Method Article

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

DOI:

10.3791/57080

⸱

December 29th, 2017

 ,  ,  , 
1Department of Biological Sciences, University of Maryland

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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.

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Protocol

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1. Pre-bind Antibody to Magnetic Beads

  1. Mix the protein A/G magnetic beads well and transfer 20 µL of magnetic beads per one immunoprecipitation (IP) sample to a 1.5 mL tube.
    NOTE: When pipetting beads, use wide-bore, low-retention tips.
  2. Place the tube with beads into a magnetic stand and allow beads to collect on side of the tube. Remove the supernatant.
  3. Wash the beads 3 times with 1 mL of cold Tris-buffered saline (TBS) (50 mM Tris-HCl pH 7.5, 150 mM NaCl).
  4. Add 200 µL of TBS to beads and appropriate amount of antibody. Rotate at 8 rpm overnight at 4 °C.
    NOTE: For many histone PTM antibodies, 1-3 µg 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.
  5. Place the beads in a magnetic stand and remove the supernatant. Wash them with 1 mL of TBS.
  6. Resuspend the beads in 20 µL of TBS per IP sample plus an additional 5 µL (in case of pipetting error) of TBS.
    NOTE: The beads can be stored short-term at 4 °C until use.

2. Grow Yeast Cells

  1. 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 °C overnight in a shaking incubator at 220 rpm.
  2. 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.
  3. Grow the cells at 30 °C in a shaking incubator at 220 rpm until they reach mid-log phase (OD600 = 0.6-0.8).

3. In Vivo Crosslinking of Proteins to DNA

  1. 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 °C (or room temperature) and shake it at 50 rpm for 15 min.
  2. 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.
  3. Transfer the cells to centrifuge bottles and spin the cells at 5800 x g for 5 min at 4 °C. Decant supernatant.
    1. 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 °C. Remove the supernatant.
    2. 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 °C).
    3. Spin the cells at 2800 x g for 3 min at 4 °C. Remove the supernatant.
      NOTE: Cells can be frozen with liquid nitrogen and stored at -80 °C until further processing.

4. Make Yeast Lysates

  1. 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 µL of glass beads.
  2. Transfer the cells to a bead beating apparatus for high speed agitation at 4 °C and bead beat 6 times for 30 s each. Keep the cells on ice for at least 1 min between each bead beating.
  3. 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 °C.
  4. Discard the supernatant and resuspend the pellet in 250 µL 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 °C) by gently pipetting up and down.
  5. Add 2.5 µL of MNase to the reactions. Mix them gently by inverting 4-6 times and immediately incubate it in a 37 °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.
  6. Immediately place the tubes on ice to stop the reaction and add 5 µL of 0.5 M EDTA for a final concentration of 10 mM EDTA. Mix them gently by inversion.
  7. Centrifuge the reaction at 15,500 x g for 15 min at 4 °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.

5. Immunoprecipitate (IP) Modified Histones

  1. 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.
    1. Prepare a stock solution of 2 mg/mL bovine serum albumin (BSA).
    2. Add the appropriate volume to achieve the following concentrations of BSA in 1 mL of Bradford reagent: 0 μg/mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL and 10 μg/mL. Add 2 μL of the MNase-released chromatin fraction to the additional cuvettes.
    3. 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.
    4. 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 μg/mL) to blank the spectrophotometer prior to taking the first measurement.
    5. 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.
  2. For each IP, add 50 µg 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.
  3. Remove 100 µL from the IP mix to process as the input sample. Freeze the input sample at -20 °C until step 7.1.
    NOTE: Any remaining chromatin sample can also be frozen at -20 °C and used for a subsequent IP if desired.
  4. Add 20 µL 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 °C.

