chromatin techniques for plant cells

TECHNIQUES FOR MOLECULAR ANALYSIS

Chromatin techniques for plant cells

Chris Bowler1,2,*, Giovanna Benvenuto1, Pierre Laflamme1,†, Diana Molino1, Aline V. Probst3, Muhammad Tariq3,‡ and

Jerzy Paszkowski3,*

1Laboratory of Molecular Plant Biology, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Naples, Italy,2CNRS/ENS FRE2433, Organismes Photosynthetiques et Environnement, Departement de Biologie, Ecole Normale Superieure,

46 Rue d’Ulm, 75230 Paris Cedex 05, France, and3Laboratoire de Genetique Vegetale, Universite de Geneve, 30 Quai Ernest-Ansermet, CH-1211 Geneve 4, Switzerland

Received 8 February 2004; revised 9 June 2004; accepted 18 June 2004.*For correspondence (fax þ33 1 4432 3935; e-mail [email protected]; fax þ41 22 379 3107; e-mail [email protected]).†Present address: Department of Biological Sciences, Brock University, 500 Glenridge Ave., St Catharines, Ont., Canada.‡Present address: Centre for Molecular Biology Heidelberg, Im Neuenheimerfeld 282, D69120 Heidelberg, Germany.

Summary

A large number of recent studies have demonstrated that many important aspects of plant development are

regulated by heritable changes in gene expression that do not involve changes in DNA sequence. Rather, these

regulatory mechanisms involve modifications of chromatin structure that affect the accessibility of target

genes to regulatory factors that can control their expression. The central component of chromatin is the

nucleosome, containing the highly conserved histone proteins that are known to be subject to a wide range of

post-translational modifications, which act as recognition codes for the binding of chromatin-associated

factors. In addition to these histone modifications, DNA methylation can also have a dramatic influence on

gene expression. To accommodate the burgeoning interest of the plant science community in the epigenetic

control of plant development, a series of methods used routinely in our laboratories have been compiled that

can facilitate the characterization of putative chromatin-binding factors at the biochemical, molecular and

cellular levels.

Keywords: chromatin, chromatin immunoprecipitation, histone code, histones, mononucleosomes, nucleo-

somes.

Introduction

The DNA of a eukaryotic cell must be compacted several

thousand-fold in order for it to fit into the nucleus. Cells are

able to package their chromosome complement in such a

way that it remains accessible to regulatory proteins that can

activate or repress specific genes, repair damage, mediate

recombination and replicate the DNA during the cell cycle.

The term chromatin is used to describe packaged DNA. The

basic unit of chromatin is the nucleosome, which contains

approximately 146 bp of DNA wrapped almost twice around

a core histone octamer, composed of two molecules each of

the histones H2A, H2B, H3 and H4 (organized into an H3–H4

tetramer and two H2A–H2B dimers) (Luger et al., 1997). Each

nucleosome has a diameter of around 10 nm, although the

least compacted chromatin appears in the electron

microscope as a 30 nm diameter fibre, which contains the

linker histone H1 that binds to the 40–70 bp of DNA that

separates each nucleosome. These 30 nm fibres appear to

be helical structures containing around six nucleosomes per

turn, an arrangement that compacts the DNA around 40-fold

(Goodrich and Tweedie, 2002).

The 30 nm fibres appear to correspond to euchromatin,

the component of a eukaryotic genome that is actively

transcribed. Heterochromatin, on the contrary, is typically

silent transcriptionally and is characterized by higher order

packaging of the nucleosomes up to several thousand-

fold. Heterochromatin can be reversibly relaxed into

euchromatin to allow activation of gene expression, or

may be permanently inactive, for example, as found in

776 ª 2004 Blackwell Publishing Ltd

The Plant Journal (2004) 39, 776–789 doi: 10.1111/j.1365-313X.2004.02169.x

gene-poor regions of the genome such as peri-centromeric

regions.

The regulation of chromatin structure has a key role in the

epigenetic control of gene expression. A central mechanism

whereby chromatin can be modulated is by recognition by

chromatin remodelling proteins of post-translational mod-

ifications of highly charged and flexible histone tails, which

protrude from the nucleosomes. The core histones can be

modified (sometimes reversibly) by acetylation, methyla-

tion, phosphorylation, ubiquitination or ADP-ribosylation

(Fischle et al., 2003; Jenuwein and Allis, 2001; Strahl and

Allis, 2000). Lysine residues within the amino-terminal tails

are the best characterized sites for these modifications. For

example, actively transcribed genes are predominantly

associated with highly acetylated histones whereas inactive

genes are often characterized by the presence of hypoacet-

ylated histones in the nucleosomes that are associated with

them. Consequently, histone acetylation and deacetylation

reactions are thought to be central for the epigenetic control

of gene expression, by altering the accessibility of the

associated DNA to transcription factors and to chromatin

modifying proteins.

Histone tail modifications are often inter-connected, for

example, deacetylation of lysine 9 of histone H3 is a

prerequisite for its subsequent methylation, which pro-

motes binding of repressor proteins such as heterochroma-

tin protein 1, that maintain heterochromatin in an inactive

state (Rice and Allis, 2001). Examples such as this have lead

to the ‘histone code’ hypothesis, whereby combinatorial

post-translational modifications on different histone tails act

as recognition sequences for binding of different chromatin-

associated factors (Fischle et al., 2003; Jenuwein and Allis,

2001; Strahl and Allis, 2000). Such dynamic and reversible

modifications considerably extend the information potential

of DNA and provide heritable (although often reversible)

mechanisms for the epigenetic control of gene expression

during development.

In addition to histone modifications, the DNA itself can be

modified, most commonly by cytosine methylation, which

often characterizes inactive genes (Martienssen and Colot,

2001; Ng and Bird, 1999). Furthermore, RNA has also been

found associated with heterochromatin (Maison et al.,

2002).

Numerous chromatin-associated factors have now been

characterized, many of which mediate post-translational

histone tail modifications or DNA methylation. Major fam-

ilies include histone acetyltransferases (HATs), histone

deacetylases (HDACs), SET-domain containing proteins

(that typically methylate histones), and DNA methyltransf-

erases. In addition, SWI/SNF complexes modulate chroma-

tin compaction by ATP-dependent repositioning of

nucleosomes. Members from each of these families have

now been characterized in plants (Fransz and de Jong, 2002;

Goodrich and Tweedie, 2002; Wagner, 2003).

In addition, a range of factors that recognize histone and

DNA modifications are also known. Well known families

include bromodomain- and chromodomain-containing pro-

teins, that recognize specific histone modifications and

methylcytosine-binding domain proteins. A comprehensive

list of chromatin-related factors identified in plants is

available at http://www.chromdb.org.

Studies of chromatin-level epigenetic effects are now an

intense area of plant research. Most notably, they have been

inferred to be involved in the regulation of flowering time,

vernalization and photomorphogenesis (He et al., 2003;

Schafer and Bowler, 2002; Sung and Amasino, 2004). For

this reason, we have compiled a series of protocols routinely

used in our laboratories that can be of use for the investi-

gation of such mechanisms. Obviously, other equally useful

techniques have been developed or optimized in other

laboratories, so this article should be considered as a useful

starting point rather than an all-inclusive reference guide.

Some of the protocols we detail are in fact rather old and

have been derived from experiments of several decades

ago, in which basic studies were made of histone modifica-

tions and histone-binding proteins. For example, in vitro

experiments that study binding of proteins or complexes to

purified mononucleosomes can now be very useful for

characterizing putative chromatin-binding proteins, as can

separation of modified histones on acetic acid-urea-triton

(AUT) polyacrylamide gels. In addition, we detail a protocol

for chromatin immunoprecipitation (ChIP), a technique that

can be used to identify genes associated with particular

modified histones or chromatin-interacting proteins, and a

protocol for fluorescent in situ hybridization (FISH), which

can be used to reveal subnuclear sites in which modified

histones or associated factors are localized. All of these

techniques have now been greatly potentiated by the

availability of a large range of antibodies (many of which

are available commercially) that can reveal specific histone

tail modifications.

