The specificity of Fabs can furthermore be verified in living cells by inhibitor treatments and/or genetic knockout experiments ( Hayashi-Takanaka et al., 2011). With FabLEM, dynamic changes in endogenous protein modification levels throughout living cells and embryos can be monitored in real time, without affecting the cell cycle or developmental processes ( Hayashi-Takanaka et al., 2009 Hayashi-Takanaka et al., 2011 Yuan and O'Farrell, 2016 Ruppert et al., 2018 Seller et al., 2019).

We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.įabLEM (Fab-based live endogenous modification labeling) is a technique to visualize transcription and histone modification dynamics using modification-specific antigen-binding fragments (Fabs) ( Hayashi-Takanaka et al., 2011 Kimura et al., 2015). When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture.

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