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n Vivo Cross-Linking and Immunoprecipitationor Studying Dynamic Protein:DNA Associationsn a Chromatin Environment

in-Hao Kuo1,2 and C. David Allis1

rticle ID meth.1999.0879, available online at http://www.idealibrary.com on

epartment of Biochemistry and Molecular Genetics and Department of Microbiology, University of Virginia,ealth Sciences Center, Charlottesville, Virginia 22908

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Chromatin structure plays important roles in regulating manyNA-templated processes, such as transcription, replication, and

ecombination. Considerable progress has recently been made inhe identification of large, multisubunit complexes dedicated tohese nuclear processes, all of which occur on nucleosomal tem-lates. Mapping specific genomic loci relative to the position ofelectively modified or unique histone variants or nonhistoneomponents provides valuable insights into how these proteinsand their modifications) function in their normal chromatin con-ext. Here we describe a versatile and high-resolution methodhich involves two basic steps: (1) in vivo formaldehyde cross-

inking of intact cells followed by (2) selective immunoprecipita-ion of protein–DNA complexes with specific antibodies. This

ethod allows for detailed analyses of protein–DNA interactionsn a native chromatin environment. Recently, this technique haseen successfully employed to map the boundaries of specificallyodified (e.g., acetylated) histones along target genes, to define

he cell cycle-regulated assembly of origin-dependent replicationnd centromere-specific complexes with remarkable precision,nd to map the in vivo position of reasonably rare transcriptionactors on cognate DNA sites. Thus, the basic chromatin immu-oprecipitation technique is remarkably versatile and has noween used in a wide range of cell types, including budding yeast,y, and human cells. As such, it seems likely that many moretudies, centered around chromatin structure and protein–DNAnteractions in its native setting, will benefit from this technique.n this article, a brief review of the history of this powerful ap-roach and a discussion of the basic method are provided. Pro-

1 To whom correspondence should be addressed. (CDA) Fax: (804)24-5069. E-mail: allis@virginia.edu. (MHK) Fax: (517) 353-9334.

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-mail: kuom@pilot.msu.edu.2 Present address: Department of Biochemistry, 309 Biochemistry

uilding, Michigan State University, East Lansing, MI 48824-1319.

046-2023/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

edures for protein recovery as well as limitations and extensionsf the method are also presented. © 1999 Academic Press

Eukaryotic genomes are packaged into chromatinithin which DNA-templated processes, such as tran-

cription, replication, recombination, and repair takelace. The intimate association between DNA and his-one proteins contained within the nucleosome, theasic unit of chromatin, and higher levels of nucleoso-al folding create a physical barrier, often antagoniz-

ng interactions between DNA and molecular machineshat use chromatin as a substrate. To fully understandow cells regulate the above activities, it is essential toake into account positive and negative effects imposedt the most fundamental level by chromatin structure.Chromatin is not a dormant, repetitive structure exist-

ng as little more than a structural platform for genomicNA organization. Instead, nucleosomal arrays areighly dynamic structures that must be reversiblypened and closed in a tightly regulated fashion to ac-ommodate seemingly contrasting states, such as moreecondensed chromatin facilitating transcriptional read-ut or more condensed chromatin permitting faithfulhromosome segregation. During metaphase of mitosis,or example, the 30-nm nucleosomal fiber (with a packingatio of about 50) becomes condensed to a fully condensedetaphase chromosome (with a packing ratio of 104).his highly constrained structure must then be rapidlyecondensed and returned to an interphase chromatinorm following completion of each cell division. Recently,

he condensation of mitotic and meiotic chromosomes haseen determined to be temporally and spatially linked toistone H3 phosphorylation (1, 2), providing a clear ex-

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426 KUO AND ALLIS

mple for how covalent modifications of histone proteins,nd presumably chromatin structure at its most basicrganizational level, can produce dramatic effects onhromosomal and nuclear functions. Indeed, ample evi-ence suggests that regulation of many DNA-mediatedrocesses involve highly conserved mechanisms of chro-atin remodeling and histone modification (3–8).It becomes of interest to know how specific genes orNA sequences are configured in a chromatin settingnd what proteins are bound to them in this environ-ent. These general issues represent a challenging

roblem to researchers as chromatin and histone pro-eins serve to package essentially the entire eukaryoticenome. An extensive literature exists of attempts toiochemically fractionate chromatin in a meaningfulay (e.g., transcriptionally active versus inactive loci)

ollowed by characterization of the associated DNAequences and chromatin components (9, 10). How-ver, because of the dynamic nature of chromatin itself,s well as the transient nature of interactions betweenNA and nonhistone proteins, it becomes a concern as

o how to preserve physiologically relevant protein:NA interactions within chromatin-based structures.ell known is the fact that DNA-binding proteins can

earrange or be lost during biochemical fractionationse.g., see (11) for references).