Methods

Isolation of mononucleosomes

Plant nuclei can be easily obtained from cauliflower florets

and used as a source of nuclear material such as nucleo-

somes. Furthermore, these nucleosomes can be depleted

of their H1 linker histones by limited nuclease digestion to

generate mononucleosomes which can then be fixed to a

chromatographic support and used for in vitro binding

studies as in Benvenuto et al. (2002). The protocol we

present here for isolating plant nuclei was adapted from

Foster et al. (1992), while the protocol for isolating mono-

nucleosomes was adapted from Schnitzler (2000). As a

close relative of Arabidopsis, cauliflower provides a con-

venient source of mononucleosomes that can be utilized

Chromatin techniques for plant cells 777

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 39, 776–789

for studying chromatin-interacting proteins from Arabid-

opsis. If, on the contrary, Arabidopsis-derived mononucle-

osomes are required, the protocol described below should

be easily adaptable provided sufficient starting material is

available.

Isolation of nuclei from plants

Equipment and reagents. Note: All operations must be

carried out at 4�C, either in a cold room or on ice. All solu-

tions should be kept at 4�C, unless stated otherwise.

Cauliflower florets from a local market

Miracloth

Refrigerated high-speed centrifuge (e.g. Beckman Avanti

J-25; Beckman Coulter, Inc., Fullerton, CA, USA) with a fixed

angle rotor (e.g. Beckman JA 14, JA 25.50; Beckman)

Phenylmethylsulphonyl fluoride (PMSF) stock solution:

prepare a 100 mM stock solution in isopropanol and store in

a dark bottle at room temperature

Glycerol, sterile

Nuclei grinding buffer (NGB): 1 M hexylene glycol, 10 mM

PIPES/KOH (pH 7.0), 10 mM MgCl2, 0.2% Triton X-100, 5 mM

2-mercaptoethanol*, 0.8 mM PMSF* (*these components

should be added immediately prior to use)

Nuclei wash buffer (NWB): 0.5 M hexylene glycol, 10 mM

PIPES/KOH (pH 7.0), 10 mM MgCl2, 0.2% Triton X-100, 5 mM

2-mercaptoethanol*, 0.8 mM PMSF* (*these components

should be added immediately prior to use)

Experimental protocol. 1. Grind 200 g of cauliflower florets

in 800 ml of NGB in a commercial blender fitted with a

customized razor blade holder.

2. Filter the mixture through four layers of Miracloth and

centrifuge filtrate at 2000 g for 10 min at 4�C. Centrifuge

without brake.

3. Decant supernatant and gently resuspend the pellet in

40 ml of NWB and centrifuge at 3000 g for 10 min at 4�C.

Repeat this washing procedure two more times.

4. Gently resuspend the nuclei, following the last wash

step, with two volumes of NWB (with respect to the pellet

volume). Add an equal volume of sterile glycerol and mix

gently to obtain a homogeneous resuspension. Divide into

equal aliquots and store nuclei at )20�C until further

processing is required. If necessary, nuclei can be further

purified on a Percoll gradient according to Slatter et al.

(1991), although Percoll-purified nuclei are not necessary for

nucleosome isolation.

Isolation of H1-depleted oligonucleosomes from plant nuclei

Equipment and reagents. Note: All operations must be

carried out at 4�C, either in a cold room or on ice. All solu-

tions should be kept at 4�C, unless stated otherwise.

Washed plant nuclei (see above)

Refrigerated high-speed centrifuge (e.g. Beckman Avanti

J-25) with a fixed angle rotor (e.g. Beckman JA 25.50)

Refrigerated ultracentrifuge with a swing-out rotor (e.g.

Beckman SW28; Beckman)

UV spectrophotometer

Agarose- and SDS-polyacrylamide (vertical, mini slab)

(e.g. Bio-Rad Mini-Protean II; Bio-Rad Laboratories, Hercu-

les, CA, USA)-gel electrophoresis apparatus

Gradient maker, capable of holding 20 ml per side with a

side outlet, to prepare linear concentration gradients with a

volume of 32 ml, with a stirring mechanism

Thinwall polyallomer tubes (2.5 · 8.9 cm)

21-G needles

Tubing (1/16¢¢ ID · 1/8¢¢ OD) (e.g. Tygon-Schlauch ST;

Saint-Gobain Performance Plastic Corporation, Akron, OH,

USA)

Peristaltic pump

Potter homogenizer

Dialysis membrane, 6–8 kDa molecular weight cut off

(MWCO)

Proteinase K stock solution: prepare a 10 mg ml)1 stock

solution in water and store in aliquots at )20�CCentricon-10 concentrators (Amicon, INC, Beverly, MA,

USA)

Slide-a-Lyzer dialysis cassettes (7 kDa MWCO) (Pierce

Biotechnology, Inc., Rockford, IL, USA)

Phenylmethylsulphonyl fluoride stock solution: prepare a

100 mM stock solution in isopropanol and store in a dark

bottle at room temperature

Pepstatin A stock solution: prepare a 1 mM stock solution

in methanol and store at )20�CLeupeptin stock solution: prepare a 1 mM stock solution in

water and store at )20�CLow salt buffer (LSB): 20 mM HEPES, pH 7.5, 0.1 M NaCl,

1 mM EDTA, 1 mM b-mercaptoethanol (b-ME)*, 0.5 mM

PMSF* (*these components should be added immediately

prior to use)

Medium salt buffer (MSB): 20 mM HEPES, pH 7.5, 0.4 M

NaCl, 1 mM EDTA, 5% (v/v) glycerol, 1 mM b-ME*, 0.5 mM

PMSF*, 1 lM pepstatin A*, 1 lM leupeptin* (*these compo-

nents should be added immediately prior to use)

High salt buffer (HSB): 20 mM HEPES, pH 7.5, 0.65 M NaCl,

1 mM EDTA, 0.34 M sucrose, 0.5 mM PMSF* (*this compo-

nent should be added immediately prior to use)

2 M NaCl

0.1 M CaCl20.5 M ethylene glycol-bis(2-aminoethyl)-N,N,N¢,N¢-tetra-

acetic acid (EGTA), pH 8.0

High salt buffer, without sucrose, containing 10% (v/v) and

40% (v/v) glycerol (HSB/glycerol), respectively, for glycerol

gradients

0.5% (w/v) SDS

25 U ll)1 micrococcal nuclease (purchased as 500 U of

solid from Sigma, St Louis, MO, USA): resuspend in 10 ll of

100 mM Tris–HCl, pH 8.0, 0.1 mM CaCl2 and once resuspend-

ed add 10 ll of sterile glycerol and mix by pipetting.

778 Chris Bowler et al.


Micrococcal nuclease solution can be stored at )20�C in this

fashion and is stable for approximately 1 year (Note: Differ-

ent suppliers define micrococcal nuclease units differently

and therefore if a supplier other than Sigma is used, one

must be sure that the same unit definition is used, or

determine the proper conversion ratio to Sigma units)

Dialysis buffer: 20 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM

b-ME*, 0.5 mM PMSF* (*these components should be added

immediately prior to use)

CNBr-activated Sepharose 4B (Amersham Pharmacia

Biotech, Piscataway, NJ, USA)

Experimental protocol. 1. Gently mix an aliquot of plant

nuclei suspension (prepared as above and stored at )20�C)

to obtain a homogeneous mixture.

