Biological Engineering and Bindley Bioscience Center, Purdue University; West Lafayette, IN USA.
Epigenetics. 2013 Oct;8(10):1089-100. doi: 10.4161/epi.25958. Epub 2013 Aug 8.
With unprecedented development in technology, epigenetics is recognized as a substantial and flexible regulatory pathway for phenotyping. Cytosine methylation and its subsequent oxidization have attracted significant attention due to their direct impact on gene regulation, in association with methyl-CpG-binding domain proteins (MBDs) and transcription related factors. In this study we record the dynamics of DNA demethylation using the recombinant MBD3-GFP protein in living cells under hypoxia and Decitabine treatment using Fluorescence Correlation Spectroscopy (FCS) by monitoring the diffusion dynamics of MBD3. Our study shows a DNA-replication-independent decrease of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) under hypoxia vs. a dependent decrease under Decitabine treatment. Further, we define a significantly faster diffusion of MBD3 in the nucleus as a precursory event for active demethylation rather than the Decitabine induced passive demethylation. By monitoring the diffusion of bound and unbound MBD3 in the nucleus we were able to identify and characterize hypoxia-sensitive cells from insensitive/tolerant cells, as well as the respective contribution to active demethylation in a time-dependent manner. Last, we quantitatively describe the concurrent decreasing trend in all of the three oxidized products of 5mC, which points to the potential involvement of ten-eleven-translocation proteins (TETs) in hypoxia induced active demethylation. Overall, for the first time we correlate the dynamic process of DNA demethylation with the biophysical properties of the corresponding DNA binding proteins in live single cells by single molecule spectroscopy.
随着技术的空前发展，表观遗传学被认为是表型的一种重要且灵活的调控途径。由于其对基因调控的直接影响，与甲基-CpG 结合域蛋白（MBDs）和转录相关因子相关的胞嘧啶甲基化及其随后的氧化受到了极大关注。在这项研究中，我们使用重组 MBD3-GFP 蛋白在缺氧和地西他滨处理下记录活细胞中的 DNA 去甲基化动力学，通过监测 MBD3 的扩散动力学来使用荧光相关光谱（FCS）。我们的研究表明，与地西他滨处理相比，缺氧时 5-甲基胞嘧啶（5mC）/5-羟甲基胞嘧啶（5hmC）的减少与 DNA 复制无关。此外，我们定义了核内 MBD3 的扩散速度明显加快，作为主动去甲基化的先兆事件，而不是地西他滨诱导的被动去甲基化。通过监测核内结合和未结合的 MBD3 的扩散，我们能够识别和表征对缺氧敏感的细胞与对缺氧不敏感/耐受的细胞，并以时间依赖性的方式对其各自对主动去甲基化的贡献进行了定义。最后，我们定量描述了所有三种 5mC 氧化产物的同时下降趋势，这表明 ten-eleven-translocation 蛋白（TETs）可能参与了缺氧诱导的主动去甲基化。总的来说，这是我们首次通过单分子光谱学将 DNA 去甲基化的动态过程与活单细胞中相应的 DNA 结合蛋白的生物物理特性相关联。
