Detection of Histone Modification by Chromatin Immunoprecipitation

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Detection of histone modification by chromatin immunoprecipitation combined zinc finger luciferasebased bioluminescence resonance energy transfer assay Wataru Yoshida, Aki Kezuka, Koichi Abe, Hironobu Wakeda, Kazuhiko Nakabayashi, Kenichiro Hata, and Kazunori Ikebukuro Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401036k • Publication Date (Web): 01 Jun 2013 Downloaded from http://pubs.acs.org on June 6, 2013

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Analytical Chemistry

Detection of histone modification by chromatin immunoprecipitation

combined

zinc

finger

luciferase-based bioluminescence resonance energy transfer assay Wataru Yoshida†, Aki Kezuka†, Koich Abe†, Hironobu Wakeda‡, Kazuhiko Nakabayashi§, Kenichiro Hata§, Kazunori Ikebukuro†*. †

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo

University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan ‡

Department of Urology, University of Miyazaki, 5200, Kihara, Kiyotake-cho, Miyazaki 889-

1692, Japan §

Department of Maternal-Fetal Biology, National Research Institute for Child Health and

Development, 2-10-1 Okura-cho, Setagaya-ku, Tokyo 157-8535, Japan

ACS Paragon Plus Environment

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ABSTRACT Epigenetic modification such as DNA methylation and histone modification have important roles in gene regulation. Epigenetic modification can be altered by environmental influences and are related to diseases. Therefore, epigenetic modifications may serve as biomarkers. In this study, we developed a convenient histone modification detection system by combining chromatin immunoprecipitation (ChIP) and bioluminescence resonance energy transfer (BRET)-based homogeneous PCR product detection system using zinc finger fused to luciferase (ZF–luciferase) with DNA intercalating dye (ChIP-ZF-BRET assay). The ChIP-ZF-BRET assay comprises the following 3 steps: (1) ChIP, (2) PCR amplification of the target genomic region, which includes a zinc-finger recognition site, and (3) homogenous detection of the PCR product by BRET using ZF–luciferase and fluorescent DNA intercalating dye. Using this system, we conveniently and accurately detected target histone modification at the androgen receptor gene promoter region in LNCaP and Du145 cells. The system can be applicable to DNA methylation detection using a methyl-CpG-binding domain protein or methylcytidine antibody instead of histone modification antibodies. Therefore, it may be useful and convenient for simultaneous detection of histone modification and DNA methylation in clinical diagnoses.

INTRODUCTION Epigenetic modifications such as DNA methylation and histone modifications play an important role in gene regulation.1-3 Aberrant epigenetic modifications are related to human diseases;4 therefore, DNA methylation and histone modifications at specific target genes are regarded as biomarkers.5-7 In general, unexpressed gene promoters are hypermethylated and associate with


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silencing histone modifications such as H3K9me3, H3K9me2, and H3K27me3,8 whereas expressed gene promoters are hypomethylated and associate with active histone modifications such as H3K4me3, H3K36me3, and H3K9,14Ac.9 DNA methylation is used for long-term gene silencing, whereas immediate gene expression state is determined by histone modifications.10 However, some genes do not show such a relationship between DNA methylation and histone modification.11,12 Moreover, histone modifying enzyme inhibitors have been approved as drugs;4 therefore, detection of histone modifications at specific target region is important for not only diagnosis but also evaluation of drug treatment. Chromatin immunoprecipitation (ChIP) assay has been widely used to analyze histone modification at target genes.13-15 In the assay, the level of histone modification is determined by the amount of target DNA region in the ChIP sample; thus, quantitative PCR (qPCR) with DNA intercalating dye such as SYBR Green has been utilized.16-18 However, the assay does not distinguish between specific PCR products and non-specific products. To specifically detect a target PCR product, qPCR using fluorescence hybridization probes such as molecular beacons and TagMan® is required.19,20 We focused on a zinc finger protein, which is a well-known transcription factor, as a molecular recognition element for the post-PCR detection system.21-23 Promoter regions are important target areas and transcription factors that recognize promoter regions may be suitable molecular recognition elements for PCR products in an epigenetic modification detection system. In particular, it has been reported that 10309 genes contain human ZIF268 recognition sites in the regions that span 10-kb upstream to 10-kb downstream of the transcriptional start site (TSS) in the human genome.24 We previously reported a DNA methylation detection system using methyl CpG-binding protein (MBD) and zinc finger fused to luciferase (ZF–luciferase).25 In the system, methylated DNA is captured by MBD and then PCR


