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TECHNICAL BRIEF Faster and easier chromatin immunoprecipitation assay with high sensitivity Hidetsugu Kohzaki 1, 2 and Yota Murakami 2 1 Venture Laboratory, Kyoto Institute of Technology, Kyoto, Japan 2 Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin- binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simulta- neously, and is more sensitive than the standard ChIP assay. Received: May 2, 2006 Revised: August 28, 2006 Accepted: September 27, 2006 Keywords: ChIP assay (chromatin immunoprecipitation assay) / Chromatin binding protein / Cross-linked protein–DNA complex / PCR 10 Proteomics 2007, 7, 10–14 A number of chromatin-associated proteins are involved in regulating chromosomal functions [1], including tran- scription [2–4], DNA replication [5–7], and recombination [8]. They also help in regulating the chromatin structure [9– 13]. To understand the role of chromatin-associated proteins in chromosomal functions, it is essential to clarify where these regulatory proteins bind to the chromatin in vivo. The chromatin immunoprecipitation (ChIP) assay was devel- oped for this purpose, and it has proved to be an exquisite tool that has been widely used in the study of chromosome function [2–11]. In the standard ChIP assay [5, 6, 10], living cells are treated with a reagent that crosslinks the protein complex to its neighboring DNA, and the crosslinked DNA is frag- mented into pieces by sonication. The resulting protein– DNA complexes are then immunoprecipitated by an anti- body against the protein of interest. The DNA fragments in the precipitates are subsequently recovered after reversing the crosslinking process and are subjected to PCR using primers that amplify specific target DNA sequences. Thus, the ChIP assay allows one to examine whether a target pro- tein binds to a particular region of the chromosome. In the ChIP assay, the most widely used crosslinking reagent is formaldehyde. To reverse formaldehyde cross- linking, the fragments are incubated overnight at 657C with TES (buffer of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS). Then the released DNA fragments have to be purified prior to PCR by another lengthy proce- dure that includes incubation with proteinase K, repeated phenol–chloroform extraction, ethanol precipitation, and treatment with RNase. This purification step on its own consumes a whole day, and these two laborious and time- consuming steps are a major limitation of the standard ChIP assay. Here, we describe an improved ChIP assay that is both rapid and simple. Hereafter, we described the improved ChIP assay as rapid ChIP assay. The procedure was based on the method described by Tanaka et al. [5]. Briefly, yeast cells were cultured in 1% yeast extract–2% polypeptone–2% dex- trose (glucose) (YPD) overnight [14], after which 20 mL of the culture was introduced into 20 mL of YPD (total, 40 mL), and the cells were grown for 1 h at 307C. The resulting yeast cells Correspondence: Dr. Hidetsugu Kohzaki, Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sho- goin, Sakyo-ku, Kyoto 606-8507, Japan E-mail: [email protected] Fax: 181-75-723-5708 Abbreviations: ChIP assay, chromatin immunoprecipitation assay; HA, hemagglutinin mAb peptide epitope (YPYDVPDYA) derived from the human influenza hemagglutinin protein; TE, buffer of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA; TES, buffer of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS; WCE, whole cell extract; YPD, 1% yeast extract–2% polypeptone–2% dextrose (glucose) DOI 10.1002/pmic.200600283 © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Faster and easier chromatin immunoprecipitation assay with high sensitivity

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Page 1: Faster and easier chromatin immunoprecipitation assay with high sensitivity

TECHNICAL BRIEF

Faster and easier chromatin immunoprecipitation

assay with high sensitivity

Hidetsugu Kohzaki1, 2 and Yota Murakami2

1 Venture Laboratory, Kyoto Institute of Technology, Kyoto, Japan2 Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan

Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We havedeveloped a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates.This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It alsorequires a less immunoprecipitating antibody, permits many samples to be tested simulta-neously, and is more sensitive than the standard ChIP assay.

