Detection of Histone Modifications in Plant Leaves

1. Crosslinking of plant material

1. Harvest 1-2g of Arabidopsis leaves (6 to 10cm rosette diameter) in a 50-ml plastic tube and fill up to 40mL with crosslinking buffer.
2. Make sure leaves stay immersed, for example by stuffing a tailored filter sponge right above buffer surface. Then place tubes in a desiccator.
3. Vacuum infiltrate for 10min. To each tube add 2.5mL of 2M glycine to stop crosslinking, and invert tubes to warrant equal dispersion of glycine.
4. Vacuum infiltrate for another 5min and discard fluid while harvesting leaves on a sieve.
5. Wash leaves twice with 1L of water in a glass beaker, then dry leaves thoroughly with paper towels.
6. Collect leaves in a fresh plastic tube and freeze in liquid nitrogen. Store leaves at -80° C until chromatin isolation.

2. Isolation of chromatin

1. Leaves were ground with mortar and pistil that were kept in liquid nitrogen until use.
2. Transfer powder to a 50-mL plastic tube and suspend in 30mL extraction buffer #1.
3. Incubate for 15min at 4°C on an overhead shaker.
4. Filtrate suspension through four layers of miracloth into a fresh 50-mL plastic tube.
5. Spin for 20min at 2,800 x g at 4°C.
6. Remove supernatant and suspend pellet with a paint brush in 1mL extraction buffer #2. First add a small volume of buffer #2, then suspend pellet with a paint brush, and then add the rest of buffer.
7. Transfer suspension to a 1.5mL microfuge tube and spin for 10min at 12,000 x g at 4°C.
8. Meanwhile, prepare 2mL-microfuge tubes containing 1.5mL extraction buffer #3.
9. Remove supernatant from the spun tube (step 2.7) and suspend pellet in 300μL of extraction buffer #3. First add a small volume of buffer #3, suspend pellet with a paint brush, and then add the remaining buffer.
10. Carefully pipet the suspended pellet (step 2.9) on top of the 1.5 mL extraction buffer #3 prepared in step 2.8. Make sure the two phases won’t mix. Spin for 1h at 16,000 x g at 4°C.
11. Remove supernatant and suspend chromatin pellet with a paint brush in 300μL of sonication buffer.
12. Sonicate chromatin with a sonotrode, or in an ultrasonic bath, to a DNA size of approximately 400bp. When sonicating, make sure chromatin will not be heated above approximately 30 °C to protect heat-sensitive crosslinks. The duration of sonication needs to be determined experimentally, because it varies significantly between different sonication devices. For guidance, settings for two different devices are provided:

For sonication with a Bandelin brand sonotrode expose chromatin in 0.6mL microfuge tube five times to 20 bursts with settings 25% power, cycle = 50. While doing so, cool sample after every 20^th bursts in an ice bath.

For sonication with a Diagenode Bioruptor ultrasonic bath, fill bath with water and ice and sonicate ten times for 30 seconds in 1.5mL microfuge tubes with setting ‘high’. After every 30 seconds, cool samples for another 30 seconds.

13. Spin sonicated sample for 5min at 16,000g at 4°C to precipitate undissolved material. The supernatant can directly be used for immunoprecipitation. Alternatively, samples can be shock frozen in liquid nitrogen and stored at -80°C until further use.

3. Immunoprecipitation

1. Prepare 2-mL microfuge tubes containing 40μL protein-A agarose and 1.8mL of antibody-binding buffer. Add 200μL of the chromatin preparation (step 2.13).
2. Incubate tubes for 1h on an overhead shaker.
3. Discard protein-A agarose beads and use supernatant for immunoprecipitation.
4. Remove a 40μL-aliquot to determine chromatin concentration (‘input’).
5. Add 30μL protein-A agarose and the amount of modification-specific antibody that is indicated in the below table of specific reagents and equipment to 400μL sonicated chromatin suspension.
6. Incubate for 2.5h or overnight on an overhead shaker at 4°C.
7. Spin down beads for 2min at 440 x g.
8. Wash bead pellet with 900μL of each wash buffer (see the below list). For each buffer, incubate beads in the buffer for 10min on an overhead shaker at 4°C, then spin for 2min at 440 x g, discard supernatant, and add the next buffer.
   1. Low salt wash buffer
   2. High salt wash buffer
   3. LiCl wash buffer
   4. TE wash buffer
9. After the final wash, completely remove any remaining buffer.

4. De-crosslinking of histones and DNA, and purification of precipitated DNA

1. Add 100μL of de-crosslinking buffer to the agarose pellet and the 40μl ‘input’ sample from step 3.4, mix for 5min on a vortex mixer, spin samples, and incubate at 65°C overnight.
2. Purify DNA with a commercial DNA or PCR purification kit.
3. Typically 5μl of a 1:5 dilution of the final column eluate are sufficient to perform conventional locus-specific real-time quantitative RT-PCR (RT-qPCR).

5. Table of buffers

6. Representative results:

Expression of the Arabidopsis thaliana PR2 defense gene encoding a β-1,3-glucanase with antimicrobial activity¹², was induced by benzothiadiazole (BTH), a synthetic analog of the plant hormone salicylic acid³. Figure 1 demonstrates that activation of PR2 expression is associated with an increase in the acetylation of lysine residues on histone H3 and H4 on the PR2 promoter. Since nucleosome density decreases during gene activation¹³, histone acetylation values were normalized for the signal obtained with an antibody to an invariant region of histone 3.

Figure 1 BTH induces histone acetylation on the PR2 promoter in Arabidopsis. Plants were treated with 100μM BTH or a wettable powder control. After 72h, leaves were collected and chromatin was isolated. The signal obtained after precipitation with acetylation-specific antibodies (as indicated in the figure) was normalized to the signal obtained by precipitation with an antibody to an invariant domain of histone H3. Precipitated chromatin was quantified by RT-qPCR. Data are standardized for histone modification levels in the absence of BTH treatment.