[23] use of chromatin immunoprecipitation assays 349

[23] The Use of Chromatin ImmunoprecipitationAssays in Genome-Wide Analyses of

Histone Modifications

By Bradley E. Bernstein, Emily L. Humphrey,Chih Long Liu, and Stuart L. Schreiber

Introduction

Systematic studies made possible by a variety of technological advancesare impacting many areas of biology. Of special relevance to the field ofchromatin are approaches that combine chromatin immunoprecipita-tion (chromatin IP) with microarrays to analyze histone modificationsgenome-wide in yeast.1,2 The resulting genomic maps provide a uniqueglobal perspective on the functions of, and inter-relationships between,different modifications.

Chromatin IP experiments use antibodies to immunoprecipitate a pro-tein of interest and associated DNA from a solubilized chromatin prepar-ation.3–7 Specialized antibodies can be used to enrich for DNA associatedwith histones that exhibit specific post-translational modifications. A con-siderable number of antibodies that recognize histones acetylated, methyl-ated, or phosphorylated at specific residues have been developed, andmany are commercially available. Chromatin IP experiments typicallyassess whether a particular gene, gene promoter, or genomic region is en-riched in the IP sample relative to a whole cell extract (WCE) control. Forexample, quantitative polymerase chain reaction (PCR) using pre-selectedprimer pairs can be used to compare the representation of specific DNAspecies in the IP and WCE. Although several regions can be queried simul-taneously, these conventional studies are limited to a subset of genes orregions chosen by the investigator.

Global studies have the potential to overcome selection bias byanalyzing comprehensively many or all elements in a system. Microarray

1 B. E. Bernstein, E. L. Humphrey, R. L. Erlich, R. Schneider, P. Bouman, J. S. Liu,

T. Kouzarides, and S. L. Schreiber, Proc. Natl. Acad. Sci. USA 99, 8695 (2002).2 D. Robyr, Y. Suka, I. Xenarios, S. K. Kurdistani, A. Wang, N. Suka, and M. Grunstein, Cell

109, 437 (2002).3 M. J. Solomon, P. L. Larsen, and A. Varshavsky, Cell 53, 937 (1988).4 M. Braunstein, A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach, Genes Dev. 7,

592 (1993).5 V. Orlando, H. Strutt, and R. Paro, Methods 11, 205 (1997).6 M. H. Kuo and C. D. Allis, Methods 19, 425 (1999).7 A. Hecht and M. Grunstein, Methods Enzymol. 304, 399 (1999).

Copyright 2004, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 376 0076-6879/04 $35.00

350 immunochemical assays of chromatin functions [23]

technology, typically used for mRNA analysis,8,9 can be adapted for thecomprehensive analysis of chromatin IP samples.10,11 These studies relyon several innovations, including:

1. The development of molecular biology protocols to amplify and labelchromatin IP DNA samples in a sequence independent manner and

2. The production of DNA microarrays that contain the open readingframes (ORFs) as well as the regulatory regions

Recently this approach has been used to map relative levels of histoneacetylation and methylation genome-wide in yeast, and to identify regionsdeacetylated by the various histone deacetylase enzymes in yeast (seeFig. 1).1,2

Protocols

The protocol for analyzing histone modifications genome-wide in yeastis presented in four sections:

1. Chromatin IP to isolate DNA associated in vivo with modifiedhistones

2. DNA amplification and labeling3. Microarray hybridization and data acquisition4. Data analysis and interpretation

Chromatin IP to Isolate DNA Associated In Vivo withModified Histones

Solutions

YPD: Yeast extract/peptone/dextrosePBS: Phosphate-buffered saline pH 7.4Buffer L: 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM

EDTA, 1% Triton X-100, 0.1% sodium deoxycholate.For 5 ml of buffer L, 20 �l of protease inhibitor cocktail(Sigma), and one mini complete protease inhibitor tablet(Boeringer Mannheim) are added just before use

8 J. L. DeRisi, V. R. Iyer, and P. O. Brown, Science 278, 680 (1997).9 L. Wodicka, H. Dong, M. Mittmann, M. H. Ho, and D. J. Lockhart, Nat. Biotechnol. 15,

1359 (1997).10 V. R. Iyer, C. E. Horak, C. S. Scafe, D. Botstein, M. Snyder, and P. O. Brown, Nature 409,

533 (2001).11 B. Ren, F. Robert, J. J. Wyrick, O. Aparicio, E. G. Jennings, I. Simon, J. Zeitlinger,

J. Schreiber, N. Hannett, E. Kanin et al., Science 290, 2306 (2000).

Fig. 1. Steps in the genome-wide analysis of histone modifications in yeast.