Received: May 2, 2006Revised: August 28, 2006

Accepted: September 27, 2006

Keywords:

ChIP assay (chromatin immunoprecipitation assay) / Chromatin binding protein /Cross-linked protein–DNA complex / PCR

10 Proteomics 2007, 7, 10–14

A number of chromatin-associated proteins are involvedin regulating chromosomal functions [1], including tran-scription [2–4], DNA replication [5–7], and recombination[8]. They also help in regulating the chromatin structure [9–13]. To understand the role of chromatin-associated proteinsin chromosomal functions, it is essential to clarify wherethese regulatory proteins bind to the chromatin in vivo. Thechromatin immunoprecipitation (ChIP) assay was devel-oped for this purpose, and it has proved to be an exquisitetool that has been widely used in the study of chromosomefunction [2–11].

In the standard ChIP assay [5, 6, 10], living cells aretreated with a reagent that crosslinks the protein complex toits neighboring DNA, and the crosslinked DNA is frag-

mented into pieces by sonication. The resulting protein–DNA complexes are then immunoprecipitated by an anti-body against the protein of interest. The DNA fragments inthe precipitates are subsequently recovered after reversingthe crosslinking process and are subjected to PCR usingprimers that amplify specific target DNA sequences. Thus,the ChIP assay allows one to examine whether a target pro-tein binds to a particular region of the chromosome.

In the ChIP assay, the most widely used crosslinkingreagent is formaldehyde. To reverse formaldehyde cross-linking, the fragments are incubated overnight at 657Cwith TES (buffer of 50 mM Tris-HCl (pH 8.0), 10 mMEDTA, and 1% SDS). Then the released DNA fragmentshave to be purified prior to PCR by another lengthy proce-dure that includes incubation with proteinase K, repeatedphenol–chloroform extraction, ethanol precipitation, andtreatment with RNase. This purification step on its ownconsumes a whole day, and these two laborious and time-consuming steps are a major limitation of the standardChIP assay.

Here, we describe an improved ChIP assay that is bothrapid and simple. Hereafter, we described the improvedChIP assay as rapid ChIP assay. The procedure was based onthe method described by Tanaka et al. [5]. Briefly, yeast cellswere cultured in 1% yeast extract–2% polypeptone–2% dex-trose (glucose) (YPD) overnight [14], after which 20 mL of theculture was introduced into 20 mL of YPD (total, 40 mL), andthe cells were grown for 1 h at 307C. The resulting yeast cells

Correspondence: Dr. Hidetsugu Kohzaki, Department of ViralOncology, Institute for Virus Research, Kyoto University, Sho-goin, Sakyo-ku, Kyoto 606-8507, JapanE-mail: [email protected]: 181-75-723-5708

Abbreviations: ChIP assay, chromatin immunoprecipitationassay; HA, hemagglutinin mAb peptide epitope (YPYDVPDYA)derived from the human influenza hemagglutinin protein; TE,

buffer of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA; TES, buffer of50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS; WCE, wholecell extract; YPD, 1% yeast extract–2% polypeptone–2% dextrose(glucose)

DOI 10.1002/pmic.200600283

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Proteomics 2007, 7, 10–14 Technology 11

(total, 5.86108) were then crosslinked with 1% for-maldehyde for 30 min at room temperature. After the addi-tion of 125 mM glycine and subsequent incubation for5 min, the cells were harvested and washed with TBS. After awash with lysis buffer (50 mM HEPES-KOH [pH 7.5],140 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% Na-deoxycholate), the cells were broken up with glass beads in500 mL of lysis buffer. The cell extracts were sonicated tofragment the DNA. The conditions for the sonication mustbe determined empirically. In our case, extracts were soni-cated 90 times at the rate of 1 s/cycle with 1 s intervals usingan ultrasonic homogenizer (Channel “4” on an AstrasonXL2020, Misonix, Farmingdale, NY, USA) and a tip (MisonixCat : 420) with a 3.2-mm diameter of stepped type, very highintensity, high strength, 205 mm amplitude, and 14 cmlength to yield DNA fragments that were on average 1500 bplong (data not shown). This step was performed on ice toavoid heating the cell preparation. The sonicated extractswere clarified by centrifugation and brought up to 500 mLwith lysis buffer. The entire preparation, except for 50 mL thatserved as the whole cell extract (WCE), was then incubatedwith an anti-hemagglutinin mAb (anti-HA (HA is hemag-glutinin mAb peptide epitope (YPYDVPDYA) derived fromthe human influenza hemagglutinin protein) clone 12CA5,Roche, 0.4 mg/mL) at 47C for 3 h.

