Development of live-cell imaging probes for monitoring histone modifications

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Abstract

The combination of histone posttranslational modifications occurring in nucleosomal histones determines 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 maintained 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.

Introduction

Despite identical genome sequences, cells acquire and maintain unique tissue-specific gene expression pattern during differentiation. 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 phenotype.1 Because transcriptionally active chromatin was tightly associated with histone acetylation, histone acetylation was proposed as an epigenetic code.2 Furthermore, Strahl and Allis proposed histone code hypothesis; combinatorial histone modifications regulate the adequate gene expression in appropriate phase.3 These histone modifications, mainly phosphorylation, acetylation, and methylation of N-terminal tails of histones, are reversibly and dynamically controlled by modifying and demodifying enzymes. Although histone methylation had been considered a stable modification, it became 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 removed during cellular events such as cell division, differentiation, and reprogramming, mainly due to the lack of imaging tools that allow monitoring the histone modifications in living cells.

Herein, we briefly introduce general imaging techniques for epigenetics focusing on histone modifications; phosphorylation, methylation, and acetylation. We include a method using an antigen binding fragment (Fab)-conjugated chemical fluorescent dye.5, 6 Finally we discuss recent advances in imaging histone modifications via Förster/fluorescence resonance energy transfer (FRET).7, 8, 9, 10 FRET has previously been used for detecting the intracellular dynamics11, 12 of Ca2+ and protein phosphorylation13, 14, 15, 16, 17, 18, 19, 20; but recently FRET-based probes have successfully visualized histone acetylation, providing an application to drug screening and evaluation.9, 10

Section snippets

Probing with an antigen-binding fragment (Fab) labeled with a fluorescent dye

An imaging tool using a Fab of IgG for endogenous histone modification in living cells has been reported in 2009.6 Two Fab fragments, 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 trimethylated 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

Probes based on Förster/fluorescence resonance energy transfer (FRET) for epigenetics

Another approach for monitoring epigenetic alternations in living cells has been designed based on the principle of FRET, which uses two distinct fluorescent molecules. FRET is the transfer of the excited-state energy from the initially excited donor to acceptor only when the two fluorophores are close together with an appropriate orientation. FRET-based probes are typically tandem fusion proteins consisting of a substrate, a flexible linker, a domain recognized post-translational

Evaluation of HDAC inhibitors in living cells using Histac probes

Epigenetic gene regulation by histone modifications is involved in diseases such as cancer. Indeed, two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and FK228 (Fig. 3), were recently approved as anti-cutaneous T-cell lymphoma drugs and several HDAC inhibitors are currently evaluated in clinical trials for anticancer therapeutics.23 Therefore, observation of a dynamic behavior of histone acetylation upon treatment with HDAC inhibitors in living cells is not only important for

Evaluation of bromodomain inhibitors in living cells using Histac probes

Histone code hypothesis consists of three steps, ‘writing’ by histone modifying enzymes, ‘erasing’ by histone demodifying enzymes, and ‘reading’ by proteins interacting with specific histone modifications.2, 27 Both writing and erasing steps have been shown to be promising targets for antitumor drug development.28 Accordingly, inhibitors of epigenetic writers and erasers have been extensively investigated. On the other hand, small molecules that target the reading step have not been explored

Prospects

Histac is the first live cell imaging probe that allows detection of dynamic change in histone acetylation by FRET in the living cell chromatin, which enabled monitoring of cellular activity of small molecules inhibiting HDAC, HAT, and interaction between bromodomains and acetylated histones. The FRET-based approach has a number of advantages over the conventional immunological methods (e.g. immunoblotting, immunofluorescence, chromatin immunoprecipitation), which are essentially inapplicable

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