Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.

Nucleic acids research (2011-05-18)
Yoko Hayashi-Takanaka, Kazuo Yamagata, Teruhiko Wakayama, Timothy J Stasevich, Takashi Kainuma, Toshiki Tsurimoto, Makoto Tachibana, Yoichi Shinkai, Hitoshi Kurumizaka, Naohito Nozaki, Hiroshi Kimura
RESUMEN

Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

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Glass beads, acid-washed, ≤106 μm (−140 U.S. sieve)

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