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is performed to amplify the target genomic region from the captured genomic DNA. The PCR products are immobilized on magnetic beads and are then sequence-specifically detected using ZF–luciferase (ZF-beads assay). We assumed that the platform would be applicable for the detection of histone modification using antibodies instead of MBD. To construct a convenient assay, a homogeneous PCR product detection system is ideal. We report herein a convenient detection system for histone modifications at target genomic regions, designated as chromatin immunoprecipitation combined zinc finger luciferase-based bioluminescence resonance energy transfer assay (ChIP-ZF-BRET assay). This system comprises the following 3 steps: (1) ChIP, (2) PCR amplification of the target genomic region, which includes a zinc-finger recognition site, and (3) homogeneous detection of the PCR product by BRET using ZF–luciferase and fluorescent DNA intercalating dye that is excited at luciferase luminescence (ZF-BRET assay) (Figure 1). Using ChIP-ZF-BRET assay, we analyzed histone modifications at the promoter region of androgen receptor in prostate cancer cell lines.


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Figure 1. Schematic representation of ChIP-ZF-BRET assay Chromatin immunoprecipitation was performed using target histone modification antibodies. The target region that contains the zinc finger recognition site was amplified by PCR. The PCR product was detected by BRET using ZF–luciferase and fluorescent DNA intercalating dye that is excited at luciferase luminescence. The emission intensity of the fluorescent DNA intercalating dye was defined as the BRET signal.


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EXPERIMENTAL SECTION Preparation of zinc-finger fused to luciferase. The Zif268–luciferase fusion protein was prepared as previous study.24 In brief, E. coli BL21 (DE3) cells were transformed by a pET30c vector encoding StrepTag-Zif268–luciferase fusion protein and the fusion protein was expressed using the Overnight Express Auto induction system at 20°C for 48 h. The StrepTag-Zif268– luciferase was affinity purified using a Strep-Tactin Superflow Plus Cartridge (QIAGEN, Hilden, Germany). The purity of the fusion proteins were confirmed by sodium dodecyl sulfatepolyacrlyamide gel electrophoresis (SDS-PAGE). The luciferase activity was measured by PicaGene (Toyo Ink, Tokyo, Japan) and ARVO MX 1420 (PerkinElmer, MA, USA). ZF-BRET assay. The 49-bp target synthetic dsDNA (Table S-1) was incubated with 1 µM BOBO™-3 (Life Technologies, CA, USA) in PBS buffer (pH 7.3) supplied with 90 µM ZnCl2 at RT for 30 min. Then, 50 nM Zif268–luciferase was added to the mixture in 100 µl of reaction volume and incubated at room temperature (RT) for 30 min. After incubation, 100 µl of PicaGene® was added. Luciferase luminescence at 550 nm and BOBO™-3 emission at 602 nm were simultaneously measured by ARVO MX 1420 after 1-min incubation at RT. In the PCR product detection, PCR was performed using 1.0 × 101 to 1.0 × 108 copies of synthetic dsDNA of the AR promoter region (Table S-1), 1.25 U Ex Taq® Hot Start Version, and 1 µM primer mix in 25 µl of reaction volume under the following conditions: 98°C for 5 min; 37 cycles at 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s. A mixture of AR F-primer and AR Rprimer was used as the primer mix (Table S-1). The PCR product (20 µl) was then treated as above to detect luciferase chemiluminescence and BOBO™-3 emission.


