Chromatin Immunoprecipitation to Study The Plant Epigenome

Abstract

Chromatin immunoprecipitation (ChIP) has been widely used for studying in vivo protein–DNA interactions for decades. ChIP is a powerful tool that is adaptable for studying epigenetic modifications at certain genomic loci or the genomic level. Given its utility in studying the epigenome and the many technical challenges, we present a detailed in-lab ChIP protocol primarily used for studying histone modifications in plants, but can be easily adapted for other chromatin targets in other species.

1 Introduction

Many aspects of plant development, including gametogenesis, seed development, and flowering time, are directly or indirectly regulated by epigenetic marks [1]. As sessile organisms, plants developed effective strategies to rapidly respond to environmental changes, mainly through epigenetic modifications . Epigenetic modifications, including DNA methylation and histone modification, regulate gene expression by altering chromatin structure. In addition, at key locations such as active genes and centromeres, canonical histones are replaced by histone variants such as H2A.Z and CENH3, which influence local chromatin structure and gene activity [2–4]. Given the significance of epigenetic modifications in plants , research focus has shifted to studying genome-wide epigenetic modifications —the epigenome [5, 6]. To study the epigenome, and in particular histone modifications, the most powerful approach is chromatin immunoprecipitation coupled with tiling microarray (ChIP-chip) or deep sequencing (ChIP sequencing), which reveals epigenetic modifications at the genomic level.

With the increasing interest in the epigenome, a number of chromatin immunoprecipitation (ChIP) techniques have been developed and optimized for studying genome-wide histone modifications [7–12]. In general, the ChIP technique is considered technically challenging. The two most critical steps that determine the success of ChIP are chromatin isolation and the antibody selection. While the chromatin is relatively stable, the interaction in the chromatin complex is compromised if harsh conditions (for example, SDS buffer and over-digestion of chromatin) are used for chromatin isolation. To prevent disassembly of the chromatin complex, cross-linking with a reagent such as formaldehyde is necessary [13]. Performing uniform cross-linking is essential to the ChIP technique as the efficiency of cross-linking varies significantly for different plant material. In addition, how much cross-linking reagent should be used and how long the cross-linking is allowed to proceed are other issues for consideration and optimization. Another important step is the antibody selection, which might be the most critical factor to ensure successful ChIP . In general, polyclonal antibodies have relatively high binding affinity but low specificity, while monoclonal antibodies have relatively high specificity but low affinity. The quality of antibodies used in ChIP is crucial. Here, we present a detailed ChIP protocol developed from previously reported ChIP protocols [11, 12]. This modified protocol includes extensive discussion on some critical steps so that it can be easily followed and adapted to individual needs.

2 Materials

• 1. Immunoprecipitation magnetic beads (such as Protein A-Dynabeads^® from Invitrogen).

• 2. Micrococcal Nuclease, MNase dissolved in 50 % filter-sterilized glycerol with a final concentration of 15 U/μl, and kept at −20 °C.

• 3. 1.5 ml graduated, low-retention microcentrifuge tubes.

• 4. Miracloth.

• 5. Glycogen solution at 20 mg/ml of molecular biology grade glycogen free of DNAses and RNases.

• 6. RNase A at 10 mg/ml.

• 7. Proteinase K at 10 mg/ml.

• 8. 100 mM PMSF solution: 174.2 mg Phenylmethylsulfonyl fluoride dissolved in 10 ml 100 % ethanol and stored in −20 °C (protect from light).

• 9. Cross-linking buffer: 0.4 M Sucrose, 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 % Formaldehyde, 0.1 mM Phenylmethylsulfonyl fluoride (PMSF).

• 10. M1 buffer: 11.9 % Hexylene glycol, 10 mM KPO₄, pH 7.0, 100 mM NaCl, 5 mM beta-mercaptoethanol, 0.1 mM PMSF (freshly added), 1× Plant protease inhibitor cocktail (freshly added).

• 11. M2 buffer: 8.85 % Hexylene glycol, 10 mM KPO₄, pH 7.0, 10 mM MgCl₂, 0.5 % Triton X-100, 5 mM beta-mercaptoethanol, 100 mM NaCl.

