Development of a Detection System for Epigenetic Modifications By Enzyme Fused Zinc Finger Protein

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 (10 2 to 10 6 copies), and detected. N