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, 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.
Highlights
We demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies
We first cloned ~1.1 kb sequences upstream and ~1.2 kb sequences downstream of the stop codon of DOT1L from HEK293 genomic DNA into pAAV-USER-Neo-LoxP-3 × Flag vector using the USER system to use as a donor plasmid [15] [16]
The double strand break (DSB) was readily repaired by homologous recombination (HR) in presence of the donor plasmid; we observed highly efficient HR occurs at the DSB
Summary
Yeast Dot (disruptor of telomeric silencing; or Kmt4) [4] or its mammalian homolog DOT1-Like (DOT1L) is the sole KMT in their respective genome with methylation activities toward H3K79 inside globular domain of histones. Both enzymes can catalyze mono-(me1), di-(me2), and trimethylation (me3) in a non-processive manner [5] [6]. DOT1L plays pivotal roles in embryonic development, hematopoiesis, and cardiac function [9]. High level of DOT1L expression and H3K79me was a predictor of poor patient survival of colorectal cancer [11]
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