• 12. Incubation buffer: 20 mM Tris–HCl, pH 6.8, 50 mM NaCl, 5 mM EDTA, 0.1 mM PMSF (freshly added), 1× Plant protease inhibitor cocktail (freshly added).

• 13. MNB buffer: 50 mM Tris–HCl, pH 8.0, 2.5 mM CaCl₂, 4 mM MgCl₂, 0.3 M Sucrose.

• 14. PK digestion buffer (10×): 50 mM EDTA, 100 mM Tris–HCl, pH 7.4, 0.25 % SDS.

• 15. Low salt washing buffer: 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, 0.1 mM PMSF (freshly added).

• 16. High salt washing buffer: 500 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, 0.1 mM PMSF (freshly added).

• 17. LiCl washing buffer: 0.25 M LiCl, 1 % NP-40, 1 % Sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.0, 0.1 mM PMSF (freshly added).

• 18. TE buffer: 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 0.1 mM PMSF (freshly added).

• 19. Elution buffer: 1 % SDS, 0.1 M NaHCO₃.

3 Methods

3.1 Chromatin Preparation

• 1. Grind 2 g of plant material of interest (for example, maize young ears or leaves, soybean seeds) in liquid nitrogen using mortar and pestle (see Notes 1 and 2 ).

• 2. Transfer the powder into 50 ml tubes, add 30 ml of freshly made cross-linking buffer containing 1 % Formaldehyde and vortex briefly.

• 3. Incubate on ice for 20 min, mix by gently shaking every 5 min (see Notes 3 and 4 ).

• 4. Add glycine to a final concentration of 0.1 M to stop cross-linking , mix by gentle shaking. Incubate on ice for another 5 min.

• 5. Filter the cross-linked chromatin through two layers of Miracloth to remove plant debris (see Note 5 ).

• 6. Spin down at 1000 × g at 4 °C for 10 min, after transferring the filtered chromatin solution into a number of 1.5 ml low-retention microcentrifuge tubes (see Note 6 ).

• 7. After carefully removing the supernatant, resuspend the pellet (containing crude chromatin) by pipetting up and down gently in 1 ml of ice-cold M1 buffer and spin at 1000 × g at 4 °C for 10 min.

• 8. Repeat step 7.

• 9. Combine all tubes of a single sample into one tube, and wash one more time with 1 ml of ice-cold M2 buffer.

• 10. Add 400 μl MNB buffer and the desired amount of MNase and incubate at 37 °C for the desired time (see Notes 7 and 8 ).

• 11. Stop MNase digestion by adding 1/10 volume of 0.5 M EDTA and keep on ice.

• 12. Spin down at 16,000 × g in a microcentrifuge at 4 °C for 10 min.

• 13. Save the supernatant containing the chromatin , resuspend the pellet with 400 μl of incubation buffer, and incubate on ice for 60 min to extract more chromatin from the pellet.

• 14. Spin down at 16,000 × g at 4 °C for 10 min, and combine two supernatant fractions, which contain the digested chromatin .

3.2 Isolation of Specific Chromatin –DNA Complex

• 1. Add 40 μl of prewashed immunoprecipitation magnetic beads in incubation buffer and 2 μl of IgG antibodies to the chromatin solution to preclear the chromatin , and rotate/mix at 4 °C for at least 2 h (see Notes 9 and 10 ).

• 2. Use a magnetic stand to remove the beads, save 1/10 of the supernatant as an input control fraction for later steps (keep at −20 °C).

• 3. Divide the rest of the supernatant into several low-retention tubes (see Note 11 ), and add a certain amount of antibodies into different tubes (see Notes 12 and 13 ), as well as 500 μl of incubation buffer (see Note 14 ). Rotate at 4 °C overnight.

• 4. Add 20 μl of prewashed immunoprecipitation magnetic beads in incubation buffer into each tube and continue to rotate at 4 °C for another 3–5 h.

