Sensing Histone Modification and Transcription Dynamics in Living Cells

Posttranslational histone modification plays an important role in epigenetic gene regulation. To understand the mechanism how histone modifications regulate transcription in vivo, we have developed methods to track posttranslational modifications of endogenous proteins in living cells. In Fab-based live endogenous modification labeling (FabLEM) technique, fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells so that the Fabs can bind to target modifications in cell nuclei. This technique allowed us to track active (phosphorylated) RNA polymerase II and various histone modifications during gene activation in response to various stimuli, including steroid hormone and heat shock, and during development. In all cases analyzed so far, histone H3 lysine 27 acetylation was upregulated before RNA polymerase II activation, while histone methylation did not exhibit drastic changes. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody), which consists of antibody single-chain variable fragment and a fluorescent protein. Mintbodies have been expressed in various organisms, including fruit fly, nematode, zebrafish, frog, and mouse, without affecting their development and fertility. These tools will be useful for understanding the transcription and epigenetic regulation in vivo.