Development of live-cell imaging probes for monitoring histone modifications Kazuki Sasaki a,b , Akihiro Ito b,c,d,⇑ , Minoru Yoshida b,c,d a Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan b Chemical Genetics Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan c Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan d Japan Science and Technology Agency, CREST Research Project, Kawaguchi, Saitama 332-0012, Japan article info Article history: Available online 21 January 2012 Keywords: Histac Imaging Fluorescence Histone Acetylation Bromodomain abstract The combination of histone posttranslational modifications occurring in nucleosomal histones deter- mines the epigenetic code. Histone modifications such as acetylation are dynamically controlled in response to a variety of signals during the cell cycle and differentiation, but they are paradoxically main- tained through cell division to impart tissue specific gene expression patterns to progeny. The dynamics of histone modifications in living cells are poorly understood, because of the lack of experimental tools to monitor them in a real-time fashion. Recently, FRET-based imaging probes for histone H4 acetylation have been developed, which enabled monitoring of changes in histone acetylation during the cell cycle and drug treatment. Further development of this type of fluorescent probes for other modifications will make it possible to visualize complicated epigenetic regulation in living cells. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Despite identical genome sequences, cells acquire and maintain unique tissue-specific gene expression pattern during differentia- tion. The concept of epigenetics, initially described by Waddington in 1942, has shed new light on the developmental phenomena above the level of genome, the gap between genotype and pheno- type. 1 Because transcriptionally active chromatin was tightly asso- ciated with histone acetylation, histone acetylation was proposed as an epigenetic code. 2 Furthermore, Strahl and Allis proposed his- tone code hypothesis; combinatorial histone modifications regulate the adequate gene expression in appropriate phase. 3 These histone modifications, mainly phosphorylation, acetylation, and methyla- tion of N-terminal tails of histones, are reversibly and dynamically controlled by modifying and demodifying enzymes. Although his- tone methylation had been considered a stable modification, it be- came clearer that methylation is also dynamically modulated when lysine-specific demethylase, LSD1, was discovered in 2004. 4 Although it is generally accepted that histone modifications serve as epigenetic marks to determine the cell fate, it remains unclear when, where, and how histone modifications are induced or re- moved during cellular events such as cell division, differentiation, and reprogramming, mainly due to the lack of imaging tools that al- low monitoring the histone modifications in living cells. Herein, we briefly introduce general imaging techniques for epi- genetics focusing on histone modifications; phosphorylation, meth- ylation, and acetylation. We include a method using an antigen binding fragment (Fab)-conjugated chemical fluorescent dye. 5,6 Fi- nally we discuss recent advances in imaging histone modifications via Förster/fluorescence resonance energy transfer (FRET). 7–10 FRET has previously been used for detecting the intracellular dynam- ics 11,12 of Ca 2+ and protein phosphorylation 13–20 ; but recently FRET-based probes have successfully visualized histone acetylation, providing an application to drug screening and evaluation. 9,10 2. Probing with an antigen-binding fragment (Fab) labeled with a fluorescent dye An imaging tool using a Fab of IgG for endogenous histone mod- ification in living cells has been reported in 2009. 6 Two Fab frag- ments, Fab311 or Fab313, were prepared from monoclonal antibodies that can recognize phosphorylated histone H3 at S10 adjacent to un-, mono-, and dimethylated H3K9 or di- and trime- thylated H3K9, respectively. The Fab fragments were conjugated with fluorescent dyes, then were loaded into cultured cells using glass beads or were injected into mouse embryos. Because a Fab fragment (50 kDa) is much smaller than whole IgG, it is able to pass through nuclear pore complex. These fluorescent dye-labeled Fab fragments allowed monitoring the phosphorylation of histone H3 at S10 in cultured cells and mouse embryos. A different spatio- temporal pattern of phosphorylation of histone H3S10 between normal cells and cancer cells was also observed using the Fab311 fragment. In addition, it was revealed that the Fab313 fragment preferentially concentrated at maternal chromosomes, while the Fab311 fragment distributed in both maternal and paternal 0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2012.01.018 ⇑ Corresponding author. Tel.: +81 48 467 9518; fax: +81 48 462 4676. E-mail address: akihiro-i@riken.jp (A. Ito). Bioorganic & Medicinal Chemistry 20 (2012) 1887–1892 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc