Development of a Detection System for Epigenetic Modifications By Enzyme Fused Zinc Finger Protein
Authors: Lee Jinhee , Yoshida Wataru , Hiraoka Daisuke , Kezuka Aki , Tasumi Atsuro , Osawa Yuko , Abe Koichi , Nakabayashi Kazuhiko , Wakeda Hironobu , Hata Kenichiro , Sode Koji , Ikebukuro Kazunori 
Published: February 2020

INTRODUCTION

Epigenetic modifications such as DNA methylation and histone modifications play an important role in gene regulation. Recently, it has been reported that aberrant modifications are related to diseases such as cancer. Therefore, these modifications are regarded as new biomarkers (1). To detect these modifications, usually, quantitative polymerase chain reaction (qPCR) is performed after protein-based precipitation. qPCR is a powerful and simple method to quantify DNA, but this method is not able to distinguish between specific PCR products and nonspecific products. Therefore, to utilize these modifications as a biomarker, more accurate detection method is required. To achieve that, we focused on zinc finger protein (ZF), a well-known DNA binding protein, because it can bind to double stranded DNA with sequence specifically. In addition, to detect DNA methylation level, we have to treat DNA with bisulfite and perform sequencing. Although this method is a standard method to detect the level, it is time consuming and laborious. To detect the level more simply and rapidly, we focused on Methyl CpG-binding domain (MBD). Using MBD, we can separate methylated DNA without bisulfite treatment in shorter time period. Thus we report simple and rapid detection system for epigenetic modifications using ZF and MBD (2,3).

METHOD

0. Design of detection system using enzyme fused ZF Our detection method comprises following three steps:1. Methyl CpG-binding domain (MBD) or histone modification antibodies based DNA collection, 2. PCR amplification of the target genomic region, which includes a zinc finger recognition site and 3. Detection of the PCR products by enzyme fused zinc finger protein (Fig.). As reporter enzymes, we chose luciferase and glucose dehydrogenase. We constructed a homogenous detection system combining Bioluminescence Resonance Energy Transfer (BRET) with luciferase fused zinc finger protein (ZF-Luc). We also constructed a simple electrochemical detection system using glucose dehydrogenase fused zinc finger protein (ZF-GDH).
1. Electrochemical detection of DNA methylation using ZF-GDH
We detected DNA methylation levels in androgen receptor (AR) promoter regions of human genomic DNA using ZF-GDH. First, we checked genomic DNA copy number dependency. We used highly methylated genomic DNA (Du145) and lowly methylated genomic DNA (LNCaP). These genomic DNA was purified and diluted to certain copy numbers (102 to 106copies), and detected.
Next, we tried to detect methylation level in 106copies of genomic DNA. Genomic DNA from LNCaP, Du145, PC3 and TSU-PR1 cells were analyzed and we compared with the results obtained by a conventional method: bisulfite sequencing.
2. Homogeneous detection of histone modifications using ZF-Luc
For detection of histone modification, we targeted H3K4me3 at GAPDH promoter region using ZF-Luc. This promoter region is reportedly associated with H3K4me3 in HeLa cells. We then analyzed histone modifications at the AR promoter region. The ARpromoter region is reportedly hypermethylated in Du145 cells and hypomethylated in LNCaP cells.

RESULTS AND DISCUSSION

1. As we expected, copy number dependent signal increase was observed only when we detected highly methylated DNA. This result means that we can detect methylated DNA specifically. The methylation level detected by our method was correlated with the result obtained by the conventional method. This shows that the methylation levels of target genomic regions can be correctly determined without bisulfite treatment and sequencing. Importantly we could detect methylation level in 106copies of genomic DNA because it is enough sensitive for a real diagnositics.
2. As the result of histone modifications detection at GAPDH, we detected high luciferase activity from the H3K4me3 sample but not the H3K9me3 sample. Also for ARregion, indeed, we detected high luciferase activity from the s3 sample for Du145 cells and the H3K4me3 sample for LNCaP cells. These results are correlated with reported results and demonstrate that histone modifications were correctly detected by our homogeneous detection method.

CONCLUSION

We developed a detection system for DNA methylation and histone modification using enzyme fused zinc finger proteins and demonstrated that the DNA methylation levels and histone modification levels can be determined by this system. We believe that DNA methylation levels and histone modification levels can be detected simultaneously by our system and it would be a useful system for the diagnosis of diseases that have a significant impact on human health.

REFERENCES

1. Gal-Yam, E. N.; Saito, Y.; Egger, G.; Jones, P. A., (2008), Annu. Rev. Med. 59, 267−280.
2. Hiraoka, D., Yoshida, W., Abe, K., Wakeda, H., Hata, K., & Ikebukuro, K. (2012). Anal. Chem., 84(19)
3. Yoshida, W., Kezuka, A., Abe, K., Wakeda, H., Nakabayashi, K., Hata, K., & Ikebukuro, K. (2013). Anal. Chem., 85(13)

Figure 1