[23] use of chromatin immunoprecipitation assays 351

Buffer W1: Buffer L with 500 mM NaClBuffer W2: 10 mM Tris-HCl, pH 8.0, 250 mM LiCl,

0.5% NP-40, 0.5% sodium deoxycholate,1 mM EDTA

TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTAProteinase K solution: TE with 0.4 mg/ml glycogen, 1 mg/ml

proteinase KElution buffer: TE, pH 8.0, with 1% SDS, 150 mM NaCl,

and 5 mM DTT

DNA is isolated using the following chromatin IP protocol, adaptedfrom published protocols by Kuo and Allis,6 and Hecht and Grunstein.7

352 immunochemical assays of chromatin functions [23]

A 180-ml culture of yeast (Saccharomyces cerevisiae) is grown in YPD at 30�

to an OD600 of 1.0. To cross-link proteins and DNA, 4.9 ml of 37% formal-dehyde is added and the culture incubated at room temperature for 15 minwith occasional shaking. The formaldehyde is quenched by the addition of9 ml of 2.5 M glycine, and the cells are left at room temperature for an ad-ditional 5 min. The yeast are pelleted by centrifugation at 2000g for 5 minat 4

�, and washed twice with 180 ml ice-cold PBS. Washed cells are divided

into four aliquots, pelleted, frozen in liquid nitrogen, and stored at �80�.

To lyse cells, one aliquot (representing 45 ml of the original culture) isresuspended in 400 �l of buffer L. Five hundred microliters of acid-washedglass beads are added and the mixture is vortexed on high for 45 min at 4

�.

With the lid of the microfuge tube open, the bottom of the tube is punc-tured with a hot 25-gauge needle and the cell lysate is spun to a new tube(5 s full speed, cut off the top of the punctured tube before spinning). Thetotal volume of lysate is then adjusted to 700 �l by addition of bufferL. Chromatin is fragmented by sonication using a Branson 250 Sonifier(4 � 20 s at setting 3 and 70% duty, with 20-s rests), and the insolublefraction precipitated by centrifugation at 14,000g for 15 min at 4

�. The

supernatant contains the solubilized chromatin and is referred to as theWCE. The size distribution of the chromatin fragments can be examinedby gel electrophoresis (see Fig. 2). A desired distribution is achieved byvarying sonication time.7

Prior to immunoprecipitation, 15 �l of the WCE are set aside for a con-trol. The remaining WCE is added to 50 �l of packed protein A beads (pre-equilibrated in buffer L) and rotated at 4

�for 1 h to remove proteins that

bind nonspecifically to the beads. The supernatant is then transferred to an-other tube that contains 20–30 �l of anti-modified histone antibody and ro-tated at 4

�for 4 h. Antibody-histone complexes are precipitated by the

addition of 50 �l of pre-equilibrated protein A beads and rotation at 4�

for 1 h. The beads are collected by centrifugation at 3000g for 30 s andwashed successively with 1 ml of buffers L (twice), W1 (twice), W2 (twice),and TE (twice) for 5 min each. The beads are then incubated in 125 �l ofelution buffer at 65

�for 10 min with frequent mixing. The beads are

pelleted by centrifugation at 10,000g for 2 min and the supernatant isretained. After a second elution, the eluted samples are combined, overlaidwith mineral oil, and left at 65

�overnight to reverse cross-links. In parallel,

15 �l of WCE is combined with 235 �l of elution buffer, overlaid, andincubated at 65

�overnight.

