Global DNA Methylation Detection System Using MBD-Fused

Aug 19, 2016 - DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute ap...
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Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay Wataru Yoshida,* Yuji Baba, and Isao Karube School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan S Supporting Information *

ABSTRACT: DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal.

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complementation assay has been reported.24 In this assay, two fusion proteins, C-terminal luciferase fragment-fused methylCpG binding domain (MBD)25 and N-terminal luciferase fragment-fused MBD, were utilized. These two proteins colocalized on DNA containing two methylated CpG sites to reassemble the luciferase fragments. The reassembled luciferase activity depended on the distance between two methylated CpG sites, suggesting that it leads to underestimation of the DNA methylation level. A previous study reported a homogeneous assay for detecting PCR products based on bioluminescence resonance energy transfer (BRET) using zinc finger protein-fused luciferase and DNA intercalating dye.26 In this assay, the target PCR product was detected by BRET between the zinc finger protein-fused luciferase and DNA intercalating dye that was excited by luciferase luminescence because the fusion protein and the DNA intercalating dye were colocalized on the target PCR product. By combining chromatin immunoprecipitation (ChIP) and the BRET assay, a convenient histone modification detection system was developed. In this study, we aimed to develop a homogeneous assay for global DNA methylation detection based on BRET using MBD-fused luciferase (MBD-luciferase) and DNA intercalating dye. We expected that the MBD-luciferase would bind to the methylated DNA, whereby BRET between the luciferase and

NA methylation is associated with the silencing of gene expression.1,2 In mammalian cells, the DNA methyltransferase family (Dnmt1, Dnmt3a, and Dnmt3b) catalyzes the transfer of a methyl group from S-adenosylmethionine to cytosine in CpG dinucleotides.3,4 Transposable elements constitute approximately 45% of the human genome.5 In normal cells, transposable elements are highly methylated to suppress retrotransposons; namely, the global DNA methylation level correlates with the methylation level of transposable elements. In cancer cells, the global DNA methylation level decreases because transposable elements are hypomethylated.6,7 Such hypomethylation has been shown to participate in reactivation of the transposable elements. Change of aberrant methylation patterns has been reported to be caused by environmental factors such as smoking, not only by aging.8−11 Therefore, the global DNA methylation level is considered a biomarker for cancer diagnostics. Several global DNA methylation level detection systems have been established, such as a high-performance liquid chromatography (HPLC)-based assay,12−15 enzyme-linked immunosorbent assay (ELISA),16 sodium bisulfite-based assays,17−19 methyl group acceptance assay,20 chloroacetaldehyde assay,21 the electrocatalytic detection system,22 and capillary electrophoresis-based immunoassay.23 In these detection systems, global DNA methylation level can be accurately detected; however, several steps are required for its detection. To simply detect the global DNA methylation level, a homogeneous assay that does not require several steps is ideal. A homogeneous assay for global DNA methylation detection based on split-luciferase © XXXX American Chemical Society

Received: July 6, 2016 Accepted: August 19, 2016

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DOI: 10.1021/acs.analchem.6b02565 Anal. Chem. XXXX, XXX, XXX−XXX