To avoid these and other potential artifacts, differenteagents and methods have been developed over theears to bind proteins covalently to DNA in situ byreating living cells or isolated structures with cross-inking agents. Among these, UV and formaldehydeHCHO) have been used very successfully in manytudies to cross-link DNA to histone and nonhistoneomponents of chromatin. Formaldehyde cross-linkings reported to occur between the exocyclic amino groupsnd the endocyclic imino groups of DNA bases and theide-chain nitrogens of lysines, arginine, and histidineas well as the a-amino groups of all amino acids)12–14). Because histones are abundant nuclear pro-eins characteristically rich in these basic amino acidsnd because these proteins evolved to “package DNA,”aking well-defined histone:histone and histone:DNA

nteractions (15, 16), the cross-linking of histones toNA is particularly favorable and efficient withCHO. Formaldehyde cross-linking as a general strat-

gy provides an important additional advantage: cross-inks formed by HCHO treatment are fully and easilyeversible, which allows for further analyses of bothroteins and DNA ((17) and see below). With the re-ewed interest in chromatin and the generation ofigh-titer, monospecific antibodies to DNA bindingroteins (including histones and their posttransla-ional modifications), as well as to convenient oligopep-ide tags, formaldehyde cross-linking combined with

mmunoprecipitation has become a method of choiceor many different topics in chromatin-related studies.

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ISTORICAL REVIEW: EARLY DEVELOPMENTS

As might be expected, development of the basicrocedures for chromatin immunoprecipitation (asescribed below) has taken a long journey, and manyesearchers have contributed to the evolution of thisncreasingly used technique. The original use ofCHO as a protein–protein, protein–DNA, androtein–RNA cross-linking reagent can be tracedack to the 1960’s (18, 19). At that time, isolatedhromatin or nuclei, frequently containing in vivoadiolabeled precursors, was fixed with HCHO forxtensive periods of time followed by CsCl gradiententrifugation for studies monitoring the distribu-ion of newly synthesized histones along newly rep-icated DNA. Extension of this technique includedhe use of HCHO to map histone– histone interac-ions within the nucleosome itself (20, 21). One of theey initial reports of reversing HCHO-induced cross-inks to release histones for detailed electrophoreticnalyses was by Jackson (17, 21) with conditionseing further refined by Varshavsky and colleagues aew years later (22). An important developmentame when Jackson and Chalkley (23, 24) reportedhe use of “whole cell” formaldehyde fixation forhromatin studies. In these pioneering studies,CHO treatment was shown to faithfully preserve

hromatin structure. By combining this techniqueith CsCl gradient analyses, these workers went on

o study the fate of newly deposited nucleosomesuring chromatin assembly and their subsequentaturation.Inspired by progress being made in in vitro stud-

es, Gilmour and Lis used UV irradiation to co-alently cross-link proteins to neighboring DNA inntact cells and fractionated the protein:DNA ad-ucts using immunoprecipitation with specific anti-odies to RNA polymerase (25) and topoisomerase I26), and this protocol was later applied by the Lisab to the study of other DNA-binding proteins (e.g.,7–30). The first use of histone antibodies to studyhe association between histones and DNA in rela-ion to transcription was reported by Varshavskynd co-workers (31), who showed that, during heathock, the highly transcribed hsp70 gene had anltered chromatin structure. Importantly, thesetudies provided some of the first evidence that his-one H4 was retained on this gene during the tran-cription process (i.e., this histone was not dissoci-ted from the DNA). Gorovsky and co-workers thenystematically simplified and improved this basicpproach (11) and applied it to demonstrate alter-tions in H1:DNA interaction associated withhanges in transcriptional activity in the ciliated

rotozoan Tetrahymena (32). As H1-type (or linker-ssociated) histones were well known to exchange in

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427PROTEIN–DNA INTERACTIONS AND CHROMATIN ENVIRONMENT

hromatin during biochemical isolation steps, thesen situ cross-linking methods provided an importantdvance in avoiding artifactual rearrangements.