To isolate DNA, the IP sample and WCE control are incubated with anequal volume of proteinase K solution at 37

�for 2 h, extracted with 1

volume phenol (twice), 1 volume chloroform/isoamyl alcohol (25:1) (once),and ethanol precipitated. The samples are centrifuged at 15,000g for

Fig. 2. WCE starting material and products amplified by R-PCR or IVT. Lanes 2–4 of a

2% non-denaturing agarose gel contain 200 ng nucleic acid. Lanes 1 and 5: 500 ng 100 bp

ladder (NEB). Lane 2: Yeast WCE. Lane 3: DNA amplified by R-PCR from WCE. Lane 4:

RNA amplified by IVT from WCE.

[23] use of chromatin immunoprecipitation assays 353

15 min at 4�, washed with cold 70% ethanol, dried, resuspended in 20 �l of

TE with 10 �g of RNase A, and incubated at 37�

for 1 h. Prior to amplifi-cation, the DNA is further purified with a MinElute PCR PurificationKit (Qiagen).

DNA Amplification and Labeling

Chromatin IP experiments typically yield low nanogram quantities ofDNA. An amplification step is required to obtain the 2–4 �g of materialnecessary for DNA microarray analysis. Amplification of a heterogeneouspopulation of fragmented genomic DNA poses unique challenges. ThePCR requires a known flanking sequence for priming. Two PCR-basedprotocols have been developed for the amplification of genomic DNA. In‘‘ligation-mediated PCR,’’11 the genomic DNA is blunt-ended, and ligatedto double-stranded linkers. Ligated DNA is then amplified by PCR usingthe linker sequence for priming. In random-primer PCR (R-PCR),10,12,13

12 S. K. Bohlander, R. Espinosa III, M. M. Le Beau, J. D. Rowley, and M. O. Diaz, Genomics

13, 1322 (1992).13 J. D. Lieb, X. Liu, D. Botstein, and P. O. Brown, Nat. Genet. 28, 327 (2001).

354 immunochemical assays of chromatin functions [23]

the genomic DNA is copied twice using a primer adaptor that has aconserved 50 sequence and a degenerate 30 sequence that anneals to theends of the genomic DNA. The copied DNA is amplified by PCR usingthe 50 conserved sequence for priming. Both methods have been used toamplify DNA obtained by chromatin IP.

Due to its exponential kinetics, PCR is particularly susceptible tosequence- and length-dependent biases. Recently, a linear amplificationstrategy has been applied to the amplification of genomic DNA.14 In thisprocedure, a T7 promoter sequence is added to the ends of the genomicDNA, creating a template suitable for in vitro transcription (IVT). Al-though this IVT protocol has the added complication of working withRNA, it preserves the species representation of the chromatin IP DNAmore effectively than the PCR methods. Here we present the R-PCRand IVT amplification protocols as we have applied them in studies ofhistone modifications.

R-PCR Amplification Protocol

Solutions

5� Sequenasebuffer (Amersham):

200 mM Tris, pH 7.5, 100 mM MgCl2,250 mM NaCl

dNTP mix: 3 mM each of dATP, dCTP, dGTP, anddTTP

Sequenase reactionmix (for 2 reactions):

2.5 �l 5� Sequenase buffer, 3.75 �l dNTPmix 1.875 �l 0.1 M DTT, 3.75 �l 0.5 mg/mlBSA, 0.75 �l Sequenase 2.0 (Amersham)

Sequenase Dilutionbuffer (Amersham):

10 mM Tris, pH 7.5, 5 mM DTT, 0.1 mMEDTA

50� aa-dNTP mix: 25 mM dATP, 25 mM dCTP, 25 mM dGTP,10 mM dTTP, 15 mM aminoallyl-dUTP

PCR buffer: 500 mM KCl, 100 mM Tris, pH 8.3Primer A: 40 pmol/�l 50-GTTTCCCAGTCACGAT-

CNNNNNNN- NN-30

Primer B: 100 pmol/�l 50-GTTTCCCAGTCA-CGATC-30

DNA from the chromatin IP sample and the WCE control are amplifiedin parallel using a variant of the Round A/Round B DNA amplificationprotocol used by Iyer et al.10 and described at www.microarrays.org. Thisvariant protocol minimizes PCR rounds in an attempt to reduce amplifica-tion bias. In Round A, 7 �l sample, 2 �l 5� Sequenase buffer, and 1 �l