To block the nonspecific binding of proteins and DNA,magnetic beads coated with Protein G (Dynal beads) werewashed three times with 1 mL of PBS and once with 1 mL oflysis buffer containing 5 mg/mL BSA. The beads were thentreated for 3 h at 47C with lysis buffer containing 10 mg/mLof salmon sperm DNA and 4.5 mg/mL BSA. The blockedmagnetic beads were added to the antibody-treated extract,mixed for 3 h at 47C, collected with a magnetic apparatus(Dynal), and then washed three times with 1 mL of lysisbuffer, three times with 1 mL of lysis buffer containing360 mM NaCl (final concentration 0.5 M), three times with1 mL of washing buffer (10 mM Tris-HCl (pH 8.0), 250 mMLiCl, 0.5% NP 40, 0.5% Na-deoxycholate, and 1 mM EDTA),and once with 1 mL of TE (buffer of 10 mM Tris-HCl(pH 8.0) and 1 mM EDTA). All buffers contained proteinaseinhibitors (100 mM 4-(2-aminoethyl)benzenesulfonyl fluo-ride, 80 mM aprotinin, 1.5 mM E-64, 2 mM leupeptin, 5 mMbestatin, and 1 mM pepstatin A) and 1 mM PMSF. Finally,the precipitates were washed twice with 1 mL of TE andsubjected either to the standard ChIP assay or the rapid ChIPassay as described below.

In the standard ChIP assay, the precipitates were incu-bated with 125 mL of TES at 657C for 15 min and the super-natant was collected. The extraction with TES was repeatedonce, and the resulting supernatant was combined with thefirst supernatant (total volume 250 mL). As a control, 5 mL ofWCE was added to 245 mL of TES. The supernatant andcontrol WCE were then incubated overnight to reverse thecrosslinking. To obtain purified DNA, the samples andcontrol WCE were added to 250 mL of water (distilled andautoclaved) and treated with 12.5 mL of 20 mg/mL protein-

ase K for 2 h, extracted with phenol-chloroform, precipitatedwith 1 mL of ice-cold ethanol, stored at –807C for 1 h, dried,suspended in 40 mL of TE, and incubated for 1 h at 377Cwith 5 mL of 10 mg/mL RNase A. One microliter of theresulting purified DNA was then subjected to PCR asdescribed below.

In the rapid ChIP assay, the precipitated beads were sus-pended in 40 mL of TE. One microliter of the resulting beadsmixture was then subjected to PCR as described below. To getcontrol WCE DNA, 5 mL of WCE was mixed with 245 mL ofTE and extracted sequentially with equal volumes of phenol,phenol-chloroform, and chloroform. The extracted samplewas precipitated with ethanol and suspended into 10 mL ofTE. PCR was carried out in a 25-mL volume containing 1 mLof the immunoprecipitated bead suspension or the controlWCE sample. For the immunoprecipitated sample, the sus-pension was first extensively mixed by vortexing just beforetaking the aliquot for the PCR.

PCR was carried out in a 25-mL volume using Taq poly-merase (AmpliTaq Gold: Applied Biosystems) and the buffersystem recommended by the suppliers.The PCR primers usedto detect ARS1 were (WT-50) 50-GGGGGTCGACTAG-CAAATTTCGTCAAAAATGCTAAGAAATAGGTTATT-30 and(K30-1) 50-GGGGAATTCGTGAAATGGTAAAAGTCAAC-CC-30, and those used to detect CYC1 were (CYC150) 50-CCGTGGAAAAGGGTGGCCCACATAAGGTTG-30 and(CYC130) 50-CGTCCCACAACACGTTTTTCTTGATATTG-GCATC-30. We avoided repeated freeze-thawing of the PCRprimers to decrease non-specific amplification. The PCR pro-tocol consisted of an initial denaturation step for 5 min at 947Cfollowed by 30 cycles of denaturation for 1 min at 947C,annealing for 1 min at 537C, and polymerization for 2 min at727C, and a final extension step of 7 min at 727C. The PCRproducts were separated in a 2.3% agarose gel and stained with0.2 mg/mL ethidium bromide. Images of the gels were print-ed or saved in electronic files (jpeg format) using Printgraph(ATTO). Quantification was carried out using a Fluor-S™ mul-tiImager and Quantity One image analyzing software(BioRad) [15, 16]. Both ChIP assays were repeated severaltimes to assess their reproducibility.