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luminescence was measured by a microplate reader (Spark 10M, Tecan). Analysis of the Binding of MBD-Luciferase to Methylated DNA. Methylated or unmethylated 20-bp dsDNA modified with biotin was synthesized (Table S1; Tsukuba Oligo Service). The DNAs (250 pmol) were added to a streptavidin-immobilized plate (Tefco). After washing the well with 200 μL of washing buffer (PBS, 0.05% Tween 20), 200 μL of MBD-luciferase (34 nM) was added and then incubated for 30 min at RT. After washing the well, luciferase activity was measured by the addition of 100 μL of PicaGene. Preparation of Methylated Plasmid. pET30c-StreptagMBD-luciferase (13 μg) was methylated by 104 U of CpG methyltransferase (NEB), followed by phenol chloroform purification. The plasmid is 7176-bp long and contains 456 CpG sites. To confirm DNA methylation, 150 ng of the methylated DNA was treated with 1 U of the methylationsensitive restriction enzyme HpaII (NEB) at 37 °C for 1 h and then analyzed using 1% agarose gel in 1× TAE buffer. Preparation of Genomic DNA. HeLa cells were cultured in a culture medium [DMEM medium (Sigma-Aldrich), 10% FBS (Sigma-Aldrich), 1× penicillin-streptomycin L-glutamine (Sigma-Aldrich)]. To prepare hypomethylated genomic DNA, HeLa cells (4.0 × 105 cells) were seeded in a 100 mm dish with the culture medium supplemented with 10, 1.0, or 0.1 μM 5Aza-2′-deoxycytidine (Tokyo Chemical Industry). After 24 h of cultivation, the medium was replaced with culture medium lacking 5-Aza-2′-deoxycytidine, followed by culture for 3 days. The HeLa genomic DNA was purified using the DNeasy Blood and Tissue Kit (Qiagen). Bisulfite Methylation Analysis. Bisulfite treatment of the HeLa genomic DNA (2.0 μg) was performed using the EpiTect Bisulfite Kit (Qiagen). PCR was performed using Ex taq HS (Takara) to amplify the LINE1 region.19 PCR primer sequences are shown in Table S1. The PCR products were purified using Wizard SV Gel and PCR Clean-Up System (Promega). The purified PCR products were treated with 2 U of TaqαI (NEB) at 65 °C for 1 h and then analyzed on a 15% polyacrylamide gel in 1× TBE buffer. The band intensities were quantified using ImageJ software. The DNA methylation level of LINE1 was determined by calculating the ratio of the digested PCR product intensity to the total PCR product intensity. BRET Analysis for Plasmid DNA. Methylated or unmethylated pET30c-Streptag-MBD-luciferase (10 or 5.0 ng/ μL) was incubated with 1.0 μM BOBO-3 (Thermo Fisher Scientific) in PBS buffer at RT for 30 min. Then, 17 nM MBDluciferase was added to the mixture in a 50 μL reaction volume and incubated at RT for 1 min. After incubation, 50 μL of PicaGene was added to measure the emission spectra at 488, 503, 518, 533, 548, 563, 578, 593, 608, 623, 638, and 653 nm using a microplate reader (Spark 10M, Tecan). In the analysis of DNA methylation frequency, the unmethylated and methylated plasmids were mixed to prepare plasmid DNAs at methylation frequencies of 0, 10, 50, and 100%. The emission spectra were measured using 10 ng/μL plasmids, as described above. BRET Analysis for HeLa Genomic DNA. HeLa genomic DNA (70 or 7.0 ng/μL) was incubated with 7.0 μM BOBO-3 in PBS buffer at RT for 30 min. Then, 8.5 nM MBD-luciferase was added to the mixture in a 150 μL reaction volume and incubated at RT for 1 min. After incubation, 50 μL of PicaGene was added to measure the emission spectra at 488, 503, 518,

DNA intercalating dye would be detected and the intensity of the BRET signal would depend on the DNA methylation level of the target genome (Figure 1). We prepared MBD-luciferase and then applied it to detect the global DNA methylation level of HeLa genomic DNA.

Figure 1. Global DNA methylation detection system using MBDluciferase and DNA intercalating dye. The MBD-luciferase binds to methylated DNA to excite the DNA intercalating dye. Luciferase and MBD are shown as yellow and light blue, respectively. Methyl group and DNA intercalating dye are shown as black circle and orange square, respectively.