ENEWED INTEREST IN CHROMATIN ANDSSOCIATED IMMUNOPRECIPITATION ASSAYS

Between the late 1980s and the mid-1990s, interestn the basic chromatin immunoprecipitation techniqueescribed above waned, in part because of the qualitynd types of antibodies available. “General” histonentibodies existed for many of the major histone typesrom various cellular sources (i.e., H1, H2A, H2B, H3,4, etc.) but in many ways, the more interesting ques-

ions relate to what underlies nucleosomal variationnd instability and not to what is constant. An exten-ive body of literature in the chromatin field documentshe fact that not all nucleosomes are created equal (9).or example, nonallelic histone variants or subtypesxist for many of the histones, and even among “stan-ard” histones, extensive and dynamic heterogeneity isrought about through posttranslational modificationf histone “tails,” including acetylation, phosphoryla-ion, etc. (see below).

The development of immunological reagents directedt these highly conserved, often invariant, histoneodifications, when combined with the chromatin im-unoprecipitation assay, has proven to be a powerfulew approach to address mechanistic questions re-arding chromatin modification and gene regulation.or example, antibodies selective for acetylated his-ones were used in immunoprecipitation experimentsusing nuclease-digested chromatin without cross-inking) aimed at examining the acetylation state ofhe chicken b-globin locus in embryonic erythrocytes33–36). These pioneering studies by Crane-Robinsonnd colleagues provided a crucial link between coreistone acetylation and transcriptionally poised chro-atin. Moreover, these studies suggested that the do-ain boundary of acetylation around the b-globin gene

xtends past the transcribed region, coinciding wellith regions of increased DNase sensitivity (35). In

ight of the recent reports that histone acetylation,ediated by coactivators containing intrinsic histone

cetyltransferase activity or corepressors containingistone deacetylase activity, is a targeted phenomenonreviewed in 37, 38), it becomes of interest to knowhere exactly histone acetylation occurs in down-

tream target genes. In this regard, the chromatinmmunoprecipitation assay is particularly well suitedsee below).

Studies of gene silencing in yeast Saccharomyceserevisiae provided additional crucial evidence demon-

trating the important roles of histone acetylation inegulating gene activity. Broach and colleagues (39)

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xed whole yeast cells with HCHO and immunopre-ipitated chromatin fragments with antibodies specificor acetylated histone H4 and showed that yeast silentoci were associated with underacetylated nucleo-omes; loss of silencing was accompanied by increasedcetylation in the desilenced loci. Subsequent studiesocumented enrichment of silent loci using antibodiesgainst unacetylated H3 but only when these loci wereilent (40). In genetic backgrounds in which these lociere derepressed, this enrichment was lost and lociere selectively enriched with acetylation-specific H3ntibodies. Using acetylation site-specific antibodies,hese authors went on to demonstrate that the mono-cetylated histone H4 at lysine 12 was preferentiallyaintained in silent loci (40), a result in excellent

greement with immunocytological and immunopre-ipitation studies which suggest that the pericentro-eric heterochromatin in Drosophila (41) and humans

42) is largely associated with this acetylated lysine 12mark”. In general, these findings are in good agree-ent with the dogma that histone hyperacetylation is

ightly linked to gene activity, although it seems likelyhat exceptions to this general rule exist and that nu-leosomes marked with different site-specific acetyla-ion patterns may function differently inside the nu-leus in ways that remain to be elucidated (40, 43–45).gain, the chromatin immunoprecipitation assay isell suited to look for these “exceptions” with respect to

pecific genes.Considerable progress has recently been made in

dentifying components of the transcription machineryhat are dedicated to the remodeling or modification ofhromatin (see 4, 46, 47 for reviews and referencesherein). These findings, along with the generation ofigh-titer, monospecific antibodies directed against his-one modifications, have rekindled interest in the basichromatin immunoprecipitation technique. Moreover,airwise sets of antisera have been developed for sev-ral of the core histones (i.e., acetylated versus unac-tylated, phosphorylated versus unphosphorylated)e.g., 39, 48). The availability of these antibodies hasllowed workers to carefully investigate the status ofhromatin and gene structure with respect to the mod-fication in question.