2. Using a pipette, resuspend approximately 2 ml of plant

nuclei suspension with 40 ml MSB, divide equally among

two centrifuge tubes and centrifuge using a fixed angle rotor

at 10 000 g for 10 min at 4�C.

3. Resuspend the nuclear pellets with 4 vol of HSB (with

respect to the pellet volume), transfer to a Potter homoge-

nizer, homogenize with 40–50 strokes and centrifuge at

10 000 g for 20 min at 4�C.

4. Remove the gelatinous supernatant and place in

dialysis tubing (6–8 kDa MWCO), previously wetted and

rinsed with distilled water followed by LSB. Clamp the

dialysis tubing securely and dialyse overnight against 4 l of

cold LSB, at 4�C with gentle stirring.

5. Collect the dialysate and gently press along the sides of

the dialysis tubing to mix and collect any precipitated

oligonucleosomes.

6. Add 0.1 M CaCl2 to 3 mM (final) and warm the sample at

37�C for 5 min.

7. Add micrococcal nuclease (25 U ll)1) to the warmed

sample of oligonucleosomes to a final concentration of

0.01 U ll)1 and incubate at 37�C for 8 min.

8. Add 0.1 vol of 0.5 mM EGTA to the reaction mix and chill

on ice. This will terminate the nuclease digestion.

9. Add 2 M NaCl to the chilled reaction mixture, while

gently vortexing, to a final NaCl concentration of 0.6 M. Keep

on ice.

10. In a cold room, prepare 32 ml linear 10–40% glycerol

gradients using the respective HSB/glycerol buffers, in thin-

wall polyallomer centrifuge tubes (2.5 · 8.9 cm) with the use

of the peristaltic pump and gradient maker. The pump must

run at a flow rate of about 1.5 ml min)1 to avoid introducing

any air bubbles in the gradient. Place the outlet of the tubing

along the inside edge at the bottom of the tube slowly

moving the tubing upwards as the gradient carefully fills the

tube. The lighter glycerol-containing buffer is at the top.

11. Remove any precipitated protein and carefully apply

2 ml of the quenched digestion reaction on top of each

gradient and centrifuge in a swing-out rotor (e.g. Beckman-

type SW28) at 45 000 g for 20 h at 4�C.

12. Harvest the gradients by piercing the bottom edge of

the tubes with a 21-G needle attached to tubing which is

attached to a peristaltic pump, making sure the pump is not

operating during this time. Holding the needle with the

bevelled edge up, carefully pierce the tube and place the tip

of the needle near the bottom of the gradient. Cover the top

of the tube with Parafilm and gently apply pressure to start

the flow. Turn on the pump with a flow rate of about

1.5 ml min)1 avoiding any air bubble formation and collect

1 ml fractions. The fractions can be stored at 4�C while they

are being analysed.

13. Determine the DNA concentration in the fractions by

diluting an aliquot 10–40-fold in 2 M NaCl and measuring the

absorbance at 260 nm.

14. Digest aliquots of fractions (‡0.5 lg) with proteinase K

(0.5 mg ml)1) in 0.5% SDS for 60 min at 50�C.

15. The proteinase K-digested reaction products can be

analysed on 1.5% agarose/TBE gels and stained with

0.1 lg ml)1 ethidium bromide (Figure 1a). A smear of over-

digested DNA along with a band of DNA at approximately

150 bp (mononucleosomes) and larger bands equal to

approximately 150 bp with multiples of approximately

200 bp (di-, tri-, tetranucleosomes, etc.) should be observed,

whereby the smaller fragments will be located in fractions

isolated towards the top of the gradient (Figure 1a). Frac-

tions containing DNA which is smaller than 150 bp has been

overdigested and should not be considered for further

experimentation.

16. Analyse 5–20 ll of the glycerol gradient fractions on

15% SDS-PAGE gels and stain with Coomassie brilliant blue

for the presence of all four core histones (H2A, H2B, H3 and

Figure 1. Qualitative analysis of samples from a plant mononucleosome

preparation.

(a) Glycerol gradient fractions (from 18 to 26) loaded on a 1.5% agarose/TBE

gel after digestion with proteinase K. M is 100 bp ladder.

(b) Fractions 20 (polynucleosomes) to 26 (mononucleosomes) loaded on 15%

SDS-PAGE and stained with Coomassie brilliant blue.



H4) and for the absence of H1 in the mono- and dinucleo-

some containing fractions (Figure 1b).

17. Pool the H1-depleted mono- and dinucleosome-

containing fractions and concentrate to approximately

1 mg ml)1 using Centricon-10 concentrators according to

the manufacturer’s instructions.

18. Dialyse the concentrated samples against 100 vol of

dialysis buffer at 4�C for a minimum of 4 h using Slide-a-

Lyzer dialysis cassettes (7 kDa MWCO).

19. Dialysed mono- and dinucleosomes can be stored at

4�C for several weeks or frozen in liquid nitrogen and stored

at )80�C for up to 2 years.

An effective way of investigating protein–nucleosome

interactions is by immobilizing nucleosomes to CNBr-acti-

vated Sepharose 4B, as was carried out in Benvenuto et al.

(2002). Approximately 1 mg of a mixture of mono- and

di-nucleosomes can be immobilized to approximately 500 ll

of CNBr-activated Sepharose 4B according to the manufac-

turer’s instructions. Coupling of the ligand to the resin and

blocking of the remaining active groups is carried out

overnight at 4�C. The blocking buffer used is 200 mM

glycine, pH 8.0. The resin can be stored as a 50% slurry in

phosphate-buffered saline (PBS) at 4�C until ready for use.

Acetic acid-urea-triton polyacrylamide gels for analysing

histone variants

Acetic acid-urea-triton polyacrylamide gels allow for the

separation of modified histone variants (derived from dif-

ferent post-translational modifications) and histone H2A,

H2B, and H3 subtypes (derived from different gene prod-

ucts) based on charge and mass. The protocol we present

here is a modification of a procedure used by Boulikas

(1985) to resolve histone variants from calf thymus nuclei.

The addition of a non-ionic detergent such as Triton X-100

to the gels causes a differential reduction in the elec-

trophoretic mobility of the different histones as a result of

the formation of mixed micelles between the detergent and

the hydrophobic moieties of histones (Zweidler, 1978).

There are several early reports on the use of AUT gels,

either in one and/or two dimensions, to resolve histone

variants from plant sources (Moehs et al., 1988; Spiker,

1982; Spiker and Ley, 1976; Waterborg et al., 1987). The

advantage of using AUT gels is that they provide a

superior separation of histone variants with respect to

standard SDS-PAGE gels (see Figure 2), and when com-

bined with immunoblotting can allow the precise identifi-

cation of different bands corresponding to specific histone

modifications.

Equipment and reagents

Note: This gel electrophoresis system does not require a

stacking gel. Gel methods are for 60 · 100 · 0.75 mm gels.

All reagents must be of highest quality.

Vertical mini slab polyacrylamide gel electrophoresis

apparatus (e.g. Bio-Rad Mini-Protean II system)

Power supply

Vacuum pump or vacuum line

Stock acrylamide: 30% (w/v) acrylamide, N,N¢-methylene-

bis-acrylamide (BIS) (37.5:1), electrophoresis grade

Water, high quality (milliQ)

Glacial acetic acid (17.4 M)

Methanol

Urea, high quality

10% (w/v) Triton X-100 solution

10% (w/v) ammonium persulphate (APS) solution in water

N,N,N¢,N¢-tetramethylenediamine (TEMED)

b-mercaptoethanol

Sample buffer (1X): 0.9 M acetic acid, 20% (v/v) glycerol,

6 M urea, 0.05% (w/v) pyronin Y, 0.7 M b-ME* (*this compo-

nent should be added immediately prior to use)

Running buffer: 0.9 M acetic acid

Silver Stain Plus (Bio-Rad)

Experimental protocol

Preparation of gel. 1. Prepare the following resolving gel

(for one 15% acrylamide gel, 0.75 mm thick):

Component For 15% acrylamide gel (0.75 mm)30% Acrylamide stock 5.0 mlGlacial acetic acid (17.4 M) 517 ll (¼0.9 M final)10% Triton X-100 370 ll (¼0.37% final)Urea 3.6 g (¼6 M final)Water (milliQ) to 10 ml10% APS 125 llTEMED 50 ll

2. Dissolve the indicated quantity of urea in the stated

volume of acrylamide. While gently agitating, add the acetic

acid.