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EXPERIMENTAL SECTION Plasmid Construction. The MBD gene coding amino acids 2−82 of MBD1 was amplified by PCR from pGEX-2THMBD27 using KOD DNA polymerase (Toyobo) with a forward primer containing an NdeI site and a Streptag sequence and a reverse primer containing an EcoRI site (Table S1, Eurofins). The PCR product was digested by NdeI (NEB) and EcoRI (Nippon Gene). pET30c-Streptag-Zif 268-luciferase23 was digested with NdeI and EcoRI to remove the Streptag-Zif 268 and then the Streptag-MBD was ligated with the pET30c-luciferase by Ligation High Ver.2 (Toyobo). Escherichia coli (E. coli) DH5α (Toyobo) was transformed by the ligated product, and then the plasmid was purified. The plasmid was sequenced using a 3730xl DNA analyzer (Thermo Fisher Scientific). Expression and Purification of the MBD-Luciferase. E. coli BL21 (DE3; Biodynamics) was transformed by pET30cStreptag-MBD-luciferase. The cells were cultured in 150 mL of LB medium at 37 °C. When the OD600 reached 0.7, 0.5 mM IPTG (Wako) was added and then incubated at 20 °C for 16 h to induce the MBD-luciferase expression. The cells were harvested by centrifugation at 2500 × g for 10 min, and then the cell pellets were lysed with BugBuster Protein Extraction Reagent (Millipore). The water-soluble fraction was prepared by centrifugation at 16000 × g and 4 °C for 20 min. The watersoluble fraction was 10-fold diluted with PBS buffer (Thermo Fisher Scientific) and then filtered with a 0.45 μm filter (Millipore). The filtered sample was loaded onto a strep-Tactin Superflow column (Qiagen) to purify the MBD-luciferase. The column was then washed with 10 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The MBD-luciferase was eluted using 10 mL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM desthiobiotin, pH 8.0). In all steps, 1 mL of each fraction was collected. Purified samples were analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel (Tefco) and visualized with Coomassie Brilliant Blue (Bio Craft). Protein concentration was measured by a DC protein assay (Bio-Rad). To measure the luciferase activity of each fraction, 100 μL of PicaGene (Toyo Ink) was added to 10 μL of the sample, and then the B

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Analytical Chemistry 533, 548, 563, 578, 593, 608, 623, 638, and 653 nm using a microplate reader (Spark 10M, Tecan). In the analysis of the genomic DNA purified from HeLa cells treated with 5-Aza-2′deoxycytidine, 70 ng/μL genomic DNA was used as target DNA, and the emission spectra were measured as described above.

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RESULTS AND DISCUSSION Expression and Purification of MBD-Luciferase. The MBD gene coding amino acids 2−82 of MBD1 was amplified by PCR with a forward primer containing the Streptag sequence from pGEX-2TH-MBD. pET30c-Streptag-Zif 268-luciferase was digested with NdeI and EcoRI to remove the Streptag-Zif 268 and then Streptag-MBD was cloned to pET30c-luciferase to construct pET30c-Streptag-MBD-luciferase (Figure S1). E. coli BL21 (DE3) was transformed by the plasmid and then the protein expression was induced by IPTG. After 16 h of cultivation, MBD-luciferase was purified from the water-soluble fraction using the Streptag system. In the purification step, the luciferase activities were monitored. In the second elution fraction, high luciferase activity was detected (Figure S2). In the SDS-PAGE analysis of each fraction, a single band corresponding to MBD-luciferase (71 kDa) was detected in the elution fraction (Figure S3). These results indicate that MBD-luciferase retaining luciferase activity was purified. Binding Assay of MBD-Luciferase to Methylated DNA. The binding ability of MBD-luciferase to methylated CpG was analyzed by a plate assay. Synthesized methylated or unmethylated 20-bp dsDNA containing one CpG site was immobilized on a streptavidin-immobilized plate and then purified MBD-luciferase was added. After washing, luciferase activity was measured. Compared with that in the unmethylated dsDNA-immobilized well, 3-fold higher luciferase activity was detected in the methylated dsDNA-immobilized well (Figure S4). These results indicate that the MBD-luciferase retains methylated CpG binding activity. Detection of Methylation Level of Plasmid DNA by the BRET Assay. MBD-luciferase showed peak emission at approximately 578 nm (Figure S5). The emission intensities at 593, 608, 623, 638, and 653 nm were 0.88-, 0.66-, 0.42-, 0.31-, and 0.24-fold of the intensity at 578 nm, respectively. BOBO-3 exhibits emission at around 600 nm; therefore, the emission intensity of BOBO-3 excited by MBD-luciferase can be calculated by subtracting the emission intensity of MBDluciferase calculated using the above factors from the total emission intensity at each wavelength. As a target DNA, 7176-bp pET30c-Streptag-MBD-luciferase, which contains 456CpG sites, was methylated by DNA methyltransferase. The plasmid was treated with the methylation-sensitive restriction enzyme HpaII and analyzed using agarose gel to confirm the DNA methylation state (Figure S6). A mixture of 17 nM MBD-luciferase, 1.0 μM BOBO-3, and the methylated or unmethylated plasmid (10 or 5.0 ng/μL) was prepared. It should be noted that the calculated CpG concentrations are 0.96 and 0.48 μM, respectively. Then, the emission spectra were measured by the addition of a luciferase substrate. The emission intensity of BOBO-3 at each wavelength was calculated by subtracting the MBD-luciferase intensity from the total intensity. Although the emission intensity of BOBO-3 was not affected in the presence of the unmethylated plasmid, it increased in the presence of the methylated plasmid (Figure 2). The intensities depended on the methylated DNA concentration. The peak emission of

