Development of live-cell imaging probes for monitoring histone modifications
Graphical abstract
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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Cited by (17)
Visualization of the dynamic interaction between nucleosomal histone H3K9 tri-methylation and HP1α chromodomain in living cells
2022, Cell Chemical BiologyCitation Excerpt :Previous studies revealed that HP1α was released from mitotic chromatin when cells entered mitosis (Fischle et al., 2005; Hirota et al., 2005). As it is difficult to continuously track the dynamics of histone modifications and the behavior of HP1 during the cell cycle using conventional methods, it is necessary to develop imaging tools to monitor histone methylation in living cells (Sasaki et al., 2012). We previously generated genetically encoded FRET-based probes for histone acetylation; Histac (Sasaki et al., 2009) and Histac-K12 (Ito et al., 2011) visualize site-specific acetylation at K5/K8 and K12 of histone H4, respectively, while Histac-H3K9/K14 (Nakaoka et al., 2016) can be used similarly for K9/K14 of histone H3.
Imaging Translational and Post-Translational Gene Regulatory Dynamics in Living Cells with Antibody-Based Probes
2017, Trends in GeneticsCitation Excerpt :This analysis identified several critical residues that contribute to the proper folding and stability of mintbody that will aid the future design of functional intracellular scFvs. Besides antibody-based probes, Förster resonance energy transfer (FRET)-based biosensors have also been developed to quantify the degree of protein modification activity within cells [92–94]. Given that these are sensors, they do not bind to endogenous modifications; instead, they contain a modifiable peptide domain fused to one fluorophore and a modification-binding domain fused to another complementary fluorophore.
The exploitation of FRET probes to track bromodomain/histone interactions in cells for bromodomain inhibitors
2016, Drug Discovery Today: TechnologiesCitation Excerpt :Fluorescence-based imaging technologies have been used to study various dynamic intracellular processes for several years. In particular, the Förster resonance energy transfer (FRET) technique has been utilized to image protein–protein interactions, protein modifications, second messengers, metabolites and membrane voltage [1–8]. FRET is a non-radiative energy transfer from an initially excited donor fluorescent protein to an acceptor fluorescent protein, which is required for the spectral overlap of the donor emission spectrum and acceptor absorption spectrum.
DNA and histone methylation detections
2015, Revue Francophone des LaboratoiresEpigenetics reloaded: The single-cell revolution
2014, Trends in Cell BiologyCitation Excerpt :Specificity for certain acetylation sites is governed by the bromodomain used. For example, the first report of a Histac used the BRDT protein, which binds to acetylated histone H4 at K5 and K8 [50], whereas a subsequent report used the BRD2 bromodomain for specificity toward acetylated H4K12 [51]. This technique can be applied to visualize the dynamics of H4 acetylation in vivo over time since the reader affinity for binding is dependent on the acetylation status.