR (qPCR) to Detect Enriched Genomic Regions
<ol>
	<li>Make a qPCR master mix for each set of primers. One reaction contains 5 &#181;L of 2x qPCR mix, 0.25 &#181;L of 20 &#181;M forward primer, and 0.25 &#181;L of 20 &#181;M reverse primer. 
	NOTE: Proper primer design and testing for qPCR experiments are described elsewhere14,15,16.</li>
	<li>Make DNA master mixes for each DNA sample. One reaction contains 0.5 &#181;L of DNA and 4.0 &#181;L of nuclease-free water.</li>
	<li>Add 5.5 &#181;L of the appropriate primer master mix and 4.5 &#181;L of the appropriate DNA master mix to each well on a 384-well qPCR plate. 
	Note: Start with adding 5.5 &#181;L of the primer mix to each well. Spin the plate at 200 x g for 1 min at room temperature before adding 4.5 &#181;L of the DNA mix.</li>
	<li>Adhere the seal to the plate and spin at 200 x g for 1 min at room temperature.</li>
	<li>Perform qPCR using a real-time qPCR system with the following conditions: 95 &#176;C for 3 min; 40 cycles of 95 &#176;C for 10 s, 55 &#176;C for 30 s; melting curve 65 &#176;C to 95 &#176;C with 0.5 &#176;C increments, 5 s.</li>
	<li>For each primer pair, calculate the percent input of the IP sample relative to the 5% input sample used in the qPCR. Use the means of the technical PCR triplicates to perform the calculations. 
	NOTE: If substantial variation is observed in the PCR triplicates, it is best to repeat the qPCR with attention to master mix composition, pipetting errors and cross-contamination between wells in the 384-well plate.
	<ol>
		<li>Adjust the Cq values for the input to 100% by subtracting the number of cycles representing the dilution factor from the raw Cq values. 
		NOTE: Five percent input represents a dilution factor of 20-fold, which equals 4.32 cycles (log2 20 = 4.32).</li>
		<li>Calculate the percent input as 2^(Cqinput - CqIP) multiplied by 100.</li>
	</ol>
	</li>
</ol>
10. Determine MNase Digest Conditions (Recommended Prior to First Full chIP Experiment)
<ol>
	<li>Follow steps 2.1 through 4.4 of the preceding protocol. Scale up the protocol to generate enough lysate for multiple samples of MNase digestion of the chromatin-containing pellet. At step 4.4, resuspend the pellets in 1.25 mL of MNase Digestion Buffer. Aliquot the samples to 5 tubes so that each contains 250 &#181;L of the chromatin pellet resuspended in MNase Digestion Buffer.</li>
	<li>Add 0, 1.5, 2.5 or 5 &#181;L of MNase to each tube. Mix them gently by inverting 4-6 times and immediately incubate the tubes in a 37 &#176;C water bath for 20 min.</li>
	<li>Stop reactions by immediately placing tubes on ice and adding 5 &#181;L of 0.5 M EDTA for a 10 mM final concentration. Mix the solution gently by inversion.</li>
	<li>Centrifuge the tubes at 15,500 x g for 15 min at 4 &#176;C and transfer the supernatant to a new 1.5 mL tube.</li>
	<li>Add SDS to 1% final concentration (25 &#181;L of 10% stock solution) and NaHCO3 to 0.1 M final concentration (25 &#181;L of 1M stock solution) to the samples in the 1.5 mL tubes.</li>
	<li>Add 20 &#181;L of 5M NaCl, 5 &#181;L of glycogen, and 12.5 &#181;L of 20mg/mL proteinase K to the samples in the 1.5mL tubes. Incubate them at 65 &#176;C overnight.</li>
	<li>Add 10 &#181;L of 10 mg/mL RNaseA. Incubate at 37 &#176;C for 30 min.</li>
	<li>Add 300 &#181;L of PCI to the aqueous solution and mix them well. Spin at 15,500 x g for 5 min at room temperature.
	<ol>
		<li>Remove aqueous layer to a new tube. Add 300 &#181;L of PCI and mix well. Spin at 15,500 x g for 5 min at room temperature. Transfer aqueous layer to a new tube.</li>
	</ol>
	</li>
	<li>Add 0.1x volume of 3 M sodium acetate (usually ~30 &#181;L) and 1 mL of cold 100% ethanol to precipitate the DNA. Invert the tube 4-6 times to mix well. Incubate the tube at -80 &#176;C for 1 h or -20 &#176;C overnight.
	<ol>
		<li>Spin the tube at 15,500 x g for 20 min at 4&#176;C. Carefully remove supernatant with a pipette.</li>
		<li>Wash the pellets in 1 mL of cold 70% ethanol. Spin at 15,500 x g for 10 min at 4 &#176;C.</li>
		<li>Let the pellets air-dry in the hood approximately 20 min (or until completely dry). Resuspend the pellets in 30 &#181;L of nuclease-free water.</li>
	</ol>
	</li>
	<li>Add the DNA loading dye and run 20 &#181;L on a 2% agarose gel to visualize digested DNA.</li>
</ol>