To assess how our rapid ChIP assay compares to thestandard ChIP assay, we chose to test two chromatin-asso-ciated proteins that bind to chromatin at the origin in thebudding yeast Saccharomyces cerevisiae, the origin recogni-tion complex (ORC) and the MCM complex. These con-served complexes have been well characterized previouslyusing the standard ChIP assay [5–7]. ORC is a six-subunitcomplex that specifically binds to replication originsthroughout the cell cycle and serves as a landing pad forreplication proteins [6], whereas the MCM complex, whichconsists of MCM2 through 7, transiently associates with theorigin during anaphase to form a prereplicative complex.The MCM complex is thought to be an initiation helicasethat is activated at the G1-S transition and dissociates fromchromatin after replication [17–20]. We chose ARS1 and thenon-origin sequence CYC1 to be the target and control DNA

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12 H. Kohzaki and Y. Murakami Proteomics 2007, 7, 10–14

Figure 1. Comparison of signal intensities obtained with the rapid and standard ChIP assays.(A) Comparison of the sensitivities of the rapidChIP and standard ChIP assays using lysates from cells expressing Orc1-3HA. The lysates were immunoprecipitated with an anti-HA anti-body and the existence of ARS1 and CYC1 DNA in the immunoprecipitates were examined by rapid ChIP assay (lane 6) or the standard ChIPassay (lane 5). Lane M, DNA size markers (200, 300, 400, and 500 bp); lanes 1–3, PCR controls with no DNA (lane 1), an ARS1 positive controlconsisting of 0.1 mg of pHK806 [14] (ARS1 P.C., lane 2), or a CYC1 positive control containing 0.1 mg of pSKScCYC1 (CYC1 P.C., lane 3). Lane 4contains the PCR with DNA prepared from the WCE of Orc1-3HA-expressing cells without crosslink removal, as described in the text. Weverified the PCR products by sequencing. (B) The crosslink removal step can be omitted. WCE DNA was used directly for PCR (phenol, lanes5 and 6) or first subjected to crosslink removal by overnight treatment at 657C in Tris/EDTA/SDS, followed by PCR (TES, lanes 3 and 4). TheWCE were from SP1 cells (lanes 3 and 5) or cells expressing Orc1-3HA (lanes 4 and 6). M, DNA size markers; lanes 1 and 2, PCR controls withno DNA (lane 1) or positive control DNA consisting of 1 mg of pHK806 (lane 2). The asterisk shows nonspecific bands or excess primers. Wealso compared PCR products from WCEs with those from plasmids: no DNA (lane 7), 0.1 mg of pHK806 (lane 8), and CYC1 positive controlcontaining 0.1 mg of pSKScCYC1 (lane 9). Lanes 10 and 11; the ARS1 and CYC1 PCR with DNA prepared from the WCE of Orc1-3HA–expressing cells without crosslink removal. (C and D) Comparison of the sensitivities of the rapid ChIP and standard ChIP assays usinglysates from a-factor-arrested (C) or nocodazole-arrested (D) cells expressing HA-tagged MCM4. M, DNA size markers; lanes 1 and 2, PCRcontrols with no DNA (lane 1) or with 0.01 mg of pHK806 DNA (lane 2). Lane 3 indicates the PCR with DNA prepared from the WCE fromMCM4-3HA expressing cells without crosslink removal. The remaining lanes show the PCRs with immunoprecipitated DNA prepared bythe standard or rapid ChIP assays using the indicated amounts of anti-HA antibody.

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Proteomics 2007, 7, 10–14 Technology 13

sequences in our ChIP assay, respectively, because previousstandard ChIP assays have demonstrated that both ORC andthe MCM complex bind to ARS1 but not to CYC1 [7].