• 5. Prepare all the washing buffers (keep at 4 °C) and make FRESH elution buffer.

• 6. Do washes in the following order: Low salt washing buffer first, followed by high salt washing buffer and finally the LiCl washing buffer. Wash twice with each buffer using 1 ml of buffer each time. For each wash do the following: briefly spin to bring the buffer down to the bottom of tubes (1000 × g in microcentrifuge at 4 °C for 10 s), place the tubes into the magnetic stand for 1 min, carefully remove the supernatant without touching the beads, put new buffer in, rotate for 5 min or longer at 4 °C.

• 7. Following the final LiCl wash, wash the beads with 1 ml of ice-cold TE buffer. Remove TE buffer after 1 min in the magnetic stand and add 200 μl of elution buffer. Invert to mix and incubate at 65 °C for 15 min or longer, shaking from time to time.

• 8. Save the supernatant to a new 1.5 ml tube after the beads attach to the magnetic stand, add another 200 μl of elution buffer to the beads and incubate again at 65 °C for 15 min (see Note 15 ). Combine the two supernatants, which should contain the eluted chromatin target complexes.

• 9. Add 16 μl of 5 M NaCl to each tube and incubate at 65 °C overnight. In addition, take out 10 % of the chromatin used for each ChIP reaction from the previously saved input fraction (step 2 of Subheading 3.2), bring the volume up to 400 μl with elution buffer, add 16 μl of 5 M NaCl, and incubate at 65 °C overnight together with the rest of samples.

3.3 DNA Isolation

• 1. Add 1 μl of RNase A (10 mg/ml) and incubate at 37 °C for 30 min.

• 2. Add 1/10 volume of 10× PK digestion buffer and 1.5 μl of 10 mg/ml Proteinase K to each tube, and incubate at 45 °C for at least 1 h.

• 3. Add equal volume of phenol:chloroform:ispropanol (25:24:1), vortex for 1 min and spin at 16,000 × g at room temperature for 10 min.

• 4. Carefully save the supernatant into a new tube and add an equal volume of chloroform, vortex for 1 min and spin at 16,000 x g in microcentrifuge at room temperature for 10 min.

• 5. Save the supernatant into new tubes, and add 1/10 volume of 3 M sodium acetate (pH 5.2), two volumes of 100 % ethanol and 0.2 μl of glycogen. Leave at −20 °C for at least 2 h (preferably overnight).

• 6. Spin at 16,000 × g at 4 °C for 30 min, wash the pellet with 1 ml of ice-cold 80 % ethanol, and spin at 16,000 × g at 4 °C for 10 min. Carefully remove as much residual ethanol as possible without disturbing the pellet.

• 7. Air-dry the pellet in the sterile hood (leave the tubes open) for 10–15 min, the pellet should have a white color but can sometimes be invisible. After drying, add 20 μl of TE buffer to dissolve DNA (see Note 16 ).

• 8. Measure DNA concentration (see Note 17 ).

• 9. ChIPed DNA can be used for real-time PCR to verify enrichment for specific genomic DNA regions (see Note 18 ), and then used for deep sequencing, such as Illumina sequencing (see Note 19 ).

4 Notes

• 1. The amount of plant material varies from 500 mg to 2 g, or can be scaled up, depending on how many ChIP reactions you want to perform. In general, 250 mg plant tissue should be suitable for one ChIP reaction.

• 2. Due to different properties of plant materials, it is very hard to do uniform cross-linking with intact plant tissues or organs, such as roots and seeds. Therefore, it becomes much easier to do cross-linking with fine plant tissue powder, which can lead to uniform cross-linking . More importantly, the optimized conditions for cross-linking with fine tissue powder can be applied to almost any plant material. Thus, it is critical to grind plant material to a very fine powder. The better samples are ground, the more uniform the cross-linking will be and the more chromatin one will get.

• 3. It is a good idea to do cross-linking for different plant samples at the same time to reduce the ChIP variation between replicates due to the cross-linking difference.