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Cell Culture. The HeLa cells and the prostate cancer cell lines (LNCaP and Du145) were cultured in RPMI 1640 medium (GIBCO, Uxbridge, U.K.) containing 10% fetal bovine serum (GIBCO, Uxbridge, U.K.), 100 µg/mL streptomycin, and 100 U/mL penicillin (Omega Scientific, CA) at 37°C in 5% CO2. Chromatin Immunoprecipitation (ChIP). ChIP was performed using MAGnify™ Chromatin Immunoprecipitation System (Life Technologies, CA, USA) with anti-histone H3 (tri methyl K4) antibody, and anti-histone H3 (tri methyl K9) antibody (Abcam, Cambridgeshire, UK). In brief, formaldehyde cross-linked cells (1 × 107) were sonicated using Bioruptor® Sonicator (Diagenode, NJ, USA). The antibody (3 µg) was immobilized on magnetic beads and the sonicated cells (1.7 × 106) were added to the beads. After washing, the beads were incubated with Proteinase K at 55°C for 15 min. DNA was purified from the supernatant, and a total of 150 µl of ChIP sample was obtained. Quantitative PCR (qPCR). qPCR was performed using 5 µl of ChIP sample, 1.25 U of Ex Taq® Hot Start Version (Takara, Shiga, Japan), 1 µM primer mix, and SYBR® Green I Nucleic Acid Stain (LONZA, Basel, Switzerland) in 25 µl of solution by MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad, CA, USA). A mixture of GAPDH F-primer and GAPDH Rprimer was used as the primer mix (Table S-1). The PCR conditions are as follows: 98°C for 5 min; 40 cycles at 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s. ChIP-ZF-beads assay. The target region was PCR-amplified using 5 µl of ChIP sample, 1.25 U Ex Taq® Hot Start Version (Takara Bio, Shiga, Japan), and 1 µM primer mix in 25 µl of reaction volume under the following conditions: 98°C for 5 min; 35 cycles at 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s. A mixture of GAPDH F-primer and the biotinylated GAPDH


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R-primer was used to amplify the GAPDH promoter region, and a mixture of AR F-primer and the biotinylated AR R-primer was used to amplify the AR promoter region (Table S-1). The PCR products were detected by Zif268–luciferase as previously reported.25 In brief, 20 µl of PCR product was captured on magnetic beads (Magnosphere MS300/Streptavidin, JSR Corporation, Tokyo, Japan) through biotin–avidin interaction. Zif268–luciferase (100 nM) was added to the beads and incubated for 15 min. After washing, 100 µl of PicaGene® (Toyo Ink, Tokyo, Japan) was added to the beads, and luminescence was measured after 1-min incubation using ARVO MX 1420 (PerkinElmer, MA, USA). All experiments were performed at RT. ChIP-ZF-BRET assay. The AR promoter region was amplified by PCR using 10 µl of ChIP sample, 1.25 U Ex Taq® Hot Start Version, and 1 µM primer mix in 25 µl of reaction volume under the following conditions: 98°C for 5 min; 35 cycles at 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s. A mixture of AR F-primer and AR R-primer was used as the primer mix (Table S1). The PCR product was detected by ZF-BRET assay as described above.

RESULTS AND DISCUSSION ZF-BRET assay for target DNA detection. We previously reported PCR product detection system by ZF–luciferase.22-24 In the system, PCR product was immobilized on magnetic beads and then detected using ZF–luciferase (ZF-beads assay). The ZF-beads assay is required for several bound/free separation steps to detect luciferase activity of ZF–luciferase bound to target PCR product. Therefore, a homogeneous assay system is required for convenient DNA detection. To construct a homogeneous DNA detection system, we focused on BRET. We assumed that BRET would occur upon co-localized ZF–luciferase and fluorescent DNA intercalating dye that


