实验概要

This protocol describes how chromatin is prepared from Arabidopsis, which can subsequently be used for chromatin immunoprecipitation (ChIP). The exact chromatin concentration should be determined before starting the X-ChIP assay. View our cross-linking chromatin immunoprecipitation (X-ChIP) protocol, which should be used after the chromatin preparation detailed below.

实验原理

Eukaryotic chromatin is a complex of DNA and associated histone proteins that 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 that serves as an interaction platform with specific domains of chromatin-associated proteins. The immunoprecipitation (IP) of cross-linked 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, the method described in used 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).

主要试剂

1. Extraction buffer 1

0.4 M Sucrose
10 mM Tris-HCl, pH 8.0
10 mM MgCl₂
5 mM β-mercaptoethanol
Protease inhibitors

2. Extraction buffer 2

0.25 M Sucrose
10 mM Tris-HCl, pH 8.0
10 mM MgCl₂
1% w/v Triton X-100
5 mM β-mercaptoethanol
Protease inhibitors

3. Extraction buffer 3

1.7 M Sucrose
10 mM Tris-HCl, pH 8.0
2 mM MgCl₂
0.15% w/v Triton X-100
5 mM β-mercaptoethanol
Protease inhibitors

4. Nuclei lysis buffer

50 mM Tris-HCl, pH 8.0
10 mM EDTA
1% w/v SDS
Protease inhibitors

5. Protease inhibitor

100 mM PMSF
One tablet per 10ml of solution of complete mini protease inhibitor cocktail tablets (Roche®)

实验材料

Arabidopsis seeds are stratified for 48 hours in 0.1% Phytablend w/v at 4°C and then sown onto soil. 1.5 g of whole, three to four week-old seedlings, are used per chromatin preparation. It is imperative to avoid contamination with soil as much as possible during harvest.

实验步骤

1. Chromatin cross-linking

    1) Harvest 1.5 g seedlings and place them into a 50 ml tube.

    2) Rinse seedlings twice with 40 ml double distilled water. Remove as much water as possible after second wash.

    3) Add 37 ml 1% w/v 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 ten minutes.

    4) Release vacuum slowly and shake exsiccator slightly to remove air bubbles. Seedlings should appear translucent.

    5) Add 2.5 ml 2 M glycine to quench cross-linking. Draw vacuum for five 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 40 ml of double distilled 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 snap-frozen in liquid nitrogen and stored at -80°C.
2. Chromatin preparation

    1) Pre-cool mortar with liquid nitrogen. Add two small spoons of white quartz sand (Sigma Cat.: 27,473-9) and plant material. Grind plant material to a fine powder.

    2) Use cooled spoon to add powder to 30 ml of extraction buffer 1 stored on ice. Vortex to mix and keep at 4°C until solution is homogenous.

    3) Incubate for 30 min at 4°C with gentle agitation.

    4) Filter extract through into a new, ice-cold 50 ml conical tube. Press to recover extract from solid material.

    5) Repeat step four.

    6) Centrifuge extract at 4000 rpm for 20 minutes at 4°C.

    7) Gently pour off supernatant and resuspend pellet in 1 ml of extraction buffer 2 by pipetting up and down. Transfer solution to Eppendorf tube.

    8) Spin in cooled benchtop centrifuge at 13000 rpm for ten minutes.

    9) Remove supernatant and resuspend pellet in 300 µl of extraction buffer 2 by pipetting up and down.

    10) Add 300 µl of extraction buffer 3 to fresh Eppendorf tube. Use pipette to carefully layer solution from step nine onto it.

    11) Spin in cooled benchtop centrifuge at 13000 rpm for one hour. In meantime, prepare 10 ml nuclei lysis buffer. Put buffers in coldroom.

    12) Remove supernatant and resuspend pellet in 300 to 500 µl of cold nuclei lysis buffer. Resuspend by pipetting up and down and by vortexing. Keep solution cold between vortexing. Incubate for 20 minutes on ice.

    13) Remove 10 µl to run on an agarose gel

    14) Sonicate for ten minutes at 4°C

Make sure that the solution does not foam during sonication, e.g., by cooling the tubes with a mix of 100% w/v ethanol in ice during the sonication step.

    15) Spin in cooled benchtop centrifuge at 13000 rpm for ten minutes. Add supernatant to new Eppendorf tube.

    16) Repeat step 14. Remove 10 µl to run on an agarose gel

     17) Separate aliquots from steps 12 and 15 on 1.5% w/v agarose gel. In the sonicated samples, DNA should be shifted and more intense compared to untreated samples and range between 200-2000 bp, centering around 500 bp.

    18) Please continue with the X-ChIP protocol from stage two (sonication)

Following step 16, the chromatin samples can be “snap-frozen” in liquid nitrogen and stored at -80°C. Repeated freezing/thawing cycles, however, should be avoided.


References:

1. Haring M, Offermann S, Danker T, Horst I, Peterhansel C, Stam M (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods, 3: 11.

2. 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.

3. 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.

4. 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.