14 C. L. Liu, S. L. Schreiber, and B. E. Bernstein, BMC Genomics 4, 19 (2003).

[23] use of chromatin immunoprecipitation assays 355

Primer A are combined in a thin-walled PCR tube. In a thermal cycler, themixture is heated at 94

�for 2 min, cooled to 10

�, and held for 5 min while

5.05 �l of Sequenase reaction mix is added. The reaction is ramped from10

�to 37

�at a rate of 0.1

�per second, held at 37

�for 8 min, and then heated

at 94�

for 2 min. It is then cooled to 10�

and held for 5 min while 1.2 �l of Se-quenase 2.0 diluted 1:4 with Sequenase Dilution Buffer is added. Finally, thereaction is ramped from 10

�to 37

�at a rate of 0.1

�per second and held at 37

�

for 8 min. The samples are diluted with water to a final volume of 60 �l.In Round B, 15 �l diluted Round A product, 8 �l 25 mM MgCl2, 10 �l

10� PCR buffer, 2 �l 50� aa-dNTP solution, 1 �l Primer B, 1 �l Taq (5 U/�l), and 63 �l nuclease-free water are combined in a thin-walled PCR tube.The following PCR program is carried out for 30 cycles: 92

�for 30 s, 40

�for

30 s, 50�

for 30 s, 72�

for 1 min. The cycles are followed by a 5-min exten-sion at 72

�. PCR product is purified using a QIAquick PCR Purification Kit

(Qiagen). Amplification is verified by spectrophotometry and gel electro-phoresis. The size distribution should approximate that of the WCE, thoughwe note that lower molecular weight species are inefficiently amplified bythis method (see Fig. 2). Products from 2–3 Round B PCR reactions arecombined to obtain the 8–10 �g of DNA required for microarray analysis.Equal quantities of DNA amplified from IP and WCE are used forsubsequent labeling and analysis.

IVT Amplification Protocol

Solutions

NEB buffer 3(New England Biolabs):

100 mM NaCl, 50 mM Tris, pH 7.9,10 mM MgCl2, 1 mM DTT

TdT buffer (Roche; notedifferent source than enzyme):

1 M potassium cacodylate, 125 mMTris, pH 6.6, 1.25 mg/ml BSA

8% nucleotide tailing solution: 4.6 �M dTTP, 0.4 �M ddCTPEco Pol Buffer

(New England Biolabs):10 mM Tris-HCl, pH 7.5, 5 mMMgCl2, 7.5 mM DTT

50� aa-dNTP mix: 25 mMdATP,25 mMdCTP,25 mMdGTP, 10 mM dTTP, 15 mM ami-noallyl-dUTP

Priming mix: 5 �g/�l 18–22-mer oligo dT (op-tional), 5 �g/�l random primer pdN6 (Invitrogen)

T7-A18B primer: 25 pmol/�l 50-GCATTAGCGGCCGCGAAATTAATACGACT-CACT ATAGGGAG(A)18[B]-30

([B] stands for any base other than A)

356 immunochemical assays of chromatin functions [23]

DNA from the chromatin IP sample and the WCE control are treatedwith calf intestinal alkaline phosphatase (CIP) to remove 30 phosphategroups prior to IVT. DNA (up to 50 ng), 1 �l NEB Buffer 3, and 0.25 �lof 10 U/�l CIP (NEB) are combined and H2O is added to a totalvolume of 10 �l. The reaction is incubated at 37

�for 1 h, and cleaned up

with a MinElute Reaction Cleanup Kit (Qiagen). The kit is used after sev-eral steps in this protocol, always according to manufacturer’s instructionsexcept with the elution volume increased to 20 �l. Terminal transferase(TdT) is used to add 30 tails of relatively uniform length to the ends ofthe genomic DNA. Ten microliters of CIP-treated DNA, 4 �l TdT Buffer,3 �l 5 mM CoCl2, 1 �l 8% nucleotide solution, and 2 �l TdT enzyme(10 U/�l) are combined, overlaid with mineral oil, and incubated at 37

�

for 20 min. The reaction is halted by the addition of 2 �l 0.5 M EDTA,pH 8.0, and subjected to the MinElute Reaction Cleanup.