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is excited by luciferase luminescence in the presence of the target PCR product. We designated the BRET assay for DNA sensing as ZF-BRET assay. We used Zif268 fused to luciferase (Zif268–luciferase) as ZF–luciferase and BOBO™-3 (maximum excitation: 570 nm, maximum emission: 602 nm) as the fluorescent DNA intercalating dye for ZF-BRET assay. The Zif268– luciferase has peak emission of approximately 550 nm (Figure S-1). We prepared a mixture of 50 nM Zif268–luciferase, 1 µM BOBO™-3, and 0 nM–100 nM synthetic double-stranded DNA (dsDNA) that contains the Zif268 recognition site. After addition of luciferase substrate, we measured emission spectra of luciferase and BOBO™-3 between 500 nm and 670 nm. Although emission at 602 nm was not increased in the presence of the non-target dsDNA, emission increased in the presence of the target dsDNA (Figure 2). Moreover, peak emission of approximately 550 nm decreased in the presence of the target dsDNA, suggesting that BRET between Zif268–luciferase and BOBO™-3 occurred specifically on the target dsDNA.


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Figure 2. Emission spectra of ZF-BRET assay for synthetic dsDNA In the presence of target dsDNA (A) or non-target dsDNA (B), emission spectra between 500 and 670 nm were measured.


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The Zif268–luciferase has broad peak emission spectra; therefore, the emission at 602 nm would contain both luciferase and BOBO™-3 emissions. To calculate BOBO™-3 emission at 602 nm, the Zif268–luciferase emission at 602 nm should be subtracted from total emission intensity at 602 nm. The emission of Zif268–luciferase at 602 nm was 0.51-fold of the intensity at 550 nm (Figure S-1); therefore, we used the following equation to obtain BOBO™-3 emission intensity: X = I602nm − 0.51 × I550nm (X = BOBO™-3 emission intensity at 602nm, I602nm = total emission intensity at 602 nm, I550nm= total emission intensity at 550 nm)

We defined the calculated BOBO™-3 emission intensity at 602 nm as BRET signal. As shown in Figure 3-A, we observed increased BRET signal with increased target dsDNA concentration. These results demonstrate that target dsDNA is detected in homogeneous solutions by ZF-BRET assay and the detection limit of the target dsDNA was 25 nM.


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Figure 3. Detection of dsDNA by ZF-BRET assay (A) Detection of synthetic 49-bp dsDNA that contains Zif268 recognition sequence. Several concentrations of the dsDNA were added to a mixture of 50 nM Zif268–luciferase and 1 µM BOBO™-3. The luciferase maximum emission (550 nm) and BOBO™-3 maximum emission (602 nm) were simultaneously measured after addition of luciferase substrate and the BRET signal was calculated (mean ± sd, n = 3). (B) Detection of PCR products amplified from synthetic DNA of the AR promoter region. Several copies of template DNA were used for PCR. The PCR products were directly added to a mixture of 50 nM Zif268–luciferase and 1 µM BOBO™-3 and the BRET signal was measured. As a background, BRET signal was measured for a PCR reaction that was performed in the absence of template DNA. Signal normalized to background is shown (mean ± sd, n = 3).


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To next investigate whether PCR product is detected by the ZF-BRET assay, we amplified Androgen receptor (AR) promoter region that contains Zif268 recognition sequence by PCR from 1.0 × 101 to 1.0 × 108 copies of synthetic dsDNA. The Zif268–luciferase and BOBO™-3 were directly added to the PCR sample and BRET signal was measured. As shown in Figure 3-B, the BRET signal increased with an increase in levels of the target template dsDNA. These results demonstrate that PCR products containing the Zif268 recognition sequence are conveniently detected by the ZF-BRET assay, and the detection limit of the target template dsDNA was 10 copies. We previously reported that the detection limit of the ZF-BRET assay was 170 pM

