Analytical Biochemistry 476 (2015) 78–80

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A fluorescence resonance energy transfer-based method for histone methyltransferases Kanchan Devkota a,b, Brian Lohse a, Camilla Nyby Jakobsen a, Jens Berthelsen c, Rasmus Prætorius Clausen a,⇑ a

Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark NNF Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark c Department of International Health, Immunology and Microbiology, University of Copenhagen, DK-2200 Copenhagen, Denmark b

a r t i c l e

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Article history: Received 22 January 2015 Accepted 12 February 2015 Available online 19 February 2015 Keywords: Methyltransferase Assay EHMT1

a b s t r a c t A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye and a quencher, respectively. When lysine-9 residues in the peptides were methylated, they were protected from cleavage by endoproteinase–EndoLysC, whereas unmethylated peptides were cleaved, resulting in an increase in fluorescent intensity. Ó 2015 Elsevier Inc. All rights reserved.

Methyltransferase-mediated methylation of histone and nonhistone targets is involved in human diseases such as cancer, inflammatory diseases, and neurogenerative disorders [1–4] and play significant roles in processes such as meiosis [5], germ cell development [5], and embryo development [6]. The roles of these methyltransferases in various human disorders make them promising targets for drug discovery efforts and treatment strategies. Various enzyme activity assays have been developed during the past few years, generating valuable information to help understand the enzymes’ specificity and characteristics and to identify potential activators or inhibitors of methyltransferases. An enzyme-coupled fluorescent assay that employs the additional enzymes S-adenosylhomocysteine hydrolase and adenosine deaminase was the first reported methyltransferase assay [7]. Several colorimetric [8], fluorometric, and spectrophotometric assays [9] have also been used to access the activities of protein lysine methyltransferases (PKMTs),1 and most of them focus on the conversion of the by-product of methylation reaction, S-adenosylhomocysteine (SAH), to other products. Radioactive assays that use radiolabeled S-adenosylmethionine (SAM) and look at the transfer of radioactive methyl groups from the SAM to the substrate, which in turn can be quantified continuously using proximity

⇑ Corresponding author. E-mail address: [email protected] (R.P. Clausen). Abbreviations used: PKMT, protein lysine methyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; EndoLysC, endoproteinase–LysC; MCE, microfluidic capillary electrophoresis; FRET, fluorescence resonance energy transfer; H3, histone 3; 5/6-FAM, 5/6-carboxyfluorescein; BHQ1, Black Hole Quencher 1. 1 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.

scintillation counting, have also been reported [10]. This assay is highly accurate and reproducible, but hazards associated with the handling of radiolabeled products and waste favor the use of fluorescent methods. Several antibody-based assays such as enzyme-linked immunosorbent assay (ELISA) [11] and LANCE Ultra assay [12] have also been widely used, in which an antibody against specific methyllysine histone marks are employed to capture certain methylation marks. Most of these methods employ expensive reagents and/or expensive specific instruments for readouts. A drawback of these assays is the variability of specificity of the antibodies used. Assays employing proteases such as endoproteinase–LysC (EndoLysC) to cleave unmethylated peptide substrates labeled with a fluorophore have also been developed. A recent method employs EndoLysC to cleave the unmodified substrates, and the separation is achieved by using microfluidic capillary electrophoresis (MCE) through a special instrument called EZ Reader II (Caliper). Although the charge of the peptide is not altered after methylation, the difference in size of cleaved and intact peptides allows for difference in charge-to-mass ratio, hence allowing the separation using MCE [13]. In addition, a fluorescence lifetime-based method, FLEXYTETM (developed by ALMAC), has been reported for EHMT2 that uses fluorophore 9-aminoacridine coupled to specific peptide fragments. The assay reports the use of an EndoLysC cleavage step where methylated peptide fragments are uncleaved. Methylation is measured through a decrease in fluorescence lifetime of approximately 5 ns between product and substrate [14]. Here, we report modification of previous assays employing EndoLysC to determine the kinetic parameters and identify potential inhibitors for methyltransferases.


Notes & Tips / Anal. Biochem. 476 (2015) 78–80 Table 1 Peptides used in the study. Peptide


H3 tail (first 20 residues) P1 P2 P3 (=P1 methylated) P4 P5 P6 P7


Note: The lysine residue (K) in bold represents Lysine 9 residue of Histone H3 which is methylated by EHMT1/2. The lysine residues (K) in italics represent other lysine residues in Histone H3.