T7 promoters are incorporated at the ends of the DNA fragments asfollows: 20 �l tailing reaction product, 0.6 �l T7-A18B primer, 5 �l 10�EcoPol buffer, 2 �l 5.0 mM dNTPs, and 20.4 �l nuclease-free water arecombined. In a thermal cycler, the samples are incubated at 94

�for 2 min,

ramped down at �1�

per second to 35�, held at 35

�for 2 min, ramped down

at �0.5�

per second to 25�

and held while 2 �l 5 U/�l Klenow (NEB) isadded. The reaction is then incubated at 37

�for 90 min, halted by the ad-

dition of 5 �l 0.5 M EDTA, pH 8, and subjected to a MinElute ReactionCleanup. (Note: mineral oil is not used at this stage.)

Prior to the IVT, the volume is reduced to 8 �l in a vacuum centrifugeat medium heat. IVT is carried out using the T7 Megascript Kit (Ambion).The samples are purified using an RNeasy Mini Kit (Qiagen) per manufac-turer’s RNA cleanup protocol, except with an additional 500-�l wash withbuffer RPE. RNA is quantified by absorbance at 260 nm and visualized ona 2% native TAE agarose gel. Yields range from 10 to 60 �g. The sizedistribution should approximate that of the WCE (see Fig. 2).

Equal quantities of RNA from the IP sample and WCE control (�2–4�g) are reverse-transcribed to generate DNA probes. In a 14.5-�lvolume, the RNA is primed by addition of 1 �l priming mix, incubationat 70

�for 10 min, and incubation on ice for 10 min. Then, 6 �l 5� RT

buffer, 3 �l 0.1 M DTT, 3 �l nuclease-free H2O, 0.8 �l 50� aa-dNTPmix, and 2.5 �l 200 U/�l Superscript II (Invitrogen) are added, and themix is incubated at 42

�for 2 h. The reaction is halted by the addition of

10 �l 0.5 M EDTA, pH 8.0, and the RNA is hydrolyzed by the additionof 10 �l 1 N NaOH and incubated at 65

�for 15 min. The solution is neu-

tralized with 25 �l 1 M HEPES, pH 7.5. The entire samples are used forlabeling and hybridization.

[23] use of chromatin immunoprecipitation assays 357

Probe Labeling

Amplified samples are fluorescently labeled using monofunctionalCy5 and Cy3 fluorescent dyes as described by Carroll et al.,15 and at:www.microarrays.org. These derivatized dyes covalently couple to the ami-noallyl groups incorporated in Round B of R-PCR, or in the RT stepfollowing IVT. DNA samples are diluted with 400 �l of nuclease-freewater, concentrated to �70 �l in a YM-30 microcon (Millipore) by centri-fugation at 11,000g, re-diluted and re-concentrated. After a final dilution,the samples are concentrated to a volume of 18–20 �l (microcons are pre-weighed, and final volume determined by weight). Samples are collected byinverting the microcon into a new microfuge tube and spinning at 4000g for5 min. One microliter of 1 M NaHCO3, pH 9, is added and the samples aretransferred to a microfuge tube containing a dried aliquot of NHS cyaninedye (Cy5 for IP sample; Cy3 for WCE control) and incubated at room tem-perature for 75 min in the dark. [The dye aliquots are prepared by dissolv-ing one vial of monofunctional Cy5 or Cy3 dye (Amersham) in 20 �lDMSO, distributing the solution into 10 microfuge tubes, and drying in avacuum centrifuge under low heat.] Reactions are diluted with 70 �l H2Oand purified using a QIAquick PCR Purification Kit.