25

; thus,

the sensitivity of the ZF-BRET assay was lower than that of the ZF-beads assay. However, 10 copies of the target dsDNA were detected using the ZF-BRET assay because the target dsDNA was amplified by PCR. This indicates that the sensitivity of the ZF-BRET assay was sufficient to detect the PCR product, which was amplified from 10 copies of target DNA. Detection of histone modification by ChIP-ZF-beads assay. We previously developed a DNA methylation detection system using MBD and Zif268–luciferase. In the assay, methylated DNA is captured by MBD and the PCR-amplified target genomic region was detected by ZFbeads assay. We expected that histone modification would be detected by the same platform as the DNA methylation detection system. Therefore, we investigated whether histone modification is detected by ChIP followed by ZF-beads assay (ChIP-ZF-beads assay). We first targeted H3K4me3 at the GAPDH promoter region that contains the Zif268 recognition site. The promoter region is reportedly associated with H3K4me3 in HeLa cells. We performed ChIP using H3K9me3 and H3K4me3 antibodies and then the amount of the GAPDH promoter region was analyzed by qPCR. We confirmed the enrichment of H3K4me3 at the GAPDH promoter


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region in HeLa cells (Figure 4-A). We next investigate whether the histone modification is detected by ChIP-ZF-beads assay. We amplified by PCR the GAPDH promoter region from the ChIP samples using biotinylated primers, and the PCR products were immobilized on magnetic beads. After washing the unbound PCR products, Zif268–luciferase was added to the beads. Finally, unbound Zif268–luciferase was removed, and the luciferase activity was measured. We detected high luciferase activity from the H3K4me3 ChIP sample but not the H3K9me3 ChIP sample (Figure 4-B). These results demonstrate that histone modifications are correctly detected by the ChIP-ZF-beads assay, similar to the DNA methylation detection system.


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Figure 4. Comparison between ChIP-qPCR and ChIP-ZF-beads assay (A) Analysis of histone modification at GAPDH promoter region in HeLa cells by ChIP-qPCR. ChIP with control IgG antibody was performed and the IP efficiency was defined as background signal. Background was subtracted, and signal is shown for H3K4me3 and H3K9me3. (B) Analysis of histone modification at GAPDH promoter region in HeLa cells by ChIP-ZF-beads assay. The biotinylated PCR product was immobilized on streptavidin-coated magnetic beads and then the quantities of PCR products were detected using Zif268–luciferase. As a control, ChIP using anti-IgG antibody was performed and normalized signal is shown for H3K4me3 and H3K9me3 (mean ± sd, n = 3).


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We next analyzed histone modifications at the AR promoter region that contains the Zif268 recognition site. The AR promoter region is reportedly hypermethylated in Du145 cells and hypomethylated in LNCaP cells.26 We also quantitatively detected the DNA methylated level of the AR promoter region by the assay using MBD and ZF–luciferase;25 thus, it was hypothesized that the region would be associated with H3K9me3 and H3K4me3 in Du145 and LNCaP cells, respectively. Indeed, we detected high luciferase activity from the H3K9me3 ChIP sample for Du145 cells and the H3K4me3 ChIP sample for LNCaP cells (Figure 5).


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Figure 5. Detection of histone modification at AR promoter region by ChIP-ZF-beads assay Detection of histone modification in Du145 cells (A) and LNCap cells (B). As a control, ChIP using anti-IgG antibody was performed and normalized signal is shown for H3K4me3 and H3K9me3 (mean ± sd, n = 3).


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Detection of histone modification by ChIP-ZF-BRET assay. We demonstrated that PCR product was sequence-specifically detected by ZF-BRET assay in homogenous solution, and histone modification was detected by the ChIP-ZF-beads assay. Therefore, we expected that histone modification would be conveniently detected by combining the two assays (ChIP-ZFBRET assay). To investigate whether histone modification was detected by ChIP-ZF-BRET assay, we performed ChIP using H3K9me3 and H3K4me3 antibodies for Du145 and LNCap cells and then the AR region was amplified by PCR from the ChIP samples. The Zif268– luciferase and BOBO™-3 were directly added to the PCR samples and then BRET signal was measured. As shown in Figure 6, we detected high BRET signal from the H3K9me3 ChIP sample for Du145 cells and the H3K4me3 ChIP sample for LNCaP cells. These results demonstrate that histone modifications at target genomic regions are conveniently and easily detected by ChIP-ZF-BRET assay.


