期刊
GENES TO CELLS
卷 26, 期 11, 页码 905-926出版社
WILEY
DOI: 10.1111/gtc.12893
关键词
cohesin; CTCF; DNA looping; FLAG tag; intracellular antibodies; live-cell imaging; synthetic zinc-finger protein; WAPL
资金
- Japan Society for the Promotion of Science KAKENHI grant [JP18H02170, JP21H04719, JP18H05527, JP21H04764]
- Japan Science and Technology CREST [JPMJCR16G1]
- Japan Agency for Medical Research and Development BIND [JP21am0101105]
- National Institutes of Health [R35GM119728, K99GM141453]
- National Science Foundation [MCB-1845761]
This study demonstrates a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins and frankenbodies for signal amplification, enabling the simultaneous observation of the dynamics of two loci in single living cells. The results show a close association between the two loci and their separation upon the triggered degradation of the cohesin subunit RAD21.
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.
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