Fig.1. Detection principle of the dye–quencher FRET-based EndoLysC assay using peptide fragments corresponding to H3 peptide fragments as substrates. The peptide fragment has only one lysine in it. BHQ1 is the quencher used, whereas 5FAM is the dye used. (A) PKMTs methylate the lysine (K) residue in the modified peptide substrate, protecting it from cleavage by EndoLysC. (B) In the presence of inhibitors, the lysine (K) residues in the modified peptide substrate do not get methylated and, hence, are cleaved by EndoLysC, which can be quantified by measurement of fluorescence intensity (excitation = 490 nM, emission = 520 nM).

In this dye–quencher fluorescence resonance energy transfer (FRET)-based EndoLysC assay (Fig. 1), peptide fragments corresponding to N-terminal histone 3 (H3) peptide chain containing a single unmodified lysine residue are coupled with a dye (6-carboxyfluorescein, 6-FAM) at one end and a quencher (Black Hole Quencher 1, BHQ1) at the other end. The quencher eclipses the fluorescence of the dye when the peptide is intact. On treatment with EndoLysC, a protease that cleaves proteins or peptides in the C-terminal side of lysine residues, the peptide fragment containing unmodified lysine residue is cleaved and the quencher is no longer able to quench the fluorescence of the dye, resulting in an increase in fluorescence intensity. In the presence of the lysine methyltransferase EHMT1 or EHMT2, the lysine residue K9 in the peptide substrate gets methylated. This modified lysine can no longer be cleaved by EndoLysC; therefore, no increase in fluorescence intensity can be detected as the peptide remains intact (uncleaved). On the other hand, in the presence of the EHMT1/2 inhibitor, methylation is prevented, which results in unmethylated lysine residues in the peptide fragments that are cleaved by EndoLysC, leading to an increase in fluorescence intensity that can easily be quantified. Assays were performed with 100 nM EHMT1 and EHMT2 final concentrations, and methylation reactions were allowed to proceed for 30 min at room temperature. Final peptide and SAM concentrations in the reaction were 500 nM and 1.5 lM, respectively, in a final volume of 25 ll. For inhibition studies, EHMT1/2 was preincubated with BIX01294 for 15 min before adding SAM and peptide. Dimethyl sulfoxide (DMSO) concentrations were maintained at 4% final. Then, 1 ll of a 10-ng/ll EndoLysC solution was added to the reaction to stop methylation and was incubated at room temperature for 30 min. Measurements were taken using a TECAN SAFIRE II, with excitation at 490 nM and emission at 520 nM. All of the reactions were done in triplicates in OptiPlate384 white opaque 384-well microplates with a final reaction

