TALEN-Mediated FLAG-Tagging of Endogenous Histone Methyltransferase DOT1L

• 作者: Cheng An Guangjing Zhu Haimou Zhang Suzanne N. Martos Xue Feng Yankai Jia Zhibin Wang
• 来源期刊: 生命科学与技术进展(英文)
• 学科: 医学
• 关键词: DOT1L FLAG TALEN KNOCK-IN
• 年，卷（期）: 2017,（9）
• 页码范围: 311-323
• 页数: 13页
• 分类号: R73

摘要

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, colorectal 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.