Figure 2. Emission intensity of DNA intercalating dye excited by MBD-luciferase in the presence of methylated or unmethylated plasmid DNA. In the assay, 17 nM MBD-luciferase, 1.0 μM BOBO-3, and the methylated or unmethylated plasmid DNA (10 or 5.0 ng/μL) were used. The emission intensity of BOBO-3 was calculated by subtracting the emission intensity of the MBD-luciferase from the total emission intensity at each wavelength (mean ± SD, N = 3).

BOBO-3 was at 623 nm; therefore, the emission intensity of BOBO-3 at 623 nm was defined as the BRET signal. MBD of MBD1 binds to unmethylated and methylated CpG with Kd values of 10−70 nM and 5 μM, respectively.28 If MBDluciferase binds to methylated and unmethylated DNA with the same binding affinity as MBD1, the concentration of MBDluciferase-methylated DNA complex would be 15.8−16.8 nM, whereas the concentration of MBD-luciferase-unmethylated DNA complex would be 2.7 nM. This suggests that the concentration of MBD-luciferase-methylated DNA complex would be 5.9−6.2-fold higher than that of MBD-luciferaseunmethylated DNA complex. On the other hand, the BRET signal for the methylated DNA was 6.3-fold higher than that of unmethylated DNA, which is compatible with the ratio of the MBD-luciferase-methylated DNA complex to the MBDluciferase-unmethylated DNA complex. These results suggest that MBD-luciferase has similar binding specificity to MBD1. To investigate whether the intensity of the BRET signal depends on the DNA methylation level, the unmethylated and methylated plasmids were mixed to prepare 0, 10, 50, and 100% methylated DNA samples and then 10 ng/μL plasmids were used as target DNA. As a result, the intensity of the BRET signal increased with increasing methylation level of the DNA (Figures 3 and S7). These results indicated BOBO-3 was excited by MBD-luciferase that bound to the methylated DNA.

Figure 3. Detection of DNA methylation of the plasmid DNA by the BRET assay. In the assay, 17 nM MBD-luciferase, 1.0 μM BOBO-3, and 10 ng/μL plasmid DNA at methylation frequencies of 0, 10, 50, and 100% were used. The emission intensities of BOBO-3 at 623 nm were calculated by subtracting the emission intensity of MBDluciferase from the total emission intensity (mean ± SD, N = 3). C

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Analytical Chemistry Detection of Global DNA Methylation Level of Genomic DNA by the BRET Assay. In the human genome, approximately 28 million CpG sites are contained in 3.2-Gbp of DNA;29 that is, the CpG density is about 7-fold lower than that of the plasmid DNA. Therefore, a 7-fold higher concentration of BOBO-3 (7.0 μM) was used for the BRET assay of the human genome. First, the BRET signal was measured in the presence of HeLa genomic DNA. A mixture of 8.5 nM MBDluciferase, 7.0 μM BOBO-3, and HeLa genomic DNA (70 or 7.0 ng/μL) was prepared. It should be noted that the calculated CpG concentrations are 0.93 and 0.093 μM, respectively. Then, the emission spectra were measured by the addition of a luciferase substrate. As expected, the intensity of the BRET signal increased with increasing concentration of the genomic DNA (Figure 4).

Figure 5. Evaluation of global DNA methylation level of HeLa genomic DNA by the BRET assay. In this assay, 8.5 nM MBDluciferase, 7.0 μM BOBO-3, and 70 ng/μL genomic DNA purified from HeLa cells treated with 5-Aza-2′-deoxycytidine were used. The emission intensity of BOBO-3 at 623 nm was calculated by subtracting the emission intensity of MBD-luciferase from the total emission intensity (mean ± SD, N = 3).

