Development of High-Throughput Assays to Study Methylases, Demethylases and Deacetylases Targeting Histone H3K4, H3K27 and H3K36 Residues

Development of High-Throughput Assays to Study Methylases, Demethylases and
Deacetylases Targeting Histone H3K4, H3K27 and H3K36 Residues
Mathieu Arcand, Mireille Caron, Julie Blouin, Claire Normand, Anne Labonté, Hendrick Plante, Lucille Beaudet & Jaime Padrós
PerkinElmer, 1744 William St., Montreal, QC H3J 1R4, Canada
3 µM
120 min
JMJD3
1 nM
H3K27me3
50 nM
2OG
1 µM
45 min
H3K36me3
LSD1
H3K4ac
2 nM
HDAC1
300 nM
H3K4me1
1 nM
2OG
+
NAD
200 nM
H3K27ac
2 µM
30 min
500 µM
30 min
N/A
3 nM
60 min
N/A
45 min
EZH2
150 ng/well
H3 (21-44)
500 nM
SAM
3 µM
180 min
JMJD3
5 nM
H3K27me3
200 nM
2OG
0.5 µM
120 min
JMJD2A
1 nM
H3K36me3
250 nM
2OG
5 µM
30 min
Enzyme Titration & Time-Course
1,000,000
750,000
500,000
250,000
0
0
30
60
90
120
150,000
EX-527
IC50 = 0.68 µM
Suramin
IC50 = 0.26 µM
75,000
0 -
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1
30
60
90
LANCE Signal (665nm)
LANCE Ultra
LANCE Signal (665 nm)
0
120
60,000
Nicotinamide
IC50 = 180 µM
40,000
EX-527
IC50 = 0.67 µM
20,000
Suramin
IC50 = 0.20 µM
0
-
1,500,000
[LSD1] (nM)
1,250,000
8
4
2
1
0
1,000,000
750,000
500,000
250,000
0
40
60
80
100
120
200,000
100,000
0
[LSD1] (nM)
5
4
3
2
1
0
150,000
100,000
50,000
0
40
60
80
Time (min)
-5
-4
-3
-2
-1
100
120
175,000
Tranylcypromine
IC50 = 74.3 µM
150,000
125,000
100,000
75,000
50,000
25,000
0
- -7
-6
-5
-4
-3
-2
-1
Log [Tranylcypromine] (M)
LSD1 Assay Buffer: 50 mM Tris-HCl pH 9.0, 50 mM NaCl, 1 mM DTT and 0.01%
Tween-20.
SAHA
EC50 = 399 nM
40,000
20,000
0
120
60,000
150
100
75
50
25
0
50,000
40,000
30,000
20,000
10,000
0
30
60
90
120
150
180
[EZH2] (ng/well)
50,000
200
150
100
75
0
40,000
30,000
20,000
10,000
0
60
90
120
150
400,000
300,000
200,000
100,000
0
80
100
120
[JMJD3] (nM)
10
30,000
5
1
2
0.5
0
20,000
10,000
0
40
60
80
Time (min)
120
0
-
-8
-7
100,000
2
1
0.5
0.25
0.125
0
80,000
60,000
40,000
20,000
0
20
40
60
80
-6
-5
-4
Log [2,4-PDCA] (M)
100
50,000
2,4-PDCA
IC50 = 1.5 µM
40,000
30,000
20,000
10,000
0
120
-
-8
-7
-6
-5
-4
Log [2,4-PDCA] (M)
Assay Robustness
AlphaLISA LSD1 assay
20,000
Sinefungin
IC50 = 420 M
10,000
0
-
-6
-5
-4
-3
-2
-1
30,000
25,000
No inhibitor
600,000
500,000
400,000
Z' = 0.80
S/B = 643
300,000
200,000
100,000
3 mM Tranylcypromine
0
0
10
20
30
40
50
AlphaLISA
LANCE Ultra
S/B
Z'
S/B
Z'
SIRT1
384
0.77
15
0.75
LSD1
643
0.80
20
0.88
HDAC1
2.9
0.69
3.6
0.80
EZH2
52
0.71
4.6
0.80
JMJD3
69
0.70
7.4
0.75
JMJD2A
144
0.85
7.3
0.74
Well #
15,000
10,000
Enzyme
700,000
20,000
Sinefungin
IC50 = 780 M
5,000
0
-6
-5
-4
-3
-2
-1
Z’-factor values and signal to background ratios (S/B) were determined for each
AlphaLISA and LANCE Ultra optimized epigenetic assay by analyzing 48 assay wells for
both total and inhibited signals. Calculated Z’-factor values were ≥ 0.69 and remained
stable after overnight incubation (not shown).
