Chromatin Immunoprecipitation Protocol to Analyze Histone Modifications in Arabidopsis thaliana

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 Harvest 1.5g seedlings and place them into a 50ml Falcon tube; Rinse seedlings twice with 40ml bidistilled water. Remove as much water as possible after second wash; 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; Release vacuum slowly and shake exsiccator slightly to remove air bubbles. Seedlings should appear translucent; Add 2.5ml 2M glycine to quench crosslinking. Draw vacuum for 5 minutes; Again, release vacuum slowly and shake exsiccator slightly to remove air bubbles; 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 Precool mortar with liquid nitrogen. Add 2 small spoons of silicon dioxide (Sigma, S9887) and plant material. Grind plant material to a fine powder; 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; Filter extract through Miracloth into a new, ice-cold 50ml Falcon tube. Rigidly press out solid material; Repeat step 3; Centrifuge extract using the Beckman JS 7.5 rotor (or equivalent) at 4000 rpm for 20 minutes at 4°C; Gently pour off supernatant and resuspend pellet in 1ml of Extraction Buffer 2 by pipetting up and down. Transfer solution to Eppendorf tube; Spin in cooled benchtop centrifuge at 13000 rpm for 10 minutes; Remove supernatant and resuspend pellet in 300µl of Extraction Buffer 2 by pipetting up and down; Add 300µl of Extraction Buffer 3 to fresh Eppendorf tube; Use pipette to carefully layer solution from step 8 onto it. 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; 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); 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); Spin in cooled benchtop centrifuge at 13000 rpm for 10 minutes. Add supernatant to new Eppendorf tube; Repeat step 13. Remove 10µl to run on an agarose gel; 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.