BRET assay. To improve the detection limit, the binding affinity of MBD-luciferase to methylated DNA has to be improved. It has been reported that tetramerized MBD bound to methylated DNA with 50-fold higher affinity than that of monomeric MBD.31 This suggests that the detection limit of the BRET assay would be improved using tetramerized MBD fused to luciferase, which would in turn improve the sensitivity of this assay. In the BRET assay, the intensity of luciferase luminescence and peak wavelength are important to excite DNA intercalating dye. In this study, wild-type luciferase was utilized, but its properties can be changed by site-directed mutagenesis.32 Moreover, peak wavelength can also be changed using synthetic analogs of luciferin.33 Numerous types of DNA intercalating dye have been commercialized; therefore, by selecting suitable luciferase, luciferase substrate, and DNA intercalating dye, the sensitivity of the BRET assay could be improved. Recently, other epigenetic DNA modifications such as hydroxymethylcytosine34,35 and N6-methyladenosine36,37 have been identified. These modifications also play important roles in the regulation of gene expression. We believe that other epigenetic modifications at the global level would be detected by replacing MBD with other epigenetic modification recognition proteins.

Figure 4. Emission intensity of DNA intercalating dye excited by MBD-luciferase in the presence of HeLa genomic DNA. In the assay, 8.5 nM MBD-luciferase, 7.0 μM BOBO-3, and HeLa genomic DNA (70 or 7.0 ng/μL) were used. The emission intensity of BOBO-3 was calculated by subtracting the emission intensity of MBD-luciferase from the total emission intensity at each wavelength (mean ± SD, N = 3).

To investigate whether the global DNA methylation level would be detected by the BRET assay, HeLa cells were treated with a DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine, to prepare hypomethylated genomic DNA. The LINE1 region was amplified from the genomic DNAs treated with sodium bisulfite by PCR. The PCR products were treated with TaqαI and then analyzed by native PAGE to determine the DNA methylation level of LINE1. The DNA methylation level of untreated HeLa genome was 63% (Figure S8), which is comparable to that in a previous report.30 By treatment with 10, 1.0, and 0.1 μM 5-Aza-2′-deoxycytidine, the methylation level decreased to 31, 43, and 51%, respectively. A mixture of 8.5 nM MBD-luciferase, 7.0 μM BOBO-3, and 70 ng/μL HeLa genomic DNA was prepared and then the emission spectra were measured by the addition of a luciferase substrate. The intensity of the BRET signals increased with increasing global DNA methylation level of the HeLa genomic DNA (Figures 5 and S9). These results demonstrate that global DNA methylation level can be detected by the BRET assay. Under the conditions of the BRET assay for genomic DNA, the background signals were higher than those for the plasmid DNA assay, indicating that free BOBO-3 would be excited by MBD-luciferase. In the assay for genomic DNA, 7-fold higher concentrations of BOBO-3 and DNA were utilized than those in the assay for the plasmid DNA because the CpG density in human genomic DNA is about 7-fold lower than that of the plasmid DNA. These results indicate that background signals would be reduced by improving the detection limit of the

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CONCLUSIONS In this study, MBD-fused luciferase protein was constructed and applied to a homogeneous assay for measuring the global DNA methylation level based on BRET between the MBDluciferase and BOBO-3. In this assay, MBD-luciferase bound to methylated DNA, whereby BOBO-3 was excited by the luciferase luminescence. The emission intensity of BOBO-3 depended on the DNA methylation level of genomic DNA. Our results demonstrate that the global DNA methylation level of the human genome was easily detected by the BRET assay.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02565. DNA sequences used in this study, luciferase activity of each fraction in the purification of MBD-luciferase, SDSPAGE of the purified MBD-luciferase, analysis of the D

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binding of MBD-luciferase to methylated dsDNA, spectrum of the MBD-luciferase bioluminescence, DNA methylation analysis of the plasmid DNA, emission intensity of DNA intercalating dye excited by MBDluciferase in the presence of methylated plasmid DNA, DNA methylation analysis of LINE1 by COBRA, and emission intensity of DNA intercalating dye excited by MBD-luciferase in the presence of genomic DNA purified from HeLa cells treated with 5-Aza-2′-deoxycytidine (PDF).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

This manuscript was written through contributions from all authors. All authors have approved the final version of this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Nakatani Foundation. REFERENCES

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