12
Inhibitor Titration
100,000
2,4-PDCA
IC50 = 6.9 µM
80,000
Signal increase assays were developed for three demethylases
(LSD1, JMJD2A and JMJD3) and the SIRT1 deacetylase taking
advantage of antibody specificity for unmodified (H3K4) or dimethylated (H3K27me2 and H3K36me2) residues.
60,000
40,000
20,000
0
100
120
- -8
-7
-6
-5
-4
-3
-2
50,000
2,4-PDCA
IC50 = 9.2 µM
40,000
The AlphaLISA and LANCE Ultra HDAC1 signal decrease assays
showed a robust Z’-factor value (0.69 for AlphaLISA and 0.80 for
LANCE Ultra), despite of S/B ratios <4.
IC50 values for known inhibitors and rank order of potency were as
expected from the literature with either detection technology.
30,000
20,000
IC50 and Z’-factor values remained stable after overnight incubation,
allowing both online and offline HTS plate reading.
10,000
0
Summary
AlphaLISA Acceptor beads and LANCE Ultra europium-labeled antimark antibodies were used for the successful optimization of robust
and sensitive epigenetic assays using histone H3-derived peptides
as substrates.
Log [2,4-PDCA] (M)
40,000
20
100
[JMJD2A] (nM)
11
30,000
Time (min)
0
80
50,000
Log [Sinefungin] (M)
5
2
1
0.5
0.25
0
60
60
100,000
40,000
- -7
[JMJD3] (nM)
40
40
120,000
0
Inhibitor Titration
180
500,000
20
20
150,000
JMJD2A Assay Buffer: 50 mM HEPES pH 7.5, 0.01% Tween-20 and 0.1 % BSA.
Note: 5 µM Fe(II) and 100 µM ascorbate were added to enzymatic reactions along
with the biotinylated Histone H3K36me3 peptide substrate.
H3K27me3 Demethylation by JMJD3
0
0
2,4-PDCA
IC50 = 1.7 µM
Time (min)
Log [Sinefungin] (M)
60,000
30
0
Log [Inhibitors] (M)
[EZH2] (ng/well)
0
200,000
- -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
Enzyme Titration & Time-Course
300,000
-6
100
70,000
9
400,000
- -7
80
60,000
EZH2 Assay Buffer: 50 mM Tris-HCl pH 9.0, 50 mM NaCl, 1 mM DTT, 0.01%
Tween-20 and 0.01% BSA.
Tranylcypromine
IC50 = 31.9 µM
500,000
60
Trichostatin A
EC50 = 4.5 nM
H3 (21-44) Methylation by EZH2
Log [Tranylcypromine] (M)
200,000
20
0
40
400,000
200,000
Time (min)
80,000
Time (min)
600,000
Time (min)
0
20,000
0
Inhibitor Titration
AlphaLISA Signal (counts)
Enzyme Titration & Time-Course
20
40,000
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1
H3K4me1 Demethylation by LSD1
0
60,000
20
600,000
Log [Inhibitors] (M)
0
0.25
0.5
1
2
3
Log [Inhibitors] (M)
LANCE Signal (665 nm)
Detection reagents, consumables and instrument. The anti-mark
AlphaLISA Acceptor beads, Alpha Streptavidin Donor beads, AlphaLISA 5X
Epigenetics Buffer 1 kit, LANCE Ultra europium-labeled anti-mark antibodies,
ULight-Streptavidin, 10X LANCE Detection Buffer, white opaque 384-well
OptiPlate™ microtiter plates, TopSeal™-A film and EnVision® Multilabel Plate
Reader were all from PerkinElmer.