HROMATIN IMMUNOPRECIPITATION:EACHING PAST HISTONES

The chromatin immunoprecipitation method haseen used successfully for different topics related tohromatin structure and chromosomal dynamics. De-ending on the antibodies chosen, this technique hasow been extended to map a remarkable variety of

rotein:DNA interactions in a wide range of organisms,ncluding budding yeast (e.g., 39, 40, 48–53), protozoa

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428 KUO AND ALLIS

11, 32), Drosophila (54, 55), human cells (56), as wells sea urchin embryos (57), and Archaea (58). As ex-ected, many studies are centered upon proteins thatre directly related to chromatin (histones or nonhis-ones). For example, using the chromatin immunopre-ipitation assay, careful dissections have now begun tonvestigate the relationships between cell signalingathways (56, 56a), the transcriptional regulation andistone modifications in mammalian cells (56), theomposition of specialized yeast nucleosomes at theentromeres (52, 53), the distribution of ubiquitinatedistones during sea urchin development (57), and thetructure of archaeal nucleosomes (58).Importantly, the basic chromatin immunoprecipita-

ion assay has also been applied successfully to situa-ions involving DNA-binding proteins or complexeshat are considerably less abundant than histones. Forxample, this assay was used to map the associationnd genomic position of specific silent information reg-lator proteins (SIR) with telomeric DNA in yeast (49)nd the genomic binding sites of Polycomb in Drosoph-la (54, 55). Similarly, the cell cycle-regulated assemblyf origin recognition complexes (ORC), in associationith MCM and Cdc6 proteins in prereplicative com-lexes, has recently been examined in considerableetail in yeast (50, 51). In mammalian T and B cellevelopment, association of Ikaros/Helios family mem-ers with silent genes at centromeric heterochromatinas been documented by cross-linking and immunoaf-nity methods (59–61). Quite recently, this procedureas been applied to analyzing transcription factorinding to endogenous genes (61a) and thus offers it-elf as an innovative approach to studying transcrip-ional regulation of key genes in an in vivo (chromatin)etting. This assay promises to be particularly usefulhen in vitro footprinting analyses suggest that mul-

iple transfactor binding sites occur in the regulatoryegion of interest, and the critical question becomeshich one(s) matter in vivo.

HROMATIN IMMUNOPRECIPITATION ANDISTONE ACETYLATION: A CASE STUDY

It is now clear that histone acetylation is intimatelyssociated with gene activity (5). In this case, acetyla-ion refers to the addition of acetyl moieties to the-amino groups of internal, highly conserved lysines inhe amino-terminal domains of core histones, H2A,2B, H3, and H4 (instead of cotranslational acetyla-

ion of the a-amino groups or N-terminal acetylation).he steady-state balance of histone acetylation ischieved by two opposing enzyme systems, histonecetyltransferases (HATs) and histone deacetylases

HDACs) (see e.g., 6 for a review). Exciting recentevelopments, particularly the realization that a sur-

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risingly large number of histone acetyltransferasesnd deacetylases were actually previously identified asroteins with roles in transcriptional regulation, haverovided critical mechanistic links between chromatinodification and transcriptional output. DifferentATs and HDACs show different substrate and acet-

lation site preferences both in vivo and in vitro (62,3, 6). It remains an attractive, yet unproved, possibil-ty that the total number of acetyllysines and/or theositions of these modified residues within each nu-leosome may both be important determinants for theonsequent “behavior” of the underlying chromatin.As histone proteins are associated with essentially the

ntire eukaryotic genome, it becomes of interest to knowow histone-modifying enzymes influence the transcrip-ion of a small number of target genes. Recent resultsrom our laboratory (48), using yeast Gcn5p as a modelAT, and those from Grunstein’s (63) and Struhl’s (64)

aboratories, using yeast Rpd3p as a model HDAC, indi-ate that these activities exert functions preferentially athe promoter region of target genes, supporting the modelf a localized, promoter-targeted chromatin modificationechanism for gene regulation.