• 4. The cross-linking conditions (such as concentration of formaldehyde and cross-linking time) must be optimized. Less cross-linking usually results in instability of chromatin during ChIP procedures and leads to low DNA yield. Without reverse cross-linking , the chromatin with sufficient cross-linking will yield very little or no DNA, which can be used as a criterion to determine whether the cross-linking is sufficient. On the other hand, excessive cross-linking will result in low DNA yield even after reverse cross-linking . These criteria can be used to optimize cross-linking conditions. In our hands, with 1 % formaldehyde 20 min on ice is the best condition for cross-linking .

• 5. Sometimes you need to squeeze the Miracloth gently in order to get most of the solution out. However, do not let the debris get into the flow-through.

• 6. Low-retention tubes are preferred for all steps in the ChIP procedure, since any chromatin and DNA attached to the tubes will reduce ChIP yield. In addition, filtered tips are recommended to reduce cross-contamination between samples and ChIP reactions.

• 7. The common approaches to shear the chromatin include physical sonication using a bioruptor (Diagenode) or sonicator, and enzymatic digestion using MNase. For the purpose of epigenomic studies, MNase digestion is preferred since it provides information about nucleosome positioning.

• 8. The amount of MNase depends on the source of MNase and the chromatin amount. The condition for MNase digestion can be optimized by running DNA from digested chromatin on a 1 % agarose gel with EtBr staining. For epigenomic studies, the length for the majority of isolated chromatin should be one to a few nucleosomes. In our hands, for the chromatin from 0.5 g of plant powder, 4.5 units of MNase (USB) at 37 °C for 20 min will digest most of chromatin into mononucleosomes.

• 9. To achieve better ChIP performance, this preclearing step is important, since it will remove most of the chromatin that are nonspecifically attached to the immunoprecipitation magnetic beads (e.g., Dynabeads) or the antibody . If the corresponding preimmune serum for the antibody is available, it should be used. Otherwise, for commercial antibodies, IgG can be used instead.

• 10. Although salmon sperm/protein A-agarose is another choice, protein A-Dynabead is preferred since the residual salmon sperm DNA after ChIP can be detected by deep sequencing, which will introduce undesired sequencing noise.

• 11. The number of tubes depends on the number of ChIP reactions and replicates that will be performed.

• 12. Many commercial antibodies recognizing histones and histone modifications designed for ChIP are available, such as antibodies from Abcam and Millipore, and most of them can be used for plants . We have successfully used antibodies against unmodified histone H3 (Abcam, ab1791), as well as histone H3 modified with trimethyl K4 (Abcam, ab8580), dimethyl K9 (Millipore, 07-212), and trimethyl K27 (Millipore, 07-449) for soybean ChIP . As a negative control, IgG antibody should be included.

• 13. The amount of each antibody used for ChIP should be optimized based on ChIP enrichment, which might be different for different antibodies. In our hands, the amount of each histone antibody used is 1 ~ 4 μg/ChIP .

• 14. To allow effective mixing of the solution in the tubes, another 500 μl of incubation buffer should be added into each tube.

• 15. The incubation time can be longer, such as 1 h.

• 16. Incubation at 37 °C for 20 min should help remove excess solution.

• 17. Due to extreme low yield of ChIP procedure, DNA quantification using standard Nanodrop is not accurate. Instead, bioanalyzer or Nanodrop coupled with picogreen staining should be used to check DNA concentration.

• 18. Real-time PCR should follow standard real-time PCR protocol with some minor modification depending on primers. To compute ChIP fold enrichment, input DNA should be included. A DNA region that is well known to be a target of the desired epigenetic modifications (targeted DNA) or not to be a target for the same epigenetic modifications (reference DNA) should be used to calculate fold enrichment. As another negative control, the IgG control sample should not give any ChIP enrichment. The formula to calculate ChIP fold enrichment is (ChIPedDNA_target/InputDNA_target)/(ChIPedDNA_reference/InputDNA_reference).

• 19. Given the low DNA yield from ChIP procedure, DNA needs to be amplified prior to deep sequencing. One can use the GenomePlex Whole Genome Amplification (WGA) kit (Sigma) to perform DNA amplification.