6. Wash IPs and Elute Histone-DNA Complexes

  1. 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.
    1. Perform each wash by rotating at 8 rpm for 5 min at 4 °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 °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 °C), 1x - TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA, stored at 4 °C), and 1x - TE .
    2. 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.
  2. Add 250 µL 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.
  3. 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.
  4. Add another 250 µL of ChIP Elution Buffer to beads. Rotate the samples at 8 rpm for 15 min at room temperature.
  5. Carefully transfer the supernatant to the tube containing the first elution. If necessary, remove a smaller volume (240 µL) for all IPs to avoid disturbing the beads.

7. Reverse Protein-DNA Crosslinks

  1. Thaw the input samples and add 400 µL of ChIP Elution Buffer to each input.
  2. To both input and IP samples, add 20 µL of 5 M NaCl, 5 µL of 20 mg/mL glycogen, and 12.5 µL of 20 mg/mL Proteinase K. Mix well by inverting or flicking tubes. Incubate the samples in a 65 °C water bath overnight.

8. Purify and Concentrate DNA

  1. Add 10 µL of 10mg/mL RNase A to the input and IP samples. Incubate the samples in a 37 °C water bath for 30 min.
  2. Add 600 µL 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.
    1. Remove the aqueous layer to a new tube. Add 600 µL 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.
  3. 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 °C overnight.
    1. Spin the tube at 15,500 x g for 20 min at 4 °C. Carefully remove the supernatant with a pipette.
    2. Wash the pellet in 1 mL of cold 70% ethanol. Spin it at 15,500 x g for 10 min at 4 °C. Carefully remove the supernatant with a pipette.
    3. Air-dry the pellets in the hood for approximately 20 min (until completely dry). Resuspend IP samples in 50 µL of nuclease-free water and resuspend input samples in 100 µL of nuclease-free water. Store the DNA samples at -20 °C until performing qPCR.

9. Quantitative PCR (qPCR) to Detect Enriched Genomic Regions

  1. Make a qPCR master mix for each set of primers. One reaction contains 5 µL of 2x qPCR mix, 0.25 µL of 20 µM forward primer, and 0.25 µL of 20 µM reverse primer.
    NOTE: Proper primer design and testing for qPCR experiments are described elsewhere14,15,16.
  2. Make DNA master mixes for each DNA sample. One reaction contains 0.5 µL of DNA and 4.0 µL of nuclease-free water.
  3. Add 5.5 µL of the appropriate primer master mix and 4.5 µL of the appropriate DNA master mix to each well on a 384-well qPCR plate.
    Note: Start with adding 5.5 µL of the primer mix to each well. Spin the plate at 200 x g for 1 min at room temperature before adding 4.5 µL of the DNA mix.
  4. Adhere the seal to the plate and spin at 200 x g for 1 min at room temperature.
  5. Perform qPCR using a real-time qPCR system with the following conditions: 95 °C for 3 min; 40 cycles of 95 °C for 10 s, 55 °C for 30 s; melting curve 65 °C to 95 °C with 0.5 °C increments, 5 s.
  6. 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.
    1. 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).
    2. Calculate the percent input as 2^(Cqinput - CqIP) multiplied by 100.