Microarray Hybridization and Data Acquisition

Microarray production, hybridization, and data acquisition are carriedout using protocols developed by Patrick Brown and colleagues, availableon-line at www.microarrays.org and recently reviewed by Gasch.16

Comprehensive coverage of the yeast genome is achieved by hybridizingamplified DNA to microarrays that contain all ORFs and all promoter-containing intergenic regions (INTs). The INTs are designated in such away that each gene promoter can be assigned to a specific INT. DNA frag-ments representing all ORFs and INTs can be amplified from genomicDNA using primers from Research Genetics. The ORFs and INTs areprinted on separate poly-lysine–coated slides by pin transfer using a roboticarrayer as described at www.microarrays.org, or using alternative protocolsdescribed elsewhere.11,17 The inclusion of INT and ORF arrays in a globalchromatin analysis can enable an experimenter to discriminate betweenpromoters and coding regions of genes.1,13

15 A. S. Carroll, A. C. Bishop, J. L. DeRisi, K. M. Shokat, and E. K. O’Shea, Proc. Natl. Acad.

Sci. USA 98, 12578 (2001).16 A. P. Gasch, Methods Enzymol. 350, 393 (2002).17 C. E. Horak and M. Snyder, Methods Enzymol. 350, 469 (2002).

358 immunochemical assays of chromatin functions [23]

Hybridization

The competitive hybridization used in DNA microarray analyses allowsflexibility in determining which samples to compare. In this presentation ofthe protocol, the relative enrichment of a particular modification is ana-lyzed genome-wide by comparing a chromatin IP sample to its WCE con-trol. However, it is also possible to query changes in modification patternsresulting from gene deletion or small-molecule treatment by comparingtwo chromatin IP samples obtained from different sources using the sameantibody.

Reagents and solutions:

WS1: 387 ml Milli-Q water, 12 ml 20 � SSC, 1 ml 10% SDSWS2: 1 ml of 20 � SSC in 399 ml of Milli-Q water

The following is adapted from the hybridization procedure describedat www.microarrays.org. Cy5-labeled chromatin IP probe and Cy3-labeledWCE probe are combined, and concentrated in a microcon YM-30 filteruntil 50 �l of solution remain on the filter. The sample is collected byinverting the microcon into a new microfuge tube and spinning at 4000gfor 5 min. Six microliters of 20� SSC, 3 �l of 10 �g/�l poly(A), and0.96 �l of 1 M HEPES, pH 7.0, are added and the solution is filtered witha pre-wet Millipore 0.45-� filter spun at 12,000 rpm for 2 min. Ten percentSDS, 0.9 �l, is added, the mixed probe is placed in a 100

�heat block for

2 min, placed at room temperature for 10 min, and then applied to the mi-croarray. Slides printed with ORFs and INTs are placed in Telechem hy-bridization chambers with the arrays facing up. One clean lifterslip (ErieScientific) is placed on each array so that the white strips face down. Halfof the mixed probe (about 20 �l) is applied to the ORF array and the otherhalf to the INT array. Each well of the hybridization chambers is filledwith 40 �l of 3� SSC, the chambers are then sealed and placed in a 60

�

water bath for 12–15 h.Four Wheaton glass tanks containing WS1 or WS2 are prepared. A

metal slide rack is placed in the first tank of each solution. The microarrayslides are removed from the hybridization chambers and turned upsidedown in the first tank of WS1. Slides are tilted to allow the lifterslips to falloff, placed in the slide rack, and plunged up and down 20 times. Thisdunking is repeated in the second tank of WS1 and in the two WS2 tanks.The slides are dried by centrifugation at 1000 rpm in a Beckman tabletopcentrifuge for 2 min.

[23] use of chromatin immunoprecipitation assays 359

Data Acquisition and Processing

Data acquisition and analysis are detailed in a variety of sources.16–19

Briefly, data are acquired by scanning the microarrays with a scanning laserdevice (Axon Instruments) that simultaneously excites and measures fluor-escence emission for the Cy5 and Cy3 dyes. Image analysis software(Axon) is used to quantify fluorescent features and associate them withthe appropriate ORF or INT. The data output consists of Cy5 and Cy3 in-tensities and deviations for each ORF or INT. The Cy5/Cy3 ratios reflectthe amount of DNA in the chromatin IP sample relative to the WCE con-trol. The data are normalized so that the relative fluorescence signal is thesame for most spots.19 Alternatively, control samples spiked into theprobes can be used for normalization.16 To eliminate questionable meas-urements, array features having a signal-to-noise ratio of less than a cut-off value (e.g., 2.5) are removed from further analysis. Replicate data setscan be merged using a weighted-averaging procedure.18