3. When the urea is completely dissolved, degas the

solution for 10 min under vacuum.

Figure 2. Differential separation of histones on SDS-PAGE and AUT gels.

Nucleosome fractions were loaded on a 15% SDS-PAGE gel and stained with

Coomassie brilliant blue (1) and on an AUT gel which was subsequently

stained with Silver Stain Plus (2).



4. Add the Triton X-100 and swirl to mix, add the water to a

final volume of 10 ml and degas for 5 min.

5. Add the APS and degas for a further 5 min.

6. Add the TEMED, gently mix the solution avoiding air

bubbles and carefully pipette the gel solution between the

assembled glass plates with the well-forming comb parti-

ally inserted. After having completely filled the space

between the glass plates, gently, but firmly, insert the

comb to its limit in the gel solution, avoiding trapping air

bubbles underneath the comb. Allow to polymerize at

room temperature for a minimum of 1 h. Store gel(s) well-

wrapped with a moist paper towel in plastic film overnight

at room temperature.

7. Remove gels from wrapping and pre-electrophorese in

running buffer, with reversed polarity, at room temperature

at 130 V until the current no longer drops (4–5 h).

Sample preparation. 1. Prepare samples to be electro-

phoresed by resuspending lyophilized histones in 5 ll of 1X

sample buffer. However, if a fairly concentrated histone

preparation is used, as in the mononucleosome fractions, 1–

2 ll of sample can be added to 9 ll of 1X sample buffer. The

quantity of histones to be electrophoresed should be in the

order of 5 lg in order to view by Coomassie staining or 1 lg

to view by silver staining.

2. Replace pre-electrophoresis buffer with fresh running

buffer.

3. Load 5 ll (or 10 ll maximum, if using a concentrated

sample, as stated above) of each sample to be analysed.

Electrophoresis conditions. 1. Run gel(s) with reversed

polarity at room temperature, with agitation, at 150 V for

the initial 20 min and then decrease voltage to 100 V

and let electrophoresis proceed overnight (approximately

16–18 h).

2. After electrophoresis, gels can be visualized by either

Coomassie brilliant blue or silver staining (see step 3) or by

electrophoretic transfer and immunoblotting, as described

by Delcuve and Davie (1992). Immunoblotting using anti-

bodies that recognize specific histone modifications can

allow specific bands to be identified.

3. Stain gel(s) for 1 h with Coomassie brilliant blue or

silver staining according to the manufacturer’s instructions.

4. Resolution can be further improved by using a larger

gel electrophoresis apparatus.

Chromatin immunoprecipitation

Chromatin immunoprecipitation combines immunoprecipi-

tation of chromatin fragments and polymerase chain reac-

tion to map sites of protein–DNA interaction in vivo. The

ChIP technique has been successfully used in Drosophila

and yeast to study histone modifications of eu/heterochro-

matin and localization of regulatory chromatin factors. The

method relies on the rapid cross-linking of protein/DNA

complexes within the nucleus of living cells, followed by

chromatin isolation, its random shearing and immunopre-

cipitation with antibodies directed towards proteins of

interest. The amount of co-immunoprecipitated DNA is

analysed by PCR. The relative enrichment or depletion of a

particular DNA fragment in the immunoprecipitated fraction

reflects its in vivo association with the examined protein

(Hecht and Grunstein, 1999; Orlando et al., 1997).

In plants, ChIP was first used to map the subnuclear

distribution of linker histone variants in Arabidopsis thali-

ana (Ascenzi and Gantt, 1999). Further studies in plants

exploiting ChIP investigated the chromatin structure of the

pea plastocyanin gene (PetE) by determination of the

acetylation states of histones H3 and H4 and the nuclease

accessibility of this gene (Chua et al., 2001). The protocols

used for Arabidopsis as well as for pea were essentially

adopted, with slight modifications, from either Drosophila

(Orlando et al., 1997) or yeast (Hecht and Grunstein, 1999).

The major difficulty encountered in plants was the limiting

amount of material, as early protocols (Ascenzi and Gantt,

1999; Chua et al., 2001) required 100 g of Arabidopsis

tissue for a single ChIP experiment. This problem was

circumvented by recent studies that successfully combined

various protocols (Gendrel et al., 2002; Johnson et al.,

2002; Tariq et al., 2003). These methods used ChIP with

antibodies directed against modified histone tails and

allowed downscaling to 1–1.5 g of Arabidopsis. Here we

describe a ChIP protocol adapted from Gendrel et al. (2002)

with slight modifications.


Miracloth

Formaldehyde (Fluka, Buchs SG, Switzerland)

Glycine 2 M

Double distilled autoclaved water

Vacuum chamber

Vortex

Rotator

Liquid nitrogen

Falcon tubes for 50 and 15 ml

Refrigerated centrifuge

Branson Sonifier 250 (Branson-sonifier, Frankfurt, Ger-

many)

Sheared salmon sperm DNA/protein A agarose mix

(Upstate Biotechnology, Lake Placid, NY, USA)

1 M Tris–HCl, pH 6.5

5 M NaCl

0.5 M EDTA

Heating block at 65�CProteinase K (14 mg ml)1; Boehringer Mannheim GmbH,

Mannheim, Germany)

Novagen pellet paint (CN Bioscience, Darmstadt, Ger-

many)



RNase A (DNase-free)

Phenol:chloroform:isoamylalcohol, 25:24:1

Chloroform

Ethanol

Extraction buffer 1 for 100 ml

0.4 M Sucrose 20 ml of 2 M stock10 mM Tris–HCl, pH 8.0 1 ml of 1 M stock5 mM b-ME 35 ll of 14.3 M stock0.1 mM PMSF 50 ll of 0.2 M stockProtease inhibitors Protease inhibitors should be added

immediately before use becausethey are quickly degraded

Aprotinin 100 ll of 1 mg ml)1 stockPepstatin A 100 ll of 1 mg ml)1

Leupeptin 100 ll of 1 mg ml)1

Antipain 100 ll of 1 mg ml)1

TPCK 100 ll of 3 mg ml)1

Benzamidine 100 ll of 1 mg ml)1


0.25 M Sucrose 1.25 ml of 2 M stock10 mM Tris–HCl, pH 8.0 100 ll of 1 M stock10 mM MgCl2 100 ll of 1 M stock1% Triton X-100 0.5 ml of 20% stock5 mM b-ME 3.5 ll of 14.4 M stock0.1 mM PMSF 5 ll of 0.2 M stockProtease inhibitors 10 ll of each as in buffer 1


1.7 M Sucrose 8.2 ml of 2 M stock10 mM Tris–HCl, pH 8.0 100 ll of 1 M stock0.15% Triton X-100 75 ll of 20% stock2 mM MgCl2 20 ll of 1 M stock5 mM BME 3.5 ll of 14.3 M stock0.1 mM PMSF 5 ll of 0.2 M stockProtease inhibitors as in buffer 1 and 2

