Chromatin Immunoprecipitation Protocol to Analyze Histone Modifications in Arabidopsis thaliana (PROT12)

Werner Aufsatz
Gregor Mendel Institute of Molecular Plant Biology
Austrian Academy of Sciences
Dr. Bohrgasse 3
A-1030 Vienna, Austria

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Updated: 09 May 2007, Author address updated.
Last reviewed: 27 July 2005 by Olivier Mathieu, Paszkowski lab, Genève

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Introduction

Eukaryotic chromatin is a complex of DNA and associated histone proteins which are involved in the higher order packaging of DNA into chromosomes. The chromatin state of a given DNA sequence influences transcriptional activity and replication timing and is regulated by potentially reversible covalent modifications of DNA and histones. Histone modifications at conserved lysine and arginine residues within the flexible N-terminal tails, such as phosphorylation, acetylation and methylation, specify a code which serves as an interaction platform with specific domains of chromatin-associated proteins. The immunoprecipitation (IP) of crosslinked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation; ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes and is already routinely used in many labs. In contrast to animal cells, however, plant cells have a rigid cell wall which poses limitations to the simple utilization of protocols established for animals. In this protocol, I describe the method used in our laboratory to study histone modifications in the plant model organism Arabidopsis thaliana. This protocol is an adapted version of the original procedure published by Lawrence and co-workers (Lawrence et al., 2004).

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Procedure

Plant Material

Arabidopsis seeds are stratified for 48 hours in 0.1% Phytablend (Cat #PTC001, Caisson Laboratories) at 4°C and then sown onto soil. 1.5g of whole, 3-4 week old seedlings, are used per chromatin preparation. It is imperative to avoid contamination with soil as much as possible during harvest.

Day 1

Chromatin Crosslinking

1. Harvest 1.5g seedlings and place them into a 50ml Falcon tube;
2. Rinse seedlings twice with 40ml bidistilled water. Remove as much water as possible after second wash;
3. Add 37ml 1% formaldehyde solution. Gently submerge seedlings at the bottom of the tube by stuffing the tube with nylon mesh. Screw on cap and poke cap with needle holes. Put in exsiccator and draw vacuum for 10 minutes;
4. Release vacuum slowly and shake exsiccator slightly to remove air bubbles. Seedlings should appear translucent;
5. Add 2.5ml 2M glycine to quench crosslinking. Draw vacuum for 5 minutes;
6. Again, release vacuum slowly and shake exsiccator slightly to remove air bubbles;
7. Remove nylon mesh, decant supernatant and wash seedlings twice with 40ml of bidistilled water; After second wash, remove as much water as possible and put seedlings between two layers of kitchen paper. Roll up paper layers carefully to remove as much liquid as possible.

At this step, plant material can be shock-frozen in liquid nitrogen and stored at -80°C. In my hands, this step provides the only possibility, where the protocol can be interrupted and still gives reproducible results.

Chromatin preparation

1. Precool mortar with liquid nitrogen. Add 2 small spoons of silicon dioxide (Sigma, S9887) and plant material. Grind plant material to a fine powder;
2. Use cooled spoon to add powder to 30ml of Extraction Buffer 1 stored on ice. Vortex to mix and keep at 4°C until solution is homogenous;
3. Filter extract through Miracloth into a new, ice-cold 50ml Falcon tube. Rigidly press out solid material;
4. Repeat step 3;
5. Centrifuge extract using the Beckman JS 7.5 rotor (or equivalent) at 4000 rpm for 20 minutes at 4°C;
6. Gently pour off supernatant and resuspend pellet in 1ml of Extraction Buffer 2 by pipetting up and down. Transfer solution to Eppendorf tube;
7. Spin in cooled benchtop centrifuge at 13000 rpm for 10 minutes;
8. Remove supernatant and resuspend pellet in 300µl of Extraction Buffer 2 by pipetting up and down;
9. Add 300µl of Extraction Buffer 3 to fresh Eppendorf tube; Use pipette to carefully layer solution from step 8 onto it.
10. Spin in cooled benchtop centrifuge at 13000 rpm for 1 hour. In meantime, prepare 10ml Nuclei Lysis Buffer and 20ml CHIP Dilution Buffer. Put buffers in coldroom;
11. Remove supernatant and resuspend pellet in 500µl of cold Nuclei Lysis Buffer. Resuspend by pipetting up and down and by vortexing (see comment 1). Remove 10µl to run on an agarose gel (see comment 2);
12. Sonicate 4x 10 seconds, 40% duty cycle and 20% power (Bandelin Sonoplus HD 2070 with MS 73 probe). Put on ice for 1 minute between sonication steps (see comment 3 and comment 4);
13. Spin in cooled benchtop centrifuge at 13000 rpm for 10 minutes. Add supernatant to new Eppendorf tube;
14. Repeat step 13. Remove 10µl to run on an agarose gel;
15. Separate aliquots from steps 11 and 14 on 1.5% agarose gel. In the sonicated samples, DNA should be shifted and more intense compared to untreated samples and range between 200-2000bp, centering around 500bp.