ETHODS

The basic flow chart of chromatin immunoprecipita-ion discussed below is illustrated in Fig. 1. To ournowledge, most currently used chromatin immuno-recipitation procedures are derived from one of twoethods developed for yeast by the Broach (39) andrunstein (49) laboratories; the two methods differainly in the way that the starting extracts are pre-

ared following fixation in situ with formaldehyde. Theormer method requires first the preparation of sphero-lasts followed by extensive wash steps to remove theajority of cytosolic materials. The resultant “nuclear

raction” is then sonicated to obtain chromatin frag-ents. In the latter procedure, in contrast, whole cell

xtract is prepared by vortexing a mixture of yeast cellsnd glass beads. Sonication is directly applied to thesetarting extracts to shear the chromatin into manage-ble size fragments. The detailed protocol presentedelow is adapted from Strahl-Bolsinger et al. (49) sim-ly because of the ease of the operation. Although therocedure was originally designed for budding yeast,nterested readers can easily adapt it for use in otherrganisms according to the references listed above.Although the current method has received wide atten-

ion due to its versatility, we note that one aspect of thisssay is underutilized. For obvious reasons, most exper-ments to date using the chromatin immunoprecipitationssay have been focused on characterizing the DNA se-

uences associated with the DNA-binding protein of in-erest. Analyses of the proteins being immunoprecipi-

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429PROTEIN–DNA INTERACTIONS AND CHROMATIN ENVIRONMENT

ated may also be of interest and, in fact, this remains onef the most rigorous ways to demonstrate antibody spec-ficity. In the following section, procedures for acetonerecipitation for protein recovery are briefly described.utside of direct examination of the proteins in the im-unoprecipitate, competition with exogenously added

roteins or peptides can be used to demonstrate specific-ty of the antibody reactions.

aterialsormaldehyde, 37%, reagent grade.lass beads, 425–600 m, acid washed.ysis buffer: 50 mM Hepes–KOH, pH 7.5, 140 mM

NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodiumdeoxycholate, 1 mM PMSF, 1 mg/ml leupeptin, 1 3

IG. 1. Schematic drawing of the basic protocol for chromatin immunoxample. “Lollipop” symbols represent hyperacetylated nucleosomes an

P wash 1: Lysis buffer containing 500 mM NaCl.P wash 2: 10 mM Tris–HCl, pH 8.0, 250 mM LiCl,

0.5% NP-40, 0.5% sodium deoxycholate, 1 mMEDTA.

P elution solution: 1% SDS, 0.1 M NaHCO3.03 proteinase K buffer: 0.1 M Tris, pH 7.8, 50 mMEDTA, 5% SDS.

roteinase K, 20 mg/ml in H2O.

ell Growth and Extract Preparation

1. Grow yeast cells in appropriate medium to de-ired density. It is convenient to count the number ofells/ml. Typically, a 50-ml culture at 1.0 OD /ml (ca.

600

–5 3 107 haploid cells/ml, depending on the strain

mg/ml pepstatin A. used) is sufficient.

precipitation methods, using antiacetylated histone antibodies as and histones.

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2. Add 1.35 ml of 37% formaldehyde to the culturefinal 5 1%). Leave the suspension at room tempera-ure, shake occasionally to mix, for 15 min.

3. (Optional). Add 0.47 g (final 5 125 mM) of glycineo the suspension to stop the fixation. Incubate at roomemperature for 5 min.

4. Harvest cells by spinning at 5000g for 5 min at°C. Wash the pellet with cold TBS (20 mM Tris, pH.4, 150 mM NaCl). Repeat the wash once. The pelletan then be stored at 280°C in a freezer until allamples have been collected.5. Suspend cells in 400 ml of Lysis buffer at 4°C.

ransfer the suspension to 1.5-ml Eppendorf tubes.6. Add 0.5 g of acid-washed glass beads. Vortex at

he highest setting at 4°C for 40 min; this should reachreater than 90% cell breakage. We typically use a-inch multiple sample platform to hold the tubes dur-ng vortexing.