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Figure 6. Detection of histone modification at AR promoter region by ChIP-ZF-BRET assay Detection of histone modification in Du145 cells (A) and LNCap cells (B). As a control, ChIP using anti-IgG antibody was performed and normalized signal is shown for H3K4me3 and H3K9me3 (mean ± sd, n = 3).


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In the ChIP-ZF-BRET assay for LNCaP cells, signals of both H3K9me3 and H3K4me3 were lower than those in the ChIP-ZF-beads assay. These results suggest that the PCR cycle was not sufficient for obtaining a large BRET signal, because the ZF-BRET assay is less sensitive than the ZF-beads assay. Thus, additional PCR cycles would improve the detection signal. In the ZF-BRET assay, we used firefly luciferase whose maximum emission is approximately 550 nm and BOBO™-3 whose maximum excitation is 570 nm. There are several types of luciferase that have different emissions and many types of DNA intercalating dye; therefore, efficient BRET signal could be obtained using suitable combination of luciferase and DNA intercalating dye. In particular, Viviani et al. reported that mutant luciferase shows a maximum emission at 565 nm,27 suggesting that highly efficient BRET signal may be obtained using the mutant luciferase with BOBO™-3. For application in clinical diagnosis, the epigenetic modification detection system should be automated. In this study, we developed a homogeneous PCR product detection system that can be easily automated. The ChIP and PCR steps can be performed automatically using commercial machines; therefore, we believe that this epigenetic modification detection system will be suitable for application in clinical diagnosis. About half of human transcription factors contain zinc finger domains,28 suggesting that zinc finger proteins could be suitable molecular recognition elements for promoter regions. In this study, we used Zif268–luciferase to detect histone modification at the AR promoter region. We have constructed SP1 and artificial zinc finger fused to luciferase protein as well. The SP1 binding site is enriched on CpG islands that are important region for epigenetic modification,29 suggesting that ChIP-ZF-BRET assay may be useful for this specific detection.


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CONCLUSIONS We developed a BRET-based homogeneous PCR product detection system using ZF–luciferase and DNA intercalating dye. By combining ChIP with the homogenous DNA detection system, we conveniently detected histone modification at the AR promoter region in LNCaP and Du145 cells. The assay can be applicable to epigenetic DNA modification such as methylcytosine and hydroxymethylcytosine using stable antibodies. Overall, histone modification and DNA modification at target genomic regions could be simultaneously and conveniently detected by our assay system.

ASSOCIATED CONTENT Supporting Information Additional table and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +81-42-388-7030. Phone: +81-42-388-7030. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for challenging Exploratory Research (22656190) from the Japan Society for the Promotion of Science (JSPS)

REFERENCES (1) Chen, R. Z.; Pettersson, U.; Beard, C.; Jackson-Grusby, L.; Jaenisch, R. Nature 1998, 395, 89−93. (2) Li, E.; Bestor, T. H.; Jaenisch, R. Cell 1992, 69, 915−926. (3) Strahl, B. D.; Allis, C. D. Nature 2000, 403, 41−45. (4) Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M. Nat. Rev. Drug. Discov. 2012, 11, 384−400. (5) Kurdistani, S. K. Br. J. Cancer 2007, 97, 1−5. (6) Gal-Yam, E. N.; Saito, Y.; Egger, G.; Jones, P. A. Annu. Rev. Med. 2008, 59, 267−280. (7) Chiam, K.; Ricciardelli, C.; Bianco-Miotto, T. Cancer Lett. 2012, [Epub ahead of print] (8) Schotta, G.; Lachner, M.; Sarma, K.; Ebert, A.; Sengupta, R.; Reuter, G.; Reinberg, D.; Jenuwein, T. Genes Dev. 2004, 18, 1251−1262. (9) Guenther, M. G.; Levine, S. S.; Boyer, L. A.; Jaenisch, R.; Young, R. A. Cell 2007, 130, 77−88. (10) Raynal, N. J.; Si, J.; Taby, R. F.; Gharibyan, V.; Ahmed, S.; Jelinek, J.; Estécio, M. R.; Issa, J. P. Cancer Res. 2012, 72, 1170−1181.