10. Determine MNase Digest Conditions (Recommended Prior to First Full chIP Experiment)

  1. 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 µL of the chromatin pellet resuspended in MNase Digestion Buffer.
  2. Add 0, 1.5, 2.5 or 5 µL of MNase to each tube. Mix them gently by inverting 4-6 times and immediately incubate the tubes in a 37 °C water bath for 20 min.
  3. Stop reactions by immediately placing tubes on ice and adding 5 µL of 0.5 M EDTA for a 10 mM final concentration. Mix the solution gently by inversion.
  4. Centrifuge the tubes at 15,500 x g for 15 min at 4 °C and transfer the supernatant to a new 1.5 mL tube.
  5. Add SDS to 1% final concentration (25 µL of 10% stock solution) and NaHCO3 to 0.1 M final concentration (25 µL of 1M stock solution) to the samples in the 1.5 mL tubes.
  6. Add 20 µL of 5M NaCl, 5 µL of glycogen, and 12.5 µL of 20mg/mL proteinase K to the samples in the 1.5mL tubes. Incubate them at 65 °C overnight.
  7. Add 10 µL of 10 mg/mL RNaseA. Incubate at 37 °C for 30 min.
  8. Add 300 µL of PCI to the aqueous solution and mix them well. Spin at 15,500 x g for 5 min at room temperature.
    1. Remove aqueous layer to a new tube. Add 300 µL of PCI and mix well. Spin at 15,500 x g for 5 min at room temperature. Transfer aqueous layer to a new tube.
  9. Add 0.1x volume of 3 M sodium acetate (usually ~30 µL) 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 °C for 1 h or -20 °C overnight.
    1. Spin the tube at 15,500 x g for 20 min at 4°C. Carefully remove supernatant with a pipette.
    2. Wash the pellets in 1 mL of cold 70% ethanol. Spin at 15,500 x g for 10 min at 4 °C.
    3. Let the pellets air-dry in the hood approximately 20 min (or until completely dry). Resuspend the pellets in 30 µL of nuclease-free water.
  10. Add the DNA loading dye and run 20 µL on a 2% agarose gel to visualize digested DNA.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Yeast ExtractResearch Products International (RPI)Y20025-1000.0
PeptoneResearch Products International (RPI)P20250-1000.0
DextroseThermoScientificBP350-1
FormaldehydeSigma-AldrichF8775
GlycineFisher ScientificAC12007-0050
TrisAmresco0497-5KG
EDTASigma-AldrichE6758-500G
NaClThermoScientificBP358-10
4-Nonylphenyl-polyethylene glycolSigma-Aldrich74385Equivalent to NP-40
MgClThermoScientificS25533
CaCl2Sigma-Aldrich20899-25G-F
LiClThermoScientificAC413271000
Sodium Dodecyl SulfateAmrescoM107-1KG
Sodium DeoxycholateSigma-Aldrich30970-100G
Sodium AcetateSigma-AldrichS2889
NaHCO3ThermoScientificS25533
PMSFSigma-AldrichP7626-5G
Yeast protease inhibitor cocktailVWR10190-076
25 Phenol:24 Chloroform:1 Isoamyl AlcoholVWR Life Science97064-824
EthanolSigma-AldrichE7023
Nuclease-Free WaterVWR100720-992
Micrococcal NucleaseWorthington BiochemicalLS004797
GlycogenThermoScientificR0561
Proteinase KResearch Products International (RPI)P50220-0.1
RNase A Sigma-AldrichR6513-50MG
 Bradford Assay ReagentThermoScientific23238
 BSA Standard 2 mg/mLThermoScientific23210
α H4EMD Millipore04-858
α H4K16acEMD MilliporeABE532
α H3Abcamab1791
α H3K4me2Active Motif39142
 High Rox qPCR MixAccuris qMax Green, Low Rox qPCR MixACC-PR2000-L-1000
Protein A/G Magnetic BeadsThermoScientific88803
magnetic stand for 1.5mL tubesFisher ScientificPI-21359
Acid-Washed Glass BeadsSigma-AldrichG8772
 Microtube HomogenizerBenchmarkD1030
2.0 mL screw-cap tubes with sealing ringsSigma-AldrichZ763837-1000EA
Gel loading tipsFisher Scientific07-200-288
CuvettesFisher Scientific50-476-476
ParafilmFisher Scientific13-374-10
50 mL conical tubesFisher Scientific14-432-22
384-Well PCR PlateFisher ScientificAB-1384W
 Gyratory Floor ShakerNew Brunswick ScientificModel G10
SpectrophotometerThermoScientificND-2000c
 Real-Time PCR Detection SystemBio-Rad1855485

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Tags

Micrococcal NucleaseQuantitative PCRYeast LysateFormaldehyde CrosslinkingMagnetic BeadsH3K4me2H4K16ac

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