7. Collect the suspension by puncturing a hole withno. 25 needle at the bottom of each tube. Pulse-spin tonother tube. Replenish PMSF to the lysate.8. Sonicate the cell lysate. We use a Branson sonifier

50 set at 30–40% output, 90% duty cycle, 5 s for siximes. The average chromatin fragment size is about00 bp under this condition. Cell lysate is chilled onrushed dry ice pellets briefly and then kept on wet iceetween each sonication.9. Centrifuge at 14,000g for 5 min to remove cell

ebris. Transfer the lysate to another tube and repeatentrifugation for another 15 min. Collect the lysate.his is the whole-cell extract (WCE) ready for immu-oprecipitation. WCE can be stored at 280°C for sev-ral months.

mmunoprecipitation

1. Add appropriate amount of antibody to the lysate.ring the volume to at least 200 ml with cold Lysisuffer supplemented with protease inhibitors.The amount of antibody used should be determined

mpirically. Example: We use 1 ml antidiacetylatedistone H3 antiserum for every 3 3 107 cells WCE. Forsingle-copy gene, WCE made from 6 3 107 cells

recipitated by this antibody yields satisfactory signalsor slot-blot hybridization. Quantitative PCR requiresuch less raw material.2. Rotate the reaction at 4°C for 4 h to overnight.3. Before adding protein A beads, “mock” spin the

ysate for 30 s and transfer the supernatant to anotherube.

4. (Optional). Add 20 mg of single-stranded salmonperm DNA as a carrier. Note: It is convenient toesolve DNA purified from the unbound fraction on angarose gel to measure the fragment size distribution.n this case, carrier DNA should not be used.

5. Equilibrate the protein A beads (e.g., protein Aepharose 4 fast flow, Pharmacia, Piscataway, NJ)

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ith Lysis buffer. Add six antiserum volumes of beads1:1 slurry, equilibrated with Lysis buffer before use).otate at 4°C for 1 h.6. Collect the beads by spinning at 14,000g for 15 s.

ave the supernatant; this is the unbound fraction.7. Wash the beads with 1.5 ml of Lysis. Rotate at

oom temperature for 5 min. Collect the beads andepeat washes sequentially with wash buffer 1, washuffer 2, and TE, pH 8.0.8. Suspend the beads in 100 ml of TE, pH 8.0, con-

aining 20 mg of DNAse-free RNAse A. Incubate at7°C for 30 min.9. Add 1 ml of TE, mix, and collect the beads.10. Add 250 ml of IP elution solution and incubate at

oom temperature for 15 min. Vortex occasionally. Col-ect the supernatant and repeat elution once. Pool theupernatant.11. Add 20 ml of 5 M NaCl to the eluent. Incubate at

5°C for 5 h to revert the cross-link.It is important to revert the cross-links from a cer-

ain volume of WCE (usually 10% of that used formmunoprecipitation) or unbound fraction at the sameime. These will be needed as the input control for laternalyses.12. Precipitate protein and DNA with the addition ofml 99% ethanol. Incubate at 220°C for several hours.13. Spin at 14,000g at 4°C for 15 min. Wash the

ellet with cold 70% ethanol. Spin and air dry theellet.14. Dissolve the pellet in 100 ml of TE, pH 8.0. (Go to

tep 15 directly if proteins are to be recovered.) Add 11l of 103 proteinase K buffer and 1 ml of 20 mg/ml ofroteinase K. Incubate at 50°C for 30 min. It may beifficult to dissolve the pellet initially; however, ithould be cleared shortly after adding proteinase K.15. Extract with phenol/chloroform/isoamyl alcohol.

ack-extract the first organic phase once with same vol-me of TE, pH 8.0. Pool the two aqueous solutions andepeat phenol extraction once. Ethanol-precipitate theNA. Dissolve the final product in 100 ml of TE, pH 8.0.16. If proteins in addition to DNA sequences are to be

urther analyzed, for example, for the identity of otheractors in the same cross-linked complexes, 10 volumes ofcetone can be added to the pooled organic phase. Chillhe mixture at 220°C overnight followed by centrifuga-ion at 14,000g for 30 min. Wash once with cold acetoneprechilled to 220°C) and air dry the pellet. The proteinellet can then be dissolved in SDS–PAGE loading bufferor electrophoresis and other analyses.

nalyses

. Slot blot hybridization:

1. Denature the DNA for blotting by first bringing

he DNA solution to 0.3 N NaOH, 100 ml, and incubatet 65°C for 30 min. Cool to room temperature and add