Nuclei lysis buffer for 5 ml50 mM Tris–HCl, pH 8.0 0.25 ml of 1 M

10 mM EDTA 24 ll of 0.5 M

1% SDS 0.25 ml of 20%PMSF and protease inhibitors as in buffer 1

ChIP dilution buffer for 10 ml

1.1% Triton X-100 550 ll of 20%1.2 mM EDTA 24 ll of 0.5 M

16.7 mM Tris–HCl, pH 8.0 167 ll of 1 M

167 mM NaCl 334 ll of 5 M

PMSF and protease inhibitors as in buffer 1

Elution buffer for 20 ml

1% SDS 1 ml of 20%0.1 M NaHCO3 0.168 g

Low salt wash buffer for 50 ml

150 mM NaCl 1.5 ml of 5 M

0.1% SDS 0.25 ml of 20%1% Triton X-100 2.5 ml of 20%2 mM EDTA 200 ll of 0.5 M

20 mM Tris–HCl, pH 8.0 1 ml of 1 M

High salt wash buffer for 50 ml

500 mM NaCl 5 ml of 5 M

0.1% SDS 0.25 ml of 20%1% Triton X-100 2.5 ml of 20%2 mM EDTA 200 ll of 0.5 M

20 mM Tris–HCl, pH 8.0 1 ml 1 M

LiCl wash buffer for 50 ml

0.25 M LiCl 3.125 ml of 4 M

1% NP-40 2.5 ml of 20%1% sodium deoxycholate 0.5 g1 mM EDTA 100 ll of 0.5 M

10 mM Tris–HCl, pH 8.0 0.5 ml of 1 M

TE buffer for 50 ml

10 mM Tris–HCl, pH 8.0 5 ml of 1 M

1 mM EDTA 100 ll of 0.5 M


Note: All steps must be carried out at 4�C, unless stated

otherwise.

Preparation of plant material. 1. Sow Arabidopsis seeds on

soil covered with miracloth. Miracloth is needed to avoid soil

contamination during the harvest.

2. After 3–4 weeks (preferably 3 weeks), harvest 1.5 g of

seedlings in a 50 ml Falcon tube.

3. Rinse seedlings twice with 40 ml of double distilled (dd)

autoclaved water by gently shaking the tube (room tem-

perature).

Formaldehyde cross-linking. 4. After thoroughly removing

the water, submerge seedlings in 37 ml of 1% formaldehyde

in cross-linking solution, extraction buffer 1 (room tem-

perature) and vacuum infiltrate for 10 min.

5. Stop the cross-linking by addition of glycine to a final

concentration of 0.125 M (2.5 ml of 2 M glycine in 37 ml of

extraction buffer 1) and application of vacuum for additional

5 min. At this stage, seedlings should appear translucent.

6. Rinse seedlings twice with 40 ml cold dd autoclaved

water.

7. Remove water as thoroughly as possible by placing

seedlings on a paper towel before transferring to a new

Falcon tube. At this stage cross-linked material can be either



frozen in liquid nitrogen and stored at )80�C or processed

further for chromatin isolation at step 8.

Isolation and sonication of chromatin. 8. Pre-cool mortar

and pestle by filling with liquid nitrogen before placing

seedlings in the rest of the liquid nitrogen and grinding them

to a fine powder.

9. Resuspend the powder in 30 ml extraction buffer 1 (4�C)

in a 50 ml Falcon tube.

10. Filter the solution through four layers of miracloth into

a new 50 ml Falcon tube.

11. Spin the filtered solution for 20 min at 2880 g at 4�C.

12. Gently remove supernatant and resuspend the pellet

in 1 ml of extraction buffer 2.

13. Transfer the solution to 1.5 ml Eppendorf tube.

14. Centrifuge at 12 000 g for 10 min at 4�C.

15. Remove the supernatant and resuspend pellet in

300 ll of extraction buffer 3.

16. Overlay the resuspended pellet onto 300 ll of extrac-

tion buffer 3 in a fresh Eppendorf tube.

17. Spin for 1 h at 16 000 g at 4�C.

18. Remove the supernatant and resuspend the chromatin

pellet in 300 ll of nuclei lysis buffer by vortexing and

pipetting up and down (keep solution at 4�C). At this stage

save a 1–2 ll aliquot for later examination. Aliquots taken at

this step as well as at step 21 (see below) must be treated as

described in ‘elution and reverse cross-linking’ procedure

(see below) before analysing on the gel.

19. Once resuspended, sonicate the chromatin solution

for 10 sec, four times on 5% power (setting 3) using

a Branson Sonifier 250, to shear DNA to approximately

0.5–2 kb DNA fragments. The sonicated chromatin solution

can be frozen at )80�C or processed to step 20 for immu-

noprecipitation.

Immunoprecipitation. 20. Spin the sonicated chromatin

suspension for 5 min at 4�C (16 000 g) to pellet debris.

21. Remove supernatant to a new tube. Use an aliquot of

1–2 ll to check sonication efficiency by reverse cross-linking

(follow from step 35) and electrophoretic determination of

the average size of DNA fragments as compared with the

aliquot from step 18 (Figure 3).

22. Split the 200 ll into two tubes with 100 ll each.

23. Add 900 ll of ChIP dilution buffer to each tube. This

dilutes the SDS to 0.1% SDS.

24. Pre-clear each chromatin sample with 40 ll of salmon

sperm-sheared DNA/protein A agarose beads for at least 1 h

at 4�C with gentle agitation. Prior to use, the beads should be

rinsed three times and resuspended in ChIP dilution buffer.

25. Spin the chromatin/beads solution at 4�C for 2 min at

16 000 g.

26. Combine the two 1 ml supernatants into a 15 ml

Falcon tube, and then split the 2 ml into three Eppendorf

tubes (666 ll each).

27. Add 4 ll of antibody, for example, anti-H3K4Me or

anti-H3K9Me to two of the three tubes (anti-histone H3

di-methylated at K4 and K9 from Upstate Biotechnology;

concentrations may vary between batches of antibodies and

the titre should be determined empirically). The third tube

without antibody should be used as mock/negative control.

28. Incubate overnight at 4�C with gentle agitation.

29. Collect immunoprecipitate with 40 ll of protein A

agarose beads (rinsed in ChIP dilution buffer) for at least 1 h

at 4�C with gentle agitation.

30. Prepare fresh elution buffer (1% SDS, 0.1 M NaHCO3).

31. Pellet beads by centrifugation (2 min, 16 000 g) and

wash them with gentle agitation for 10 min at 4�C each

wash, using 1 ml of buffer per wash followed by pelleting

the beads. Apply the following washes in the order listed

below:

(a) Low salt wash buffer (one wash).

(b) High salt wash buffer (one wash).

(c) LiCl wash buffer (one wash).

(d) TE buffer (two washes).

After the final wash, remove TE thoroughly.

Elution and reverse cross-linking of chromatin. 32. Release

bead-bound complexes by adding 250 ll of elution buffer

(made fresh at step 30) to the pelleted beads.

33. Vortex briefly to mix and incubate at 65�C for 15 min

with gentle agitation.

Figure 3. Analysis of purified DNA from plantlets cross-linked (X-linked) for

10 min to check sonication efficiency. Lanes 1 and 2: Aliquots of cross-linked

chromatin taken at step 18 (before sonication) were reverse cross-linked and

purified DNA was analysed on an agarose gel. DNA before sonication is

visible as a high molecular weight fraction (white arrow). Lanes 3 and 4:

Purified DNA from aliquots taken at step 21 (after sonication). The smear

between 0.5 and 3 kb shows the sheared DNA after sonication. M, marker kb

ladder; ), before sonication; þ, after sonication.



34. Spin beads and carefully transfer the supernatant

(eluate) to a fresh tube and repeat elution of beads. Combine

the two eluates.

