Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

被引:20
作者
Liu, Yang [1 ]
Zhao, Ning [2 ]
Kanemaki, Masato T. [3 ,4 ]
Yamamoto, Yotaro [5 ]
Sadamura, Yoshifusa [5 ]
Ito, Yuma [1 ]
Tokunaga, Makio [1 ]
Stasevich, Timothy J. [2 ,6 ,7 ]
Kimura, Hiroshi [1 ,6 ,7 ]
机构
[1] Tokyo Inst Technol, Sch Life Sci & Technol, Yokohama, Kanagawa 2268501, Japan
[2] Colorado State Univ, Dept Biochem & Mol Biol, Ft Collins, CO 80523 USA
[3] Res Org Informat & Syst, Dept Chromosome Sci, Natl Inst Genet, Mishima, Shizuoka, Japan
[4] SOKENDAI, Dept Genet, Mishima, Shizuoka, Japan
[5] Fujifilm Wako Pure Chem, Life Sci Res Labs, Amagasaki, Hyogo, Japan
[6] Tokyo Inst Technol, Cell Biol Ctr, Yokohama, Kanagawa, Japan
[7] Tokyo Inst Technol, World Res Hub Initiat, Yokohama, Kanagawa, Japan
基金
美国国家卫生研究院; 日本学术振兴会; 美国国家科学基金会;
关键词
cohesin; CTCF; DNA looping; FLAG tag; intracellular antibodies; live-cell imaging; synthetic zinc-finger protein; WAPL; GENETICALLY ENCODED PROBE; LIVE-CELL; CHROMATIN INTERACTIONS; DNA-SEQUENCES; T-CELLS; COHESIN; REVEALS; GENERATION; EXPRESSION; DYNAMICS;
D O I
10.1111/gtc.12893
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG- and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.
引用
收藏
页码:905 / 926
页数:22
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