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B., 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=An+Interactive+Database+for+the+Assessment+of+Histone+Antibody+Specificity.">An Interactive Database for the Assessment of Histone Antibody Specificity.</a> Mol Cell. 59 (3), 502-511 (2015).</li></ol>

The authors have nothing to disclose.

The procedure described here allows for the efficient recovery of DNA associated with modified histones in yeast cells by immunoprecipitation. This is followed by qPCR using primers which amplify regions of interest to determine local enrichment or depletion of specific histone modifications. Despite being developed as a method almost 20 years ago, chIP remains the defining assay for investigating histone modification status at different genomic regions and under diverse conditions. Although chIP coupled to next generati...

The dynamic post-translational modification (PTM) of histones is a key regulatory mechanism for many DNA-templated processes, including transcription, replication and DNA repair1,2. The ability to determine the abundance and precise localization of modified histones concomitant with these processes is therefore critical to understanding their regulation under different conditions in the cell. The development of chromatin immunoprecipitation (chIP) as a method largely stemmed from biochemical studies of the interactions of proteins with DNA, particularly in vitro methods using chemical crosslinkers, coupled with the need to evaluate the dynamic nature of protein-DNA interactions in vivo and at specific regions of the genome3,4,5. The advancement of quantitative PCR (qPCR) and sequencing technologies has also expanded the ability to perform chIP experiments with quantitative comparisons and across whole genomes, making it a powerful tool for dissecting DNA-protein interactions at multiple levels.
Currently, chIP is a required method for any research group interested in chromatin-mediated regulation of the genome as there are no comparable methods for directly interrogating the physical link between a modified histone and a specific genomic locus in vivo. Although variations of this method using next generation sequencing to map histone modifications throughout the genome6,7 are available, these approaches may address different scientific questions and their scale, cost and technical resources may be limiting for some research groups. Additionally, targeted chIP-qPCR is necessary to complement these approaches by providing methods to both optimize the chIP protocol prior to sequencing and to validate results from the epigenomic datasets. Mass spectrometry based approaches for identifying the full complement of histone marks associated with genomic regions have also emerged8,9,10,11, however, these approaches have some limitations regarding which regions of the genome can be probed and they require technical expertise and instrumentation that will not be available to all research groups. Therefore, chIP remains a foundational method for analyzing the abundance and distribution of histone modifications under diverse conditions for all research groups interested in epigenetics, chromatin and the regulation of genomic functions.
Here, we describe a method for chIP using the budding yeast model Saccharomyces cerevisiae (S. cerevisiae) to investigate the distribution of histone PTMs at chromatin. This approach relies on a number of core components of chIP protocols developed in yeast and also applied to diverse model systems12,13. Interactions between modified histones and DNA in the cell are preserved by crosslinking with formaldehyde. Following lysate preparation, chromatin fragments are solubilized into uniformly-sized fragments by digestion with micrococcal nuclease. Immunoprecipitation of the modified histones is performed with either commercial or lab-generated antibodies and any associated DNA is isolated and analyzed for enrichment at particular genomic regions using qPCR (Figure 1). For many histone modifications, the quantity of DNA obtained from this protocol is sufficient for testing more than 25 different genomic loci by qPCR.
This chIP method is highly versatile for monitoring the distribution of a single histone modification across multiple mutant strains or environmental conditions, or for testing multiple histone modifications in wildtype cells at a number of genomic loci. Furthermore, numerous components of the protocol are easily adjustable to optimize detection of either highly- or lowly-abundant histone marks. Finally, performing chIP of modified histones in budding yeast provides the opportunity to use key controls for antibody specificity that are largely unavailable in other systems. Namely, yeast strains can be generated that carry point mutations in histone residues that are targeted for modification, and, in some cases, there is only a single enzyme that catalyzes modification on a particular histone residue (e.g. histone lysine methyltransferases). Therefore, chIP can be performed in either the histone mutant or enzyme deletion strains to assay the extent to which non-specific binding of the antibody may be occurring and generating false positive results. This control is particularly valuable for newly-developed antibodies, and may even be used to validate antibody specificity for conserved histone modifications prior to their use in other systems. This approach complements other methods to test antibody specificity that distinguish among different modification states (such as mono-, di- and tri-methylation), including probing arrays of modified peptides and performing western blots of histones or nucleosomes with defined modifications. Overall, chIP in budding yeast is a powerful method for assessing the dynamics of histone PTMs throughout the genome and dissecting the mechanisms governing their regulation.