35. Add 20 ll 5 M NaCl to the eluate to reverse the cross-

links by an overnight incubation at 65�C.

36. Add 10 ll of 0.5 M EDTA, 20 ll 1 M Tris–HCl, pH 6.5,

and 1.5 ll of 14 mg ml)1 proteinase K to the eluate and

incubate for 1 h at 45�C.

37. Extract DNA by phenol/chloroform (equal volume) and

precipitate with ethanol in the presence of Novagen pellet

paint (CN Bioscience). Wash pellets with 70% ethanol.

38. Resuspend the pellet in 40–50 ll of TE supplemented

with 10 lg ml)1 RNase A (Roche Pharmaceuticals, Nutley,

NJ, USA). The immunoprecipitated and purified DNA is then

used in PCR reactions to amplify examined target sequence

in relation to a reference sequence (internal control), prefer-

ably in a multiplex PCR in order to quantify the enrichment or

depletion of target(s) as compared with the reference and

mock control. If multiplex PCR does not work because of

different melting temperatures for primers, the DNA precipi-

tated in ChIP can be normalized by using a primer

pair (Johnson et al., 2002) specific for the 5¢ end of

ACTIN2/7 (a constitutively expressed gene assumed to be

euchromatic).

39. Use 0.5 ll for a 25 ll PCR reaction. The amount of

recovered templates may vary between experiments

depending upon efficiency of immunoprecipitation, thus

PCR conditions, for example, number of cycles may need

adaptation. The real time PCR quantification may be used as

an alternative.

ChIP on microarray (ChIP on chip). Studies in yeast and

Drosophila have combined ChIP and DNA microarrays

commonly called ChIP on chip (Iyer et al., 2001; Nal et al.,

2001; Ng et al., 2002). The possibility to apply the immuno-

precipitated DNA to a microarray allows the determination

of genome-wide patterns of chromatin protein associations

and/or to examine the global distribution of particular his-

tone modifications. After standard ChIP protocol, the puri-

fied DNA is amplified linearly by ligation-mediated PCR and

labelled with fluorophores (Cy3 or Cy5) (Iyer et al., 2001).

ChIP on chip has been successfully used in yeast and animal

systems (Iyer et al., 2001; Nal et al., 2001; Ng et al., 2002),

and recently it has also been described in plants to identify

specific regions of tobacco genes associated with acetylated

histones (Chua et al., 2004).

Fluorescence in situ hybridization

Fluorescence in situ hybridization allows the specific detec-

tion of DNA sequences within cytological preparations.

Hybridizations to metaphase or (with higher resolution)

pachytene chromosomes can be used to map multicopy and

unique sequences relative to euchromatin and heterochro-

matin regions (Fransz et al., 2000). FISH on interphase

nuclear spreads has been employed to study the organiza-

tion of heterochromatin and euchromatin in the interphase

nucleus and to examine the effects of mutations affecting

epigenetic inheritance and/or chromatin structure (Probst

et al., 2003; Soppe et al., 2002).

Plants with larger genomes and/or large numbers of

chromosomal repeats (wheat, barley, rye) have been pref-

erentially used in DNA FISH experiments (Nkongolo et al.,

1993; Pedersen and Linde-Laursen, 1994). Technical

improvements over the years have allowed studies of plants

with smaller genomes such as the model plant A. thaliana

with a genome size of only 130 Mb and a nuclear size of only

approximately 5 lm (Bauwens et al., 1991; Fransz et al.,

1998; Maluszynska and Heslop-Harrison, 1991; Murata and

Motoyoshi, 1995). Because its genome is fully sequenced

and many genetic resources are available, we exclusively

focus this part of the methodology on Arabidopsis.

Nuclei of diploid Arabidopsis contain 10 chromosomes,

and each of them displays a conspicuous heterochromatin

region, called chromocenter, marking the position of cen-

tromeric and peri-centromeric DNA (Figure 4a–d). In addi-

tion, nucleolus organizing regions present on chromosomes

2 and 4 form the clearly distinct nucleolus (Figure 4b). The

centromeric DNA consists of the core repeats (180 bp

repeats, Figure 4a), flanked by peri-centric heterochromatin

containing remnants of transposons and other dispersed

repeats (Haupt et al., 2001). Euchromatic loops of one

chromosome emanate from its defined chromocenter and

mark a chromosome territory (Fransz et al., 2002).

On interphase spreads, the DNA-FISH technique has so far

been mainly applied to analyse the structural organization of

repetitive sequences associated with chromocenters. How-

ever, recent reports prove the feasibility to use selected BAC

clones (Fransz et al., 2002) or a combination of BACs

representing a chromosome to visualize whole chromoso-

mal territories (Lysak et al., 2001, 2003).


Glass staining jars

Moist chamber

Microscope slides, coverslips (24 · 32 mm; Menzel-Gla-

ser, Braunschweig, Germany)

Heating plate with precise temperature settings

Waterbath

Fluorescence microscope with camera system (e.g. Zeiss;

Zeiss, Feldbach, Switzerland) and fluorescence filters for

4¢,6-diamidino-2-phenylindole (DAPI), FITC and TRITC

Acetic acid (Fluka)

Ethanol (Fluka)

Citrate buffer (10 mM Na-citrate, pH 4.5)

Cellulase, pectolyase, cytohelicase (Sigma)

Biotin-dUTP, digoxigenin-dUTP (Roche)

DIG and Biotin Nick translation mix (Roche)



0.5 M EDTA, pH 8.0

20x SSC (sodium chloride/sodium citrate buffer)

1 M Tris–HCl, pH 7.5

1.5 M NaCl

Phosphate-buffered saline, pH 7.4

Paraformaldehyde (PFA) (Sigma)

RNase A (DNase-free; Gibco, Langley, OK, USA)

1 M Sodium phosphate, pH 7.0

Deionized formamide (Sigma)

Dextran sulphate (Sigma)

Tween-20 (Merck, Darmstadt, Germany)

Non-fat dry milk (Bio-Rad)

Blocking reagent (Roche)

Texas Red-conjugated avidin (Vector Laboratories, Peter-

borough, UK)

Biotinylated goat-anti-avidin antibody (Vector Laborator-

ies)

Mouse anti-digoxigenin antibody (Roche)

FITC-conjugated rabbit anti-mouse antibody (Sigma)

Alexa 488 conjugated goat anti-rabbit antibody (Molecular

Probes, Leiden, the Netherlands)

Vectashield mounting medium (Vector Laboratories)

DAPI


Plant material. The nuclear architecture in interphase nuclei

is best studied in the tissue of young rosette leaves (1–

1.5 cm) that are not expected to display any mitotic activity

(Donnelly et al., 1999). If wild type and mutants are to be

compared, it is important that leaf samples are collected

from plants grown under identical conditions and of the

same age or at comparable developmental stage (best

before bolting). Multiple mutant and control plants

should be processed simultaneously in order to assess

variation between individuals.

Metaphase chromosome spreads can be obtained from

root tips of young seedlings and pachytene chromosomes

from young flower buds with the appropriate meiotic stages

(Ross et al., 1996).

Preparation of leaf nuclear spreads for analysis of interphase

nuclei. 1. Fix young rosette leaves (1–1.5 cm) in freshly

prepared ethanol/acetic acid solution mixed in a proportion

of 3:1, change the solution at least three times till the tissue

is completely white. It is possible to store the fixed material

at )20�C until use.

2. Wash the leaves twice with ddH2O and twice with citrate

buffer.

3. Cut the leaves with a scalpel into three to four pieces.

4. Incubate the tissue with cellulase, pectolyase and

cytohelicase (all at 0.3% w/v) in citrate buffer for 3 h at

37�C. Use just enough volume to cover the tissue.