50,000
6
AlphaLISA Signal (counts)
Reagents and inhibitors. S-(5’-adenosyl)-L-methionine chloride (SAM), αketoglutaric acid potassium salt (2OG), (+) sodium L-ascorbate, ammonium
iron(II) sulfate hexahydrate (Fe(II)), nicotinamide adenine dinucleotide (NAD+),
trans-2-phenylcyclopropylamine (tranylcypromine), trichostatin A, sinefungin,
2,4-pyridinedicarboxylic acid (2,4-PDCA) and nicotinamide were from SigmaAldrich. Ethylenediaminetetraacetic acid (EDTA) was obtained from Invitrogen,
suberoylanilide hydroxamic acid (SAHA) from Cayman Chemical, and suramin
from EMD Chemicals.
100,000
80,000
SIRT1 Assay Buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 0.01%
Tween-20 and 0.01% BSA.
LANCE Signal (665 nm)
Enzymes and biotinylated peptide substrates. Recombinant enzymes LSD1,
SIRT1, JMJD2A, JMJD3 and the EZH2/EED/SUZ12/RbAp48/AEBP2 protein
complex were from BPS Bioscience. HDAC1 was obtained
from Cayman
Chemical. Biotinylated peptides were from AnaSpec.
2.5
1
0.5
0.25
0.1
0
- -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
Time (min)
100,000
Time (min)
AlphaLISA
Materials
150,000
0
LANCE Ultra
3
[SIRT1] (nM)
120
[HDAC1] (nM)
Log [Inhibitors] (M)
200,000
100
Enzyme Titration & Time-Course
300,000
225,000
80
80,000
8
375,000
Nicotinamide
IC50 = 260 µM
60
HDAC1 Assay Buffer: 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.01%
Tween-20 and 0.01% BSA.
AlphaLISA
[SIRT1] (nM)
5
2.5
1
0.5
0.25
0
1,250,000
40
0
Time (min)
Inhibitor Titration
1,500,000
20
0
H3K4ac Deacetylation by SIRT1
Time (min)
In AlphaLISA and LANCE Ultra epigenetics proximity assays, biotinylated histone
H3-derived peptide substrates are incubated in enzymatic reactions in the
presence of the required cofactors (see Methods). Detection of reaction products
occurs via mark-specific antibodies coupled to Acceptor beads (AlphaLISA) or
labeled with europium chelate (LANCE Ultra). The biotin moiety of the histone
H3-derived peptide substrates is captured by streptavidin coupled to a Donor
bead (AlphaLISA) or labeled with the ULight™ acceptor dye (LANCE Ultra). In
both assay technologies, irradiation of the captured reaction products triggers an
energy transfer leading to light emission proportional to the enzyme activity.