<table><tbody><tr><td>Yeast Extract</td><td>Research Products International (RPI)</td><td>Y20025-1000.0</td><td></td></tr><tr><td>Peptone</td><td>Research Products International (RPI)</td><td>P20250-1000.0</td><td></td></tr><tr><td>Dextrose</td><td>ThermoScientific</td><td>BP350-1</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>AC12007-0050</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>0497-5KG</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758-500G</td><td></td></tr><tr><td>NaCl</td><td>ThermoScientific</td><td>BP358-10</td><td></td></tr><tr><td>4-Nonylphenyl-polyethylene glycol</td><td>Sigma-Aldrich</td><td>74385</td><td>Equivalent to NP-40</td></tr><tr><td>MgCl</td><td>ThermoScientific</td><td>S25533</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>20899-25G-F</td><td></td></tr><tr><td>LiCl</td><td>ThermoScientific</td><td>AC413271000</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Amresco</td><td>M107-1KG</td><td></td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>30970-100G</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>ThermoScientific</td><td>S25533</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626-5G</td><td></td></tr><tr><td>Yeast protease inhibitor cocktail</td><td>VWR</td><td>10190-076</td><td></td></tr><tr><td>25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td><td>VWR Life Science</td><td>97064-824</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>VWR</td><td>100720-992</td><td></td></tr><tr><td>Micrococcal Nuclease</td><td>Worthington Biochemical</td><td>LS004797</td><td></td></tr><tr><td>Glycogen</td><td>ThermoScientific</td><td>R0561</td><td></td></tr><tr><td>Proteinase K</td><td>Research Products International (RPI)</td><td>P50220-0.1</td><td></td></tr><tr><td>RNase A&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R6513-50MG</td><td></td></tr><tr><td>&amp;nbsp;Bradford Assay Reagent</td><td>ThermoScientific</td><td>23238</td><td></td></tr><tr><td>&amp;nbsp;BSA Standard 2 mg/mL</td><td>ThermoScientific</td><td>23210</td><td></td></tr><tr><td>&amp;alpha; H4</td><td>EMD Millipore</td><td>04-858</td><td></td></tr><tr><td>&amp;alpha; H4K16ac</td><td>EMD Millipore</td><td>ABE532</td><td></td></tr><tr><td>&amp;alpha; H3</td><td>Abcam</td><td>ab1791</td><td></td></tr><tr><td>&amp;alpha; H3K4me2</td><td>Active Motif</td><td>39142</td><td></td></tr><tr><td>&amp;nbsp;High Rox qPCR Mix</td><td>Accuris qMax Green, Low Rox qPCR Mix</td><td>ACC-PR2000-L-1000</td><td></td></tr><tr><td>Protein A/G Magnetic Beads</td><td>ThermoScientific</td><td>88803</td><td></td></tr><tr><td>magnetic stand for 1.5mL tubes</td><td>Fisher Scientific</td><td>PI-21359</td><td></td></tr><tr><td>Acid-Washed Glass Beads</td><td>Sigma-Aldrich</td><td>G8772</td><td></td></tr><tr><td>&amp;nbsp;Microtube Homogenizer</td><td>Benchmark</td><td>D1030</td><td></td></tr><tr><td>2.0 mL screw-cap tubes with sealing rings</td><td>Sigma-Aldrich</td><td>Z763837-1000EA</td><td></td></tr><tr><td>Gel loading tips</td><td>Fisher Scientific</td><td>07-200-288</td><td></td></tr><tr><td>Cuvettes</td><td>Fisher Scientific</td><td>50-476-476</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-10</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>384-Well PCR Plate</td><td>Fisher Scientific</td><td>AB-1384W</td><td></td></tr><tr><td>&amp;nbsp;Gyratory Floor Shaker</td><td>New Brunswick Scientific</td><td>Model G10</td><td></td></tr><tr><td>Spectrophotometer</td><td>ThermoScientific</td><td>ND-2000c</td><td></td></tr><tr><td>&amp;nbsp;Real-Time PCR Detection System</td><td>Bio-Rad</td><td>1855485</td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