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431PROTEIN–DNA INTERACTIONS AND CHROMATIN ENVIRONMENT

ame volume of 2 M ammonium acetate. Add 150 ml of2O and 150 ml of 203 SSC (final 5 63). Keep on ice.DNA samples blotted to each membrane should in-

lude the immunoprecipitation bound fraction, totalnput DNA, and (optional) the unbound fraction. It isritical to include several doses of two- or threefolderially diluted input DNA as the standard dose curveor later quantitation. The amount of total input DNAsed varies in each case, depending on the efficiency of

mmunoprecipitation. For example, we typically immu-oprecipitate about 10% of transcriptionally activatedIS3 gene using the anti-diacetylated histone H3 an-

ibody (48). Therefore, the input DNA blotted is usually0% of that used for immunoprecipitation.2. Prepare two pieces of membrane (nitrocellulose,

ylon, etc.) and blot denatured DNA samples to theembrane according to the instruction of the dot or

lot blotter used. It is convenient to load 90% of theNA to one membrane and the rest of the material tonother membrane. The former is to be repetitivelyrobed with specific gene probes and the latter withotal genomic probes (i.e., use sheared genomic DNA ashe template for probe synthesis). Probing withenomic DNA gives a good estimate of the percentagef total chromatin being precipitated by the test anti-ody.3. Any standard hybridization procedures can then

e used to detect and quantify the genes of interest.

. Quantitative PCR:It is essential to first test conditions for quantitative

CR. The final PCR products should show a linear doseesponse; that is, the final yields of each PCR fragmentre proportional to the relative input amounts. To dohis, different doses of the immunoprecipitated andnput DNA should be tried. Equally important is choos-ng an appropriate internal control. For example, whentudying histone acetylation and gene activation, wese the 39 end of the ACT1 gene as an internal controlor quantitative PCR (M.H.K. and C.D.A., manuscriptn preparation). The expression of ACT1 is constitutivend is independent of Gcn5p, and most importantly,he acetylation state of this region does not vary whilecn5p histone acetyltransferase activity is altered.he following conditions are our typical quantitativeCRs assaying histone acetylation status of yeastIS3 gene: 1/800 of immunoprecipitated DNA frag-ents (from 1.5 3 108 cells) are added to 50-ml PCR

eaction mixture (10 mM Tris–HCl, pH 9.0, at 25°C, 2M MgCl2, 50 mM KCl, 1% Triton X-100, 0.2 mM each

NTP, 0.9 mM each primer, 2.5 U Taq polymerase).he amplification parameters are as follows: 94°C 4in, 50°C 1 min, 72°C 20 s (varies with expected DNA

ragment size), 2 cycles; 94°C 1 min, 50°C 1 min, 72°C0 s, 24 cycles; 72°C 2 min. To visualize the products,

e usually phenol-extract and ethanol-precipitate theCR fragments followed by resolving in appropriate gel

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atrix. For quantitation, we use an AlphaImager dig-tal camera (Alpha Innotech Corp.) to capture thethidium bromide-staining image and quantify withhe AlphaEase program. Alternatively, we end-labelhe primer (only one of each pair) with T4 polynucle-tide kinase and add 105 cpm (about 30 fmol at 5 3 108

pm/mg) to each reaction in addition to the original 0.9M unlabeled primers. Quantitation can then be donesing PhsphorImager (Molecular Dynamic) after dry-

ng the gels.

IMITATIONS AND CAUTIONS WITH THE BASICSSAY

Obviously, the success of these methods is likely toe protein- and antibody-dependent but the method’sresent track record suggests that this technique isxtremely versatile and widely applicable to problemseing studied in a diverse group of organisms. How-ver, we stress that these procedures give, at best,elative enrichment of DNA fragments and not abso-ute purification. Thus, the likelihood of success usinghis method depends upon the questions being asked,he abundance of the desired protein-DNA adductsnder investigation, the quality and affinity of thentibodies being used, and, perhaps most importantly,he size and complexity of the genome being assayed.n some cases, subcloning and identifying DNA frag-ents that are coimmunoprecipitated may be an im-

ortant goal. For example, Lis and colleagues recentlyemonstrated, following UV cross-linking, the purifi-ation and identification of DNA sequences bound byNA pol II in vivo (65). In this study, Law et al. showed