Pre-clearing and immuno precipitation (IP)

1. Transfer two 150µl aliquots of chromatin solutions to separate Eppendorf tubes. Add 1350µl of CHIP Dilution Buffer per tube;
2. Prepare Protein A agarose beads pre-absorbed with sheared salmon sperm DNA (Upstate, Cat. 16-157) by rinsing 40ml 3 times with 1ml CHIP Dilution Buffer in an Eppendorf tube. Spin in cooled benchtop centrifuge for 30 seconds at 13000rpm between the washes to pellet the beads;
3. Add 1.5ml diluted chromatin solution from step 1 per 40µl of equilibrated beads. Rotate for 1 hour at 4°C (see comment 5);
4. Spin in cooled benchtop centrifuge for 30 seconds at 13000 to pellet the beads. Combine chromatin of identical genotypes in 14 ml Falcon tube (e.g. 3ml in this example, see step 1). Be careful not to contaminate solution with carry over of beads;
5. Store a 60µl aliquot of pooled chromatin at -20°C. This will serve as input control (see comment 6);
6. Add 600µl of pooled chromatin solution per IP to an Eppendorf tube with an appropriate antibody. We use ~10µg of antibody per IP. Also set up 600µl of chromatin solution without antibody, which serves as mock IP. Rotate overnight at 4°C.

Day 2

Collection, washes and elution of immune complexes

1. Prepare fresh CHIP Dilution Buffer and store it at 4°C;
2. Prepare Protein A agarose beads pre-absorbed with sheared salmon sperm DNA (Upstate, Cat. 16-157) by rinsing 40µl 3 times with 1ml CHIP Dilution Buffer in an Eppendorf tube. Prepare one aliquot of beads per IP. Spin in benchtop centrifuge for 30 seconds at 13000rpm between the washes to pellet the beads;
3. Add IPs and rotate for 1 hour at 4°C. In the meantime, prepare Elution Buffer and place it at 65°C (see comment 7);
4. Spin in cooled benchtop centrifuge at 5000 rpm for 30 seconds to collect beads and discard supernatant (see comment 8);
5. Add 1ml of Low Salt Wash Buffer per tube. Rotate for 5 minutes at 4°C;
6. Spin in cooled benchtop centrifuge at 5000 rpm for 30 seconds to collect beads and discard supernatant;
7. Add 1ml of High Salt Wash Buffer per tube. Rotate for 5 minutes at 4°C;
8. Spin in cooled benchtop centrifuge at 5000 rpm for 30 seconds to collect beads and discard supernatant;
9. Add 1ml of LiCl Wash Buffer per tube. Rotate for 5 minutes at 4°C;
10. Spin in cooled benchtop centrifuge at 5000 rpm for 30 seconds to collect beads and discard supernatant;
11. Add 1ml of TE Buffer per tube. Rotate for 5 minutes at 4°C;
12. Spin in cooled benchtop centrifuge at 5000 rpm for 30 seconds to collect beads and discard supernatant;
13. Repeat TE wash. Spin in cooled benchtop centrifuge at 5000 rpm for 30 seconds to collect beads and discard supernatant;
14. Elute immune complexes by adding 250µl of Elution Buffer. Vortex briefly to mix and incubate at 65°C for 15 minutes. Spin in benchtop centrifuge at 13000 rpm for 30 seconds and transfer supernatant to a fresh Eppendorf tube;
15. Repeat elution and finally combine the two elutes;
16. Add 460µl of Elution Buffer to the 60µl input control aliquoted on Day 1 (comment 9).