volume of 25 ll. The methylation buffer used was 50 mM Tris– HCl (pH 8.0), 50 mM NaCl, 1 mM dithiothreitol (DTT), and 0.01% Tween 20. Analyses of the results were done by using GraphPad Prism software (version 6.0). For data analysis, refer to the Online supplementary material (Section S2). The peptides were labeled in the N-terminal end with the dye 5-carboxyfluorescein (5-FAM) or 6-FAM and in the C-terminal with lysine modified at the distal amino group with the quencher BHQ1. A list of peptides used in the study is given in Table 1. Both P1 and P2 showed large increases in fluorescence intensity on treatment with EndoLysC for P1 (see Section S3 in Supplementary material). The shorter peptide P1 was modified by introducing a methyl group to the lysine that is cleaved to show that this was protected from EndoLysC cleavage, and indeed no difference in fluorescence intensity was observed before and after EndoLysC treatment (see Section S3 in Supplementary material). This demonstrated that EndoLysC could indeed be used to discriminate dye–quencher peptides with methylated lysines from non methylated peptides. We then subjected peptides 1 and 2 to methylation by EHMT1 and EHMT2 at the same conditions as in the LANCE assay, followed by the addition of EndoLysC. However, no change in fluorescence intensity was observed, suggesting that these peptides were not methylated. Hence, longer peptides P5 and P6 were synthesized, extending the amino acids in either side of peptide fragment P2. P5 contained lysine residue (K14) of H3 tail, whereas P6 contained lysine residue (K4) of H3 tail, and to protect this extra lysine from proteolysis by EndoLysC, it was methylated. As a control peptide, P4 was synthesized and treated with EndoLysC to confirm that K4Me would be protected. Both peptides P5 and P6 could be cleaved by EndoLysC, and indeed P6 also acted as a very good substrate for EHMT1 and EHMT2 (Fig. 2), leading to a decrease in the fluorescence intensity observed after treatment with EndoLysC. P7 was tested (modified P6 with K4 substituted with alanine [K4A]), and it acted as a very week substrate for EHMT1 and EHMT2 (see Section S4 in Supplementary material). As seen in Fig. 2A, EHMT1 (red) and EHMT2 (blue) incubated with peptide P6 as substrate and SAM as cofactor, and followed by EndoLysC treatment (E+P+S+C), were undergoing methylation reaction and, thus, showed lower fluorescence intensity compared with experiments containing methylation mixture without SAM (E+P+C) or the peptide alone (P+C), followed by treatment with EndoLysC. For EHMT2 the fluorescence level for the fully treated peptide (E2+P+S+C) reached the level for the pure peptide (P), whereas for EHMT1 the level was slightly higher, suggesting that P6 had not been fully converted. To optimize the assay conditions and enzymatic methylation kinetics for EHMT1/2, enzyme kinetics (see Section S5 in Supplementary material) and SAM titration were performed using the EndoLysC assay. The apparent Km for SAM (K app m SAM ) was found to be 2.9 lM for EHMT1 and 1 lM for EHMT2 (see Section S6 in Supplementary material). This is similar to the K app m SAM for


Notes & Tips / Anal. Biochem. 476 (2015) 78–80

Fig.2. (A) Proof of assay concept using EHMT1 (E1, red), EHMT2 (E2, blue), and modified peptide (substrate). E1, EHMT1; E2, EHMT2; P, modified peptide; S, SAM, cofactor; C, EndoLysC. (B) Dose–response curves for BIX01294 against EHMT1 (red) and EHMT2 (blue) using the EndoLysC assay. The IC50 value for EHMT1 is 0.2 lM (0.04–0.8 lM) and for EHMT2 is 0.3 lM (0.2–0.5 lM). Values in parentheses indicate 95% confidence intervals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

EHMT1 and EHMT2 found by using LANCE Ultra G9a assay from PerkinElmer (0.5 and 2.2 lM, respectively) [15]. Although K app m values obtained from different experiments cannot be directly compared due to the difference in assay procedures, reagents, and concentrations, the results support the suitability of this assay in determination of EHMT1/2 activity. To further determine the feasibility of identifying potential inhibitors of EHMT1/2 with this assay, experiments were carried out to determine the inhibition of EHMT1/2 by the known EHMT1/2 inhibitor BIX01294. Using this assay, we demonstrated that BIX01294 inhibits EHMT1 and EHMT2 in a dose-dependent manner with IC50 values of 0.2 and 0.3 lM, respectively (Fig. 2B). Although these differ somewhat from the IC50 values determined by the LANCE Ultra G9a assay (see Section S7 in Supplementary material), it is in the range of other results in the literature. In addition, IC50 analysis of Sinefungin analogue 4d, recently reported by our group as an inhibitor of EHMT1 and EHMT2, showed similar values as measured by the LANCE Ultra assay [15] (see Section S8 in Supplementary material). The assay uses simple reagents and common standard fluorescence plate readers for readouts, making it inexpensive and easy to handle. The peptide fragments can be modified, making this assay suitable for other lysine methyltransferases and demethylases as well, including arginine methyltransferase inhibitors, by using similar peptide fragments together with EndoArgC that cleave at arginine residues. Acknowledgments We are grateful to the Novo Nordisk Foundation Center for Protein Research and the Faculty of Health and Medical Sciences, University of Copenhagen, for K.D.’s PhD Fellowship. The University of Copenhagen Programme of Excellence and the Danish Cancer Society are gratefully acknowledged for financial support. J.B. is funded by the ISIM CAR project. Jesper L. Kristensen is gratefully acknowledged for valuable input and strategic discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at

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A fluorescence resonance energy transfer-based method for histone methyltransferases.

A simple dye-quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic par...
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