Journal of Molecular Biology
Volume 428, Issue 20, 9 October 2016, Pages 3885-3902
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A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation

https://doi.org/10.1016/j.jmb.2016.08.010Get rights and content
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Highlights

  • A histone H4K20me1-specific live-cell probe, H4K20me1-mintbody, was developed.

  • Dynamics and replication timing of Xi were visualized.

  • Critical amino acids for the stability and/or folding of the mintbody were revealed.

Abstract

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

Abbreviations

H4K20me1
histone H4 Lys20 monomethylation
Mintbody
modification-specific intracellular antibody
scFv
single-chain variable fragment
PCNA
proliferating cell nuclear antigen
FR
framework region
Xi
inactive X chromosome

Keywords

Histone modification
Inactive X chromosome
Intracellular antibody
Live-cell imaging

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Present address: R. Arai, Department of Anatomy and Histology, Fukushima Medical University, School of Medicine, Hikarigaoka, Fukushima 960-1295, Japan.