5. Stop the incubation by dilution with 1 ml of citrate

buffer.

6. Move one piece of leaf to a clean slide (add a small drop

of citrate buffer if necessary) and tap the tissue with forceps,

so that single cells are released (the suspension should not

dry out). If the suspension still contains larger cell clumps,

the digestion time needs to be extended.

7. Add 20 ll of 60% acetic acid to the suspension and stir

for 1 min on a heating plate set to 45�C. At this step the

nuclei are spread on the glass slide and freed from acid-

soluble proteins and cytoplasmic components.

8. Surround the acetic acid spot with ethanol/acetic acid

solution (3:1, use approximately 500 ll) and mix by tilting

the slide. Wash the slide in ddH2O, post-fix for 5 min in 2%

PFA in PBS, wash again with ddH2O and air dry. All washing

Figure 4. Visualization of different structures within interphase nuclear

spreads of wild type Arabidopsis thaliana. The DAPI staining (left panels)

reveals intensely stained regions termed chromocenters. They can be

visualized using a biotin-labelled centromere-specific 180 bp probe (a, right

panel). The nucleolus organizer regions (NORs) on chromosomes 2 and 4,

encoding rRNA, are likewise mainly heterochromatic and contribute to four

chromocenters (b, right panel). In contrast to the Rabl configuration observed

in wheat and barley (Abranches et al., 1998; Jasencakova et al., 2001),

centromeres in Arabidopsis thaliana do not accumulate at one pole of the

nucleus (a, b), but are distributed at the nuclear periphery. The telomeres

assemble around the nucleolus (c, right panel). To study the distribution of

methylated DNA, an antibody raised against 5metC (Eurogentec) can be

applied to spread nuclei. DNA methylation levels are higher in heterochro-

matin of chromocenters (d, right panel).



steps are performed by submerging the slides into a staining

jar filled with the appropriate solution.

Preparation of labelled probes by PCR. 9. Clone the

sequence of interest (180 bp centromeric repeat can be

used in initial experiments, it results in a strong FISH signal,

Figure 4a) into a PCR vector (e.g. pBluescript) – the length of

the product should be between 200 and 500 bp.

10. Use 10 ng of the vector in a standard 20 ll PCR

reaction with 0.1 mM dATP, dCTP, dGTP, 0.065 mM dTTP

and 0.035 mM biotin-dUTP or digoxigenin-dUTP.

11. Check successful amplification on an agarose gel. The

size of the band should be shifted slightly compared with a

PCR product generated from a reaction with a standard

dNTP mix.

Preparation of labelled probes by nick translation. 12. Use

1 lg of the DNA of interest (plasmids, BACs, PCR products),

add ddH2O up to a volume of 16 ll and add 4 ll of DIG or

Biotin Nick translation mix.

13. Incubate for 2 h at 15�C and stop reaction by adding

1 ll of 0.5 M EDTA (pH 8.0) and heating to 65�C for

10 min.

14. Check product size on an agarose gel (1 ll is enough),

the DNA should be in the optimal range of 200–500

nucleotides.

Pretreatment of slides before hybridization. 15. Incubate

slides 30 min at 60�C on a heating block.

16. Place the slides in a moist chamber with the tissue

soaked in 2x SSC and pipette 60 ll of RNase A (DNase-free,

100 lg ml)1 in 2x SSC) on the slide, cover with a 24 · 32 mm

coverslip and incubate at 37�C for 1 h. This step avoids

background hybridization to residual RNA.

17. Remove RNase by three washes in 2x SSC.

18. Optionally, the slides can be additionally treated with

pepsin (10 lg ml)1 in water at pH 2.0) at 37�C for 20 min,

followed by three washes with 2x SSC.

19. The slides are then dehydrated by submerging them in

staining jars filled with an ethanol series (70, 90, 100%, 2 min

each).

Hybridization in formamide buffer. 20. For the hybridization

mixture add an appropriate amount of labelled probe (1 ll of

PCR amplified 180 bp probe or 3 ll of the nick translation

mix) directly to the hybridization solution (50% formamide,

2x SSC, 50 mM sodium phosphate pH 7.0, 10% dextran sul-

phate) to a final volume of 20 ll.

21. Place hybridization mixture in the centre of the

preparation and cover with a 24 · 32 mm coverslip.

22. Denature both nuclear DNA and labelled probe by

incubating the slides on a heating plate at 80�C for 2 min.

23. Incubate the slides in a moist chamber with the paper

soaked in 2x SSC for at least 15 h to several days (for long

hybridization times it might be necessary to seal the

coverslip with rubber cement).

Washing of slides after hybridization. 24. Remove slides

from the moist chamber and remove the coverslip by tilting

the slide or dipping the slide into a beaker with 2x SSC.

25. Wash the slides in a staining jar at 42�C for 5 min in 2x

SSC, 5 min in 0.1x SSC, 3 min in 2x SSC and at room

temperature 5 min in 2x SSC/0.1% Tween-20 (formamide-

containing buffers (50% formamide, 2x SSC pH 7.0) allow a

more stringent wash).

Detection of the biotin- or digoxigenin-labelled probes. All

washing steps are performed by submerging the slides in a

staining jar with wash buffer for 5 min at room temperature.

All antibody incubations are performed in a moist chamber

with 2x SSC at 37�C for 30 min; 60 ll of diluted antibody is

placed in the middle of the preparation and covered with a

24 · 32 mm coverslip.

26. Pipet 60 ll of blocking buffer 1 (5% non-fat dry milk, 4x

SSC) on the slide, cover with a 24 · 32 mm coverslip and

incubate for 30 min at 37�C.

27. Remove the coverslip by tilting the slide and wash

once in 4x SSC.

28. Incubate with Texas Red-conjugated avidin (5 lg ml)1

in blocking buffer 1).

29. Wash slides twice in 4x SSC, 0.05% Tween-20 and once

in 100 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20.

30. Incubate slides with biotinylated goat-anti-avidin

antibody (5 lg ml)1) and mouse anti-digoxigenin antibody

(0.2 lg ml)1), both diluted in blocking buffer 2 (100 mM Tris–

HCl, pH 7.5, 150 mM NaCl, supplemented with 0.5% blocking

reagent).

31. Wash three times in 100 mM Tris–HCl, pH 7.5, 150 mM

NaCl, 0.05% Tween-20.

32. Incubate slides with Texas Red-conjugated avidin and

rabbit anti-mouse antibody coupled to FITC (28 lg ml)1) in

blocking buffer 2.



34. Incubate with an Alexa 488-conjugated goat anti-rabbit

antibody (10 lg ml)1) in blocking buffer 2.



36. Dehydrate the slide in an ethanol series, place 8 ll of

Vectashield mounting medium containing DAPI (2 lg ml)1)

in the centre of the preparation and cover with 24 · 32 mm

coverslip (the dehydration step is optional).

The distribution of DNA methylation can be studied on the

interphase or metaphase nuclear preparations described

above using an antibody directed against 5-methyl cytosine

(Eurogentec, Seraing, Belgium). The signal of the anti-5metC

antibody (diluted 1:50 in blocking solution 2) can be ampli-

fied as described using the FITC-conjugated anti-mouse and



the Alexa 488-conjugated anti-rabbit antibodies as described

above (see also Figure 4d). The detection of methylated DNA

can be combined with FISH for one DNA sequence using a

biotin-labelled probe.

Immunostaining

The DNA-FISH procedure described allows study of the DNA

organization in the nucleus and to perform cytological

mapping. Nuclear proteins, however, can only be detected

using alternative fixation methods that better preserve the

nuclear structure and the distribution of nuclear proteins, for

example, using cross-linkers such as (para)formaldehyde.