One key component of this protocol is optimizing the concentration of micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments, as outlined in Step 10. This is critical for obtaining high resolution data regarding the distribution of histone modifications at genomic regions of interest. An MNase titration should be performed to determine the most suitable concentration to achieve primarily mono-nucleosomes with a smaller amount of di-nucleosomes in the soluble chro...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

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12.6K Views.  University of Maryland. Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome. 

Video: Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

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

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

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

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

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 ...

Biochemistry

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Neuroscience

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Biology

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...

Detecting Histone Modifications in Yeast Cells with Neurodegenerative Protein Overexpression

To lyse the cells for western blot analysis, thaw the yeast cell pellets on ice before resuspension in 100 microliters of distilled water. Add 300 microliters of 0.2 molar sodium hydroxide, and 20 microliters of 2-Mercaptoethanol to each cell sample. And resuspend the pellets by pipetting.After a 10-minute incubation on ice, pellet the cells by centrifugation, and resuspend the samples in 100 microliters of loading dye. Then boil the samples for 10 minutes on a heating block. While the samples are being lysed, place two gels in a gel holder, and fill the inner chamber to the top with running buffer and the outside chamber to the two-gel line mark.When the samples are ready, load 15 microliters each cell lysis solution into each well of the 10-well 12% polyacrylamide gel and add 5 microliters of protein ladder to the protein ladder lane well. Run the gel for approximately 45 minutes at 150 volts or until loading dye front reaches the bottom of the gel.While the gel is running, soak two fiber pads per gel in transfer buffer, and one polyvinylidene fluoride or PVDF membrane per gel in methanol. When the gel is ready, rinse membrane and transfer buffer. And place one pre-soaked fiber pad on the bottom of a semi-dry transfer apparatus cell, followed by the PVDF membrane, the gel, and the second pre-soaked fiber pad.Then set the power to 150 milliamps for 1 hour. At the end of the protein transfer, remove the membrane from the transfer apparatus for a brief rinse with distilled water. Place the rinsed membrane protein side up in a small staining box, and cover the membrane with Tris-buffered saline or TBS blocking buffer for a 1 hour incubation at room temperature with gentle rocking.At the end of the blocking incubation, incubate the membrane overnight with an anti-yeast histone and modification specific antibody in TBS at 4 degrees Celsius, and a proper nuclear loading control antibody.The next morning, wash the membrane four times in TBS, supplemented with 0.1% polysorbate 20 for five minutes with rocking at room temperature per wash. After the last wash, incubate the blot with the appropriate secondary antibodies for 1 hour at room temperature, followed by four 5-minute washes in TBST and one five minute wash in TBS alone with rocking.Then image the blot on a fluorescent western blot imaging system for 2 minutes.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Summary

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