hat a significant portion of transcriptionally engagedNA pol II molecules pause near the 59 end of genes.oward this end, we suggest that the antibodies usedhould be first tested to obtain the optimal “signal-to-oise” ratio of final DNA products. To further reduceonspecific retention of unwanted contaminating DNAequences, reiterating the immunoprecipitation stepsefore thermal-reversing the cross-links may need toe considered.It is worth noting that certain histones, particularly

he amino-terminal tail domain of H3 (containing mostf the known acetylation sites), is extremely sensitiveo proteolysis. Of course, this may also be true for otherroteins and their modifications. Thus, even thoughells have been treated with formaldehyde, it is pru-ent to include appropriate protease inhibitors. Simi-arly, in using antisera directed at other potentiallyabile modifications (i.e., phosphorylation, etc.), work-rs need to consider conditions compatible with main-

aining the modification under examination. Along thisine, cross-linking and buffer compositions may affect

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432 KUO AND ALLIS

he conformation and exposure of some antigenic de-erminants.

ONCLUSIONS AND FUTURE DIRECTIONS

Current studies clearly demonstrate the versatilityf the chromatin immunoprecipitation technique, andhe potential utility of this technique seems limitless.ne example will suffice. It is well known, for example,

hat one of the female X chromosomes is inactivateduring early development and that immunocytologicaltudies show that this condensed chromosome is gen-rally underacetylated with respect to histones (e.g.,1, 42, 66, 67). However, some genes are known toscape X inactivation (e.g., 68, 69). It remains an in-eresting question to determine whether these genesre embedded in “islands” of hyperacetylation in antherwise underacetylated chromosome. The chroma-in immunoprecipitation technique presented here notnly helps to test this possibility but may also providetool to identify those loci escaping X inactivation for

urther analyses. The same rationale may be applica-le to identification of novel DNA sequences andenomic loci that are associated with a given DNAinding protein/complex.Numerous regulatory mechanisms are achieved via

ransient, yet stringently controlled, changes in bio-hysical characteristics of proteins, such as posttrans-ational modification and protein–protein as well asrotein–DNA interactions. The use of formaldehyde forxation provides a “snapshot view” of these eventsccurring within their native chromatin context. Anppropriately chosen antibody allows for specific en-ichment for proteins of interests and, importantly, thessociated DNA sequences. By combining the chroma-in immunoprecipitation assay with recent advances inenome-wide expression analyses (70, 71), the possibil-ties for gaining critical new information at a genomicevel seem endless. Moreover, success has been ob-ained using commercially available monoclonal anti-odies against HA- and myc-tagged proteins, clearlyndicating that many more proteins directly or indi-ectly involved in DNA binding can be convenientlynalyzed in situ by chromatin immunoprecipitationssays. In sum, we have been witnessing the widepplicability of this method to different organisms androteins, and we anticipate that more biologically rel-vant questions will be addressed through the use ofhis powerful assay.

CKNOWLEDGMENTS

Numerous workers have contributed to the development of the inivo formaldehyde fixation and chromatin immunoprecipitation

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ethod. In our historical review, we have tried to acknowledge somef the key papers that pioneered these methods and helped to makehem generally applicable. We apologize to those whose work was notighlighted or mentioned due to space limitations. Our work in thiseneral area has been supported by research grants from the NIHGM53512). We are also grateful to assistance from Upstate Biotech-ology Inc. (Lake Placid, NY) in the continued development ofodification-specific histone antibodies. Several of the antibodies

escribed in this report (i.e., acetylation and phosphorylation-specificistone antibodies) are commercially available from them.

Note added in proof. Since this paper was prepared, severalapers have appeared putting the chromatin immunoprecipitationssay to elegant use. Importantly, proteins such as yeast Ada2p thatarticipate in multiunit complex and are brought to chromatin vianteraction with other DNA-binding transcription factors can also bessayed by standard chromatin immunoprecipitation methods. In-erested readers should consult the following publications: Cosma,. P., Tanaka, T., and Nasmyth, K. (1999) Cell 97, 299–311; Krebs,

. E., Kuo, M.-H., Allis, C. D., and Peterson, C. L. (1999) Genes Dev.3, 1412–1421; Coffee, B., Zhang, F., Warren, S. T., and Reines, D.1999) Nat. Genet. 22, 98–101.

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