The experimental procedure should preserve the antige-

nicity of the target proteins, provide maximal accessibility of

the antibody to its antigen and reduce autofluorescence to a

minimum. In previous studies nuclei have been directly

released from the fixed tissue into isolation buffer (Mayr

et al., 2003), which guarantees immediate preservation of

their structure. However, in our experience this method

results in low amounts of nuclei and thus may require

enrichment by flow sorting. We routinely apply a protocol

adapted from Arnim et al., from the 1996 Cold Spring Harbor

Arabidopsis Course (http://www.arabidopsis.org/info/proto-

cols.jsp), which involves isolation of leaf mesophyll

protoplasts. This method guarantees a sufficient number

of nuclei per preparation with low autofluorescence levels

(Figure 5a,b). It must be remembered that the nucleus may

undergo structural changes during protoplast isolation.

However, in our experiments in which we compare the

nuclear organization of wild type and gene silencing

mutants, the results are comparable with other methods

(Probst et al., 2003; Soppe et al., 2002).


Tilting and horizontal shakers

50 and 100 lm meshes

Benchtop centrifuge (e.g. from Hettich, Universal II;

Hettich, Tuttlingen, Germany)

Snap-cap tubes

Moist chamber

Glass staining jars

Microscope slides, coverslips (24 · 32 and 24 · 24 mm;

Menzel-Glaser)

Vacuum pump and vacuum chamber

Fluorescence microscope with deconvolution option or

confocal microscope

Bovine serum albumin (BSA)

Paraformaldehyde (PFA)

Whatman paper

Digestion solution (1% cellulase, 0.25% macerozyme,

10 mM MES, pH 5.7, 0.4 M mannitol, 30 mM CaCl2, 5 mM b-

ME and 0.1% BSA)

Wash solution (4 mM MES, pH 5.7, 2 mM KCl and 0.5 M

mannitol)

PHEM buffer (6 mM PIPES, 25 mM HEPES, 10 mM EGTA,

2 mM MgCl2, pH 6.9)

Phosphate-buffered saline, pH 7.4

Poly-lysine solution (0.1% w/v in water; Sigma)

NP-40 (Sigma)

Methanol (Fluka)

Acetone (Fluka)

Antibody against the protein of interest

Fluorescently labelled secondary antibody [e.g. Alexa 488-

coupled goat anti-rabbit antibody (Molecular Probes) or

Alexa 488-coupled goat anti-mouse antibody (Molecular

Probes)]

Vectashield mounting medium (Vector Laboratories)

DAPI


Isolation of protoplasts. 1. Harvest young rosette leaves

from soil-grown plants and cut the tissue in digestion solu-

tion into 3–4 pieces.

2. Apply vacuum for 2 min to facilitate uptake of the

digestion solution into the tissue.

3. Incubate the leaf tissue for 3–3.5 h on a tilting shaker

(15 rpm, Polymax 1040; Heidolph Instruments, Cinnamin-

son, NJ, USA) at room temperature.

4. Shake the tissue at 100 rpm on a horizontal shaker

(Adolf Kuhner AG, Birsfelden, Switzerland) for 10 min to

release protoplasts.

5. Filter suspension through a 100 lm and a 50 lm mesh.

6. Pellet cells in a 15 ml snap-cap tube in the protoplast

centrifuge at low rpm for 5 min and remove the digestion

solution.

7. Resuspend the protoplasts in 10 ml of wash solution

and invert tube gently several times.

Figure 5. Distribution of histone H3K4 methylation (a) and histone H3K9

methylation (b) in wild type Arabidopsis thaliana nuclei. The nuclei have been

analysed using a Deltavision deconvolution microscope and single layers of a

deconvoluted image stack were selected. Blue DAPI staining (left panels) and

immunodetection with antibodies directed against di-methylated H3K4

(Upstate, middle upper panel) and di-methylated H3K9 (Upstate, middle

lower panel). H3K4 methylation as a mark for transcriptionally active

euchromatin is predominantly found outside chromocenters, while H3

methylated at K9 localizes predominantly to the heterochromatic chromoc-

enters. Right panels show merged images.



8. Repeat washing and centrifugation.

9. To generate poly-lysine coated slides, flame slides

quickly, cool to room temperature and distribute 10 ll

of poly-lysine on the slide, air-dry and flame the slide again.

10. Gently resuspend the protoplast pellet in approxi-

mately 200 ll of wash solution and place a drop on the

poly-lysine coated slides placed in a moist chamber with

PBS-soaked tissue – let the protoplasts settle for about 1 h.

Fixation of protoplasts. 11. Carefully remove non-adherent

protoplasts by tilting the slide.

12. Fix the protoplasts in a staining jar in freshly prepared

2% PFA in PHEM buffer (to dissolve the PFA, the fixation

solution can be vortexed and heated to maximum 55�C) for

10 min at room temperature, permeabilize them for 5 min in

0.5% NP-40 in PHEM buffer at room temperature, and post-

fix in methanol:acetone 1:1 at )20�C for 10 min.

Immunodetection. 13. Rehydrate the protoplasts with three

washes (5 min each) in PBS (all washing steps are per-

formed by submerging the slides in a staining jar with PBS

or PBS þ NP-40 for 5 min at room temperature).

14. Pipet 60 ll of 2% BSA in PBS on the slide, cover with a

24 · 32 mm coverslip and incubate for 30 min at 37�C in a

wet chamber.

15. Incubate the slides with 40 ll of the primary antibody

diluted in 1% BSA in PBS overnight at 4�C in a moist

chamber and cover with a 24 · 24 mm coverslip.

16. Wash the slides once in PBS, once in PBS with 0.1%

NP40 and again with PBS.

17. Incubate with fluorescently labelled secondary anti-

body [e.g. Alexa 488-coupled goat anti-rabbit antibody (use

20 lg ml)1) or Alexa 488-coupled goat anti-mouse antibody

(use 10 lg ml)1)] diluted in 0.5% BSA in PBS for 45 min at

37�C.

18. Wash as in step 16.

19. Wipe the slides from the back, add 6 ll of Vectashield

mounting medium containing DAPI (2 lg ml)1) to the centre

of the preparation and cover with a 24 · 24 mm coverslip,

remove excessive washing solution carefully with a small

strip of Whatman paper.

If it is desired to combine immunostaining and FISH on

the same nucleus, the slides have first to be processed as for

immunostaining. After incubation with the fluorescently

labelled secondary antibody, the slides are dehydrated in an

ethanol series (2 min in 70%, 2 min in 90% and 2 min in

100%), air-dried and baked at 60�C for 30 min on a heating

block. Following an RNase treatment (100 lg ml)1 in 2x SSC)

for 1 h at 37�C, the slides are washed in PBS, post-fixed in 4%

PFA in PBS for 20 min at 4�C, washed again in PBS,

dehydrated in an ethanol series and air-dried. Hybridization,

washing and detection of the labelled probe are then carried

out as for FISH on spread nuclei from step 21 (Jasencakova

et al., 2003).

Acknowledgements

CB would like to thank Steven Spiker for supplying purified wheathistones and anti-H2B antibodies, which have been very useful forestablishing the methods reported here. JP would like to thank PaulFransz, Ingo Schubert and Steve Jacobsen for help and discussionsduring establishment of ChIP and cytology methodologies. PL wassupported in Naples by a postdoctoral fellowship from the Fondspour la Formation de Chercheurs et l’Aide a la Recherche (FCAR)(Quebec, Canada). CB acknowledges funding from the EuropeanUnion (contract QLK5-CT-2000-00357), the Italian Ministry forResearch and Education (FIRB contract RBNE01CFKB) and theItalian Ministry of Agriculture and Forestry (contract EcoPom).

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