TITLE:
TALEN-Mediated FLAG-Tagging of Endogenous Histone Methyltransferase DOT1L
AUTHORS:
Cheng An, Guangjing Zhu, Suzanne N. Martos, Xue Feng, Haimou Zhang, Yankai Jia, Zhibin Wang
KEYWORDS:
DOT1L, Flag, TALEN, Knock-In
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.8 No.9,
September
22,
2017
ABSTRACT: 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.