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(11) Kondo, Y.; Shen, L.; Cheng, A. S.; Ahmed, S.; Boumber, Y.; Charo, C.; Yamochi, T.; Urano, T.; Furukawa, K.; Kwabi-Addo, B.; Gold, D. L.; Sekido, Y.; Huang, T. H.; Issa, JP. Nat. Genet. 2008, 40, 741−750. (12) Bachman, K. E.; Park, B. H.; Rhee, I.; Rajagopalan, H.; Herman, J. G.; Baylin, S. B.; Kinzler, K. W.; Vogelstein, B. Cancer Cell 2003, 3, 89−95. (13) Boyd, K. E.; Farnham, P. J. Mol. Cell. Biol. 1997, 17, 2529−2537. (14) Wathelet, M. G.; Lin, C. H.; Parekh, B. S.; Ronco, L. V.; Howley, P. M.; Maniatis, T. Mol. Cell 1998, 1, 507−518. (15) Parekh, B. S.; Maniatis, T. Mol. Cell 1993, 3, 125−129. (16) Wittwer, C. T.; Ririe, K. M.; Andrew, R. V.; David, D. A.; Gundry, R. A.; Balis, U. J. Biotechniques 1997, 22, 176−181. (17) Bengtsson, M.; Karlsson, H. J.; Westman, G.; Kubista, M. Nucleic Acids Res. 2003, 31, e45. (18) Giglio, S.; Monis, P. T.; Saint, C. P. Nucleic Acids Res. 2003, 31, e136. (19) Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. Proc. Natl. Acad. Sci. USA. 1991, 88, 7276−7280. (20) Pattyn, F.; Speleman, F.; De Paepe, A.; Vandesompele, J. Nucleic Acids Res. 2003, 31, 122−123. (21) Osawa, Y.; Ikebukuro, K.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K. Nucleic Acids Res. 2008, 36, e68. (22) Osawa, Y.; Ikebukuro, K.; Kumagai, T.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K. Biotechnol. Lett. 2009, 31, 725−733. (23) Abe, K.; Kumagai, T.; Takahashi, C.; Kezuka, A.; Murakami, Y.; Osawa, Y.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K.; Igimi, S.; Ikebukuro, K. Anal. Chem. 2012, 84, 8028−8032.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

(24) Tang, C.; Shi, X.; Wang, W.; Zhou, D.; Tu, J.; Xie, X.; Ge, Q.; Xiao, P. F.; Sun, X.; Lu, Z. Electrophoresis 2010, 31, 2936−2943. (25) Hiraoka, D.; Yoshida, W.; Abe, K.; Wakeda, H.; Hata, K.; Ikebukuro, K. Anal. Chem. 2012, 84, 8259−8264. (26) Kinoshita, H.; Shi, Y.; Sandefur, C.; Meisner, L. F.; Chang, C.; Choon, A.; Reznikoff, C. R.; Bova, G. S.; Friedl, A.; Jarrard, D. F. Cancer Res. 2000, 60, 3623−3630. (27) Viviani, V.; Uchida, A.; Suenaga, N.; Ryufuku, M.; Ohmiya, Y. Biochem. Biophys. Res. Commun. 2001, 280, 1286−1291. (28) Razin, S. V.; Borunova, V. V.; Maksimenko, O. G.; Kantidze, O. L. Biochemistry (Mosc) 2012, 77, 217−226. (29) Cawley, S.; Bekiranov, S.; Ng, H. H.; Kapranov, P.; Sekinger, E. A.; Kampa, D.; Piccolboni, A.; Sementchenko, V.; Cheng, J.; Williams, A. J.; Wheeler, R.; Wong, B.; Drenkow, J.; Yamanaka, M.; Patel, S.; Brubaker, S.; Tammana, H.; Helt, G.; Struhl, K.; Gingeras, T R. Cell 2004, 116, 499−509.


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Detection of Histone Modification by Chromatin Immunoprecipitation

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