Data Analysis and Interpretation

Microarray experiments generate vast amounts of data, and it can bechallenging to convert these data into informative observations. Each his-tone modification data set contains Cy5/Cy3 ratios for each microarray fea-ture. These ratios reflect the degree to which histones associated with thecorresponding ORF or INT exhibit a particular modification. A high ratioindicates a high level of modification. A ratio of around 1 indicates a modi-fication level not significantly different from the genome average. Alow ratio suggests a low level of modification, with the caveat that somegenomic regions may be less available to IP. (To control for this potentialcaveat, antibody against an invariant region of a histone can be used.) It ispossible to assign p-values that reflect the probability that the modificationlevel of histones associated with a particular ORF or INT is significantlyhigher (or lower) than the genome average.

The data can be used as a screen to identify genes or gene promotersassociated with highly modified histones. These can be verified by conven-tional chromatin IP and followed up by other biological means. Alterna-tively, since the genomic location of each ORF and INT is known,modification status can be visualized across the yeast genome usingchromosome mapping programs or plotted with respect to distance fromtelomere ends. The continuity of a modification along adjacent genomic

18 C. J. Roberts, B. Nelson, M. J. Marton, R. Stoughton, M. R. Meyer, H. A. Bennett, Y. D. He,

H. Dai, W. L. Walker, T. R. Hughes et al., Science 287, 873 (2000).19 J. Gollub, C. A. Ball, G. Binkley, J. Demeter, D. B. Finkelstein, J. M. Hebert, T. Hernandez-

Boussard, H. Jin, M. Kaloper, J. C. Matese et al., Nucleic Acids Res. 31, 94 (2003).

360 immunochemical assays of chromatin functions [23]

regions can be assessed using statistical methods, though such analysesmust account for DNA fragments in the chromatin IP experiment that spanadjacent ORFs and INTs.

Data sets that assess different modifications can be compared in severalways. The Pearson correlation provides a single measure of similarity be-tween the global patterns of two modifications (this can be visualized byplotting pairwise relationships between the modifications). Alternatively,lists of outliers (e.g., features with high ratios) can be collated andcompared. The significance of an observed overlap can be calculated usinga hypergeometric probability model.20 Two-dimensional hierarchical clus-tering, commonly used in expression profiling, can be performed andvisualized using Cluster and Treeview.21 Clustering can be used to identifysets of genes that exhibit similar modification patterns. These comparativeanalyses can also be extended to other microarray-based data sets to iden-tify functional relationships. This kind of cross-comparison was used toidentify associations between H3 Lys4 methylation and transcription,1

and between histone deacetylation and repression.1,2,22 Although theseanalyses have revealed valuable insights into roles of histone modifications,a realization of the full potential of the histone modification data setsawaits implementation of more sophisticated analytical approaches.

Conclusions and Future Directions

Methods for analysis of histone modifications genome-wide in yeasthave been developed and applied toward a limited subset of site-specificacetylation2 and methylation1 marks. Future studies will assess the poten-tial of this systematic approach to provide more comprehensive insight intothe functions of, and inter-relationships between, the many other modifica-tions that occur on histones in yeast. Furthermore, the development of mi-croarrays able to query regulatory regions of the human genome23,24

should allow for global analyses of histone modifications in higher organ-isms. The recent development of a method for linear amplification ofchromatin IP samples should facilitate this transition.

20 S. Tavazoie, J. D. Hughes, M. J. Campbell, R. J. Cho, and G. M. Church, Nat. Genet. 22, 281

(1999).21 M. B. Eisen, P. T. Spellman, P. O. Brown, and D. Botstein, Proc. Natl. Acad. Sci. USA 95,

14863 (1998).22 B. E. Bernstein, J. K. Tong, and S. L. Schreiber, Proc. Natl. Acad. Sci. USA 97, 13708

(2000).23 A. S. Weinmann, P. S. Yan, M. J. Oberley, T. H. Huang, and P. J. Farnham, Genes Dev. 16,

235 (2002).24 B. Ren, H. Cam, Y. Takahashi, T. Volkert, J. Terragni, R. A. Young, and B. D. Dynlacht,

Genes Dev. 16, 245 (2002).