Reverse crosslinking

1. Add 20µl of 5M NaCl to samples. Incubate overnight at 65°C.

Day 3

DNA cleanup

1. Add 10µl of 0.5M EDTA, 20µl 1M Tris-HCl pH6.5 and 1µl of 20mg/ml proteinase K per tube. Incubate for 1 hour at 45°C (see comment 10);
2. Extract samples with 1 volume of phenol-chloroform. Spin in cooled benchtop centrifuge at 13000 rpm for 15 minutes and transfer supernatant to 2ml reaction tube (see comment 11);
3. Precipitate DNA with 1/10 volume 3M NaOAc pH5.2 and 3 volumes absolute ethanol. Add 4µl glycogen (Roche, Cat. 901393) per precipitation. Incubate at -20°C for at least 1 hour;
4. Spin in cooled benchtop centrifuge at 13000 rpm for 15 minutes. Wash pellet with 1ml 70% ethanol and spin again. Discard supernatant and vacuum-dry pellet for 10 minutes. Dissolve DNA in 50µl 10mM Tris-HCl pH7.5. Proceed to PCR reactions.

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Materials & Reagents

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Reviewer Comments

Reviewed by: Olivier Mathieu, Paszkowski lab, Genève

1. We preclear each chromatin sample (in 15ml Falcon tubes, see comment #2) by adding 70µl of Protein A Agarose beads. Rotate 1 hour at 4°C.
2. We resuspend in 300µl of Nuclei Lysis Buffer by pipetting up and down and vortexing (keep solution cold between vortexing). Incubate for 20 minutes on ice.
3. Following this step, the chromatin solution can be frozen at -20°C.
4. We dilute 10 times with CHIP Dilution Buffer in 15ml Falcon tubes (add 2.7ml of CHIP Dilution Buffer). The point here is to dilute the 1% SDS to 0.1% SDS.
5. The solution should not foam during sonication. Cool the tubes with a mix of 100% ethanol in ice during the sonication step.
6. Supernatant of the "mock" sample can also be used as the input control (see comment #8).
7. Collect immune complexes with Protein A Agarose beads for 3 hours at 4°C.
8. Save 500µl supernatant of the "mock" sample. This will serve as the input control.
9. Do not add Elution Buffer to the Input control saved at comment #8.
10. We incubate for 3 hours at 45°C, with shaking.
11. After proteinase K treatment, the immunoprecipitated DNA can be purified using a silica-gel membrane (e.g. Qiagen PCR purification kit), eluated in 50µl and directly analyzed by PCR.

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Figures

Figure 1. Verification of this protocol at Arabidopsis loci with known histone modifications. At4g03770 and At4g03800 represent retrotransposons which are located within the heterochromatic knob on chromosome 4 and are transcriptionally silent (Lippman et al., 2004). Both loci are associated with di-methylated H3K9. At4g04040 is an active gene and is associated with tri-methylated H3K4, respectively (Xie et al., 2004). Chromatin immunoprecipitation has been performed in duplicates, using wild type plants (WT1, WT2). The a-di-methyl H3K9 antibody was kindly provided by the Jenuwein group, the a-tri-methyl H3K4 antibody was obtained from Abcam Ltd., Cambridge, UK (ab8580).

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References

1. Lawrence RJ, Earley K, Pontes O, Silva M, Chen ZJ, Neves N, Viegas W, Pikaard CS (2004) A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol Cell, 13: 599-609.
2. Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, Carrington JC, Doerge RW, Colot V, Martienssen R (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature, 430: 471-476.
3. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol., 2: 642-652.

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