To compare the sensitivities of the rapid ChIP assay andthe standard ChIP assay, we performed them side by sideusing asynchronously growing cells that express Orc1 taggedwith three copies of the HA epitope (Fig. 1A). As expected,the ARS1 fragment but not the CYC1 fragment was detectedin the precipitates prepared with both assays (lanes 5 and 6).The bands were quantified with a Fluor-S multiImager andQuantity One image analyzing software (BioRad, Hercules,CA). The amounts of PCR products obtained with thestandard ChIP assay and the rapid ChIP assay relative to thatobtained from WCE were 0.0573 6 0.0386 (n = 9, p,0.0001),and 0.9112 6 0.1139 (n = 6, p,0.0001), respectively. Thus,the signal obtained with the rapid ChIP assay was about fif-teen times stronger than the signal generated by the stand-ard ChIP assay.

We found that we could omit the crosslink reversal stepfrom the procedure used to isolate DNA from the WCE, aswe could still detect both ARS1 and CYC1 fragments whenPCR was performed on WCE DNA prepared without cross-link reversal (lane 4 of Fig. 1A). We then tested this directlyby performing PCR on yeast WCE DNA prepared with orwithout crosslink removal (Fig. 1B). Equivalent levels ofARS1 and CYC1 PCR products were generated from thetreated and untreated WCEs. Moreover, we could not detect adifference between the PCR products from WCEs and con-trol plasmid DNAs (Fig. 1B, compare lane 8 with lane 10 andlane 9 with lane 11). We also directly sequenced the observedPCR products on an ABI PRISM 3100 Genetic Analyzerusing a DNA sequencing kit (Big Dye Terminator). We con-firmed that the observed bands were ARS1 and CYC1. Thus,it is not necessary to remove the crosslinks before PCRamplification in some cases, perhaps because the PCR heat-ing step partially removes the crosslinking.

We also performed the standard and rapid ChIP assayswith cells expressing HA-tagged MCM4. The yeast cellswere synchronized at G1 or M phase with a-factor andnocodazole, respectively. Both the standard and rapid ChIPassays showed that the MCM complex was loaded ontochromatin in the a-factor-arrested cells (Fig. 1C), but not inthe nocodazole-arrested cells (Fig. 1D) as indicated by pre-vious studies [5, 6]. However, the rapid ChIP assay requireda smaller amount of antibody than the standard ChIPassay: 0.5 mL of antibody was sufficient to clearly detect theARS1 fragment with the rapid ChIP assay, whereas 8 mLwas needed in the standard ChIP assay (Fig. 1C, lanes 9and 11). The amounts of PCR products obtained with therapid ChIP assay with 0.5 mL of antibody and the rapidChIP assay with 1.0 mL of antibody relative to that obtainedwith the standard ChIP assay with 8mL of antibody are4.110 6 1.446 (n = 7, p,0.05), and 5.343 6 1.966 (n = 7,p,0.05), respectively. Therefore, the rapid ChIP assayrequires at least 16 times less antibody for the same resultas the standard assay.

In summary, we have shown that several time-consum-ing steps of the standard ChIP assay can be omitted by di-rectly using the immunoprecipitates as PCR templates. Withthis method, we detected both constitutive (ORC) and tran-sient (MCM4) binding of proteins to chromatin. Recently,Nelson et al. [21] developed a “fast chromatin immunopreci-pitation assay”, in which they omitted the overnight incuba-tion step for crosslink reversal as well as the phenol–chloro-form extraction step by using Chelex-100 resin to extractDNA from the immunoprecipitates. However, their proce-dure included several steps such as repeated boiling step forChelex extraction, a step for the addition of a specific peptide,and a proteinase K digestion step. In comparison, our rapidChIP assay is a simpler and more time-efficient method thatdoes not require any special reagents.

Thus, our method allows for the speedy detection ofspecific DNA-protein interactions. It has the added advan-tage that only small amounts of antibodies are needed andthat several samples can easily be assayed at the same time.This assay is also likely to be useful in the analysis of pro-tein–chromatin interactions for organisms other than yeast.Recently, genome-wide distribution analysis became avail-able using ChIP-on-Chip [22, 23]. We expect that our mod-ification will lead to a significant improvement in the ChIPassay.

We thank H. Araki for the Orc1-3HA and Mcm4-3HAexpressing yeast strains. We are also grateful to Y. Kawasaki, A.Abiko, H. Kato, H. Fukui, I. Matsunaga, M. Itoh, S. Nishino, K.Kamei, S. Hara, and M. Sugita for helpful discussions.

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