A Timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

David Bending, Paz Prieto Martin, Alina Paduraru, Catherine Ducker, Erik Marzaganov, Marie Laviron, Satsuki Kitano, Hitoshi Miyachi, Tessa Crompton, Masahiro Ono

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)
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Abstract

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyse in vivo. We establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky [toki], time in Japanese). Tocky uses the Fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue-to-red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR)-signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a Tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.
Original languageEnglish
Pages (from-to)2931-2950
JournalJournal of Cell Biology
Volume217
Issue number8
Early online date25 Jun 2018
DOIs
Publication statusPublished - 6 Aug 2018

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