An Cheng, Zhu Guangjing, Martos Suzanne N, Feng Xue, Zhang Haimou, Jia Yankai, Wang Zhibin
Guang'an Men Hospital, China Academy of Chinese Medical Sciences, Beijing, China.
Laboratory of Human Environmental Epigenomes, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
Adv Biosci Biotechnol. 2017 Sep;8(9):311-323. doi: 10.4236/abb.2017.89023. Epub 2017 Sep 22.
Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, co-lorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.
包括H3赖氨酸79甲基化（H3K79me）在内的组蛋白修饰在基因转录和DNA损伤修复过程中起着关键作用。DOT1L是H3K79me三种状态的唯一甲基转移酶，与白血病、结直肠癌和扩张型心肌病有关。然而，由于处理DOT1L蛋白存在困难，对DOT1L和H3K79me在这些途径和疾病发病机制中的了解一直有限。例如，基于染色质免疫沉淀（ChIP）的方法揭示的DOT1L的位点特异性或全基因组结合位点对于推断其功能是必要的，但目前尚无高质量的ChIP级抗体。在此，我们开发了一种敲入方法，在其C末端结构域用3×Flag标记内源性DOT1L，以进行功能分析。通过使用转录激活样效应因子核酸酶（TALENs）在内源性DOT1L处诱导靶向双链断裂，以刺激该位点的局部同源重组，促进了敲入。通过不同方法分离并验证了成功敲入的单细胞克隆。我们还证明，标记的DOT1L在甲基化方面保持其正常功能，并且工程细胞将对进一步研究非常有用。