Anti-H3K4
(unmodified)
Anti-H3K4
(unmodified)
Anti-H3K27ac
AntiH3K27me2-1
AntiH3K27me2-1
AntiH3K36me2
0
LANCE Signal (665 nm)
0.5 nM
100 nM
0
200,000
Time (min)
AlphaLISA Signal (counts)
0.5 nM
200,000
[JMJD2A] (nM)
1
0.5
0.25
0.125
0
AlphaLISA Signal (counts)
SAM
Inhibitor Titration
LANCE Signal (665 nm)
100 nM
AlphaLISA Signal (counts)
H3 (21-44)
400,000
800,000
LANCE Signal (665 nm)
150 ng/well
600,000
SAHA
EC50 = 161 nM
AlphaLISA
EZH2
400,000
800,000
Trichostatin A
EC50 = 3.2 nM
LANCE Ultra
30 min
1,000,000
H3K36me3 Demethylation by JMJD2A
Enzyme Titration & Time-Course
AlphaLISA Signal (counts)
N/A
AlphaLISA Signal (counts)
3 nM
LANCE Signal (665 nm)
H3K27ac
AlphaLISA Signal (counts)
1 nM
600,000
LANCE Signal (665 nm)
HDAC1
800,000
AlphaLISA Signal (counts)
60 min
0
0.125
0.25
0.5
1
2
Inhibitor Titration
LANCE Signal (665nm)
N/A
AlphaLISA Signal (counts)
80 nM
[HDAC1] (nM)
1,000,000
LANCE Signal (665 nm)
H3K4me1
1,200,000
AlphaLISA Signal (counts)
30 min
AlphaLISA
800 µM
LANCE Ultra
AlphaLISA
NAD+
2 nM
SIRT1
LANCE Ultra
200 nM
LSD1
JMJD2A
5
H3K4ac
AlphaLISA
LANCE Ultra
1 nM
Anti-H3K4
(unmodified)
Anti-H3K4
(unmodified)
Anti-H3K27ac
AntiH3K27me2-1
AntiH3K27me2-1
AntiH3K36me2
10
H3K27ac Deacetylation by HDAC1
Enzyme Titration & Time-Course
LANCE Ultra
AlphaLISA
SIRT1
AlphaLISA Signal (counts)
Assay Principles
Enzyme
AlphaLISA
2
OPTIMIZED ASSAY CONDITIONS
Reaction Detection
Substrate
Cofactor
time
reagent
LANCE Ultra
Several assay methods have been developed for quantifying the activity of
histone deacetylases (HDACs and sirtuins), histone methyltransferases (HMTs),
and histone demethylases (HDMs). These include radioactive assays, enzymelinked immunoassays (ELISA), mass spectrometry, and enzyme-coupled detection
of fluorescent peptides or reaction co-products (e.g. S-adenosylhomocysteine,
formaldehyde, hydrogen peroxide). These assays suffer from various drawbacks
such as low throughput, lack of sensitivity, generation of hazardous waste,
requirement for expensive equipment, or artifacts associated with the use of nonphysiological fluorescent moieties or enzyme-coupled assays (generation of false
positives/negatives).
In this study, we describe the development and optimization of homogeneous
antibody-based assays for measuring the catalytic activity of a series of
epigenetic lysine-modifying enzymes acting on histone H3 Lys4 (SIRT1
deacetylase and LSD1 demethylase), Lys27 (HDAC1 deacetylase, EZH2
methyltransferase and JMJD3 demethylase) and Lys36 (JMJD2A demethylase).
Two different non-radioactive, no-wash technologies were used for detection of
the enzymatic reaction products: amplified luminescent proximity homogeneous
(AlphaLISA®) assay and time-resolved Förster energy transfer (LANCE® Ultra)
assay.
Results demonstrated that all assays were sensitive, rapid and robust (Z’ factors
≥ 0.69), requiring only nanomolar concentrations of enzyme and peptide.
Furthermore, profiling of known inhibitors for each epigenetic enzyme showed the
expected potency with either technology. These assays will therefore be ideal for
the identification of selective small molecule inhibitors. The approach described
here is broadly suitable for measuring the catalytic activity of other histonemodifying enzymes by combining the appropriate biotinylated histone-derived
peptides and mark-selective antibodies.
7
Methods
LANCE Signal (665 nm)
4
Abstract
AlphaLISA Signal (counts)
1
- -8
-7
-6
-5
-4
-3
-2
Log [2,4-PDCA] (M)
JMJD3 Assay Buffer: 50 mM HEPES pH 7.5, 0.01% Tween-20 and 0.01 % BSA.
Note: 5 µM Fe(II) and 100 µM ascorbate were added to enzymatic reactions along
with the biotinylated histone H3K27me3 peptide substrate.
A comprehensive description of these assays and their optimization
is available on our website at www.perkinelmer.com/epigenetics.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com
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