High-Throughput, Homogeneous Histone Demethylase JARID1A
®
and JARID1C Enzymatic applications with HTRF Technology
Koji Adachi 1, Chikashi Tokuda 1, Thomas Roux ², Eric Trinquet ² and François Degorce ²
1
Sceti Medical Labo K.K. Tokyo, Japan - 2 Cisbio Bioassays, Codolet, France
Introduction
Assay Principle
Histone methylation is a post-translational modification
that occurs on arginine and lysine residues. On histone
H3, lysines #4, 9, 27, 36 and 79 can be mono, di or trimethylated, and represent a biologically relevant readout
for methyltransferases (HMT) and demethylases (HDM)
activity. As shown in the table on the right, the current
selection of EPIgeneous™ highly specific monoclonal
antibodies conjugated to Europium cryptate can be
used to optimize HTRF screening and profiling assays
for a these enzymes which are viewed as promising
therapeutic targets in drug discovery.
to function as a general transcriptional repressor,
JARID1C is known to play an important role in the
brain. This poster highlights optimization data for the
JARID1A and JARID1C H3K4 demethylation assays
as well as antibodies cross-reactivity profiles. We also
present IC50 resulting from several HMT or HDM
enzyme inhibition studies.
EPIgeneous™ Europium cryptate-labeled antibodies
against Methylated Histone H3 Residues
Histone H3
Me0
Me1
Me2
Me3
The JARID (Jumonji/AT-rich interactive domaincontaining protein) family contains four members:
JARID1A (RBP2), JARID1B (PLU-1), JARID1C
(SMCX) and JARID1D(SMCY). Each of them displays
a demethylase activity on mono-, di-, and tri-methyl
lysine residues via an oxidative reaction that requires
iron and α-ketoglutarate (α-KG). JARID1A is suggested
K4
•
•
•
•
K9
K27
K36
•
•
•
•
•
•
HTRF Enzymatic assays established with
Eu(K) labeled Ab
Materials & Methods
Both JARID1A and JARID1C demethylation assays were
developed using EPIgeneous™ anti-dimethyl-Histone H3K4
specific monoclonal antibody labeled with europium cryptate
(donor) and a biotinylated trimethyl-Histone H3K4 peptide
as both substrates. After the enzymatic reaction, this labeled
antibody and streptavidin-acceptor (SA-XL665) were added as a
detection mixture, as shown in the right figure.
4000%
3000%
pH 8.0
1200%
pH 7.5
DF%
DF%
1600%
pH 7.0
800%
pH 6.5
400%
pH 8.0
pH 7.5
2000%
pH 7.0
pH 6.5
1000%
0%
0%
Time course & enzymatic titration
100 ng/well
1000%
30
60
90
2000%
1000%
0%
0
6.25 ng/well
3000%
12.5 ng/well
25 ng/well
2000%
50 ng/well
30
60
300
400
90 120 150 180
JARID1C
4000
2000
Vmax=2980
KM=8.01 µM
1000
0
reaction(RT,min)
reaction(RT,min)
200
3000
0
0
100
H3K4me3 -biotin peptide (nM)
DF%
4000%
120
3000%
3000
0%
0
JARID1C
JARID1A
1000%
0%
JARID1A
Determination of Km (α-KG)
DF%
50 ng/well
2000%
DF%
DF%
25 ng/well
Detection buffer (20 µl)
5 µl Streptavidin-XL665
+ 5 µl anti-dimethyl H3 K4Cryptate
JARID1A enzymatic reaction was 25 ng/well for 30min, and JARID1C one was
70 ng/well for 120min with 50 µM Fe, 2 mM ascorbic acid, 1 mM α-KG at RT.
For all of H3K4 dimethyl peptide detections, biotin/SA ratio was fixed at 4/1. And
for the following experiments, substrate peptides for both assays were 100 nM.
5000%
12.5 ng/well
Enzymatic step (10 µl)
2 µl H3K4me3 substrate-biotin
+ 4 µl compounds
(or enzymatic buffer)
+ 2 µl enzyme
+ 2 µl cofactors
4000%
JARID1C
JARID1A
3000%
+ enzyme = maximum signal
5000%
pH8.0 : 50 mM Tris pH8, 50 mM NaCl, 0.01% tween20, pH7.5 : 50 mM Tris pH7.5, 50 mM NaCl, 0.01% tween20
pH7.0 : 50 mM HEPES pH7, 50 mM NaCl, 0.01% tween20, pH6.5 : 50 mM MES pH6.5, 50 mM NaCl, 0.01% tween20
We used for 50 mM MES pH 6.5 as a JARID enzymatic buffer.
4000%
No enzyme = signal extinction
6000%
JARID1A (50 ng/well) assay was used 100 nM H3K4me3biotin peptide with 50 µM Fe, 2 mM ascorbic acid, 1 mM α-KG
containing buffers below at RT for 90min.
JARID1C (25 ng/well) assay was used 200 nM H3K4me3biotin peptide with the same condition as JARID1A except for
enzymatic reaction time of 120min.
The reactions were stopped to add SA-XL665/anti-H3K4me2-K
mixture in the detection buffer (cat.#62SDBRDD) containing
EDTA.
JARID1C
2000%
Me(2)
Optimization of substrate
DF%
JARID1A
FRET
Me(3)
For the enzymatic step ; substrate, recombinant JARID1 enzymes
(BPS bioscience), inhibitor and cofactor mixture were dissolved
in the enzymatic buffer consisting of 50 mM MES-KOH pH 6.5
, 0.01% Tween20 and added to 384 small volume microplates.
After incubation at RT, the enzymatic reaction was stopped and
detection performed by adding 5 µL of each detection reagent
(SA-XL and anti-H3K4me2-Eu(K)) for a final assay volume of
20 µL. After 1 hr incubation at RT, the TR-FRET signal was
measured using a PHERAstar FS reader (BMG LABTECH). For
each well, fluorescence was measured at 620nm and 665nm and
the HTRF ratio (665nm/620nm) calculated.
Optimization of enzymatic pH
JARID1A or JARID1C
NO FRET
100
200
2000
1000
300
400
0
500
Vmax=3613
KM=1.09 µM
0
10
20
40
50
60
s,a-KG [µM]
s,a-KG [µM]
Both JARID1 assays used 100 nM H3K4me3-biotin peptide with 50 µM Fe, 2 mM ascorbic acid, 1 mM α-KG at RT.
H3K4 dimethyl specific HTRF signals with high titration of JARID1C or long time enzymatic reaction were decreased
because the H3K4me2 peptide was more demethylated to H3K4me1 peptide.
30
Each of the Kms was calculated using the Michaelis-Menten plot. JARID1A and JARID1C showed 8.0 µM and 1.1
µM , respectively. For the following experiments, each of α-KG was selected at 10 µM for JARID1A , and at 2 µM for
JARID1C.
For the following experiments, enzymatic doses and reaction times were 70 ng/well , 120min for JARID1A and 10
ng/well, 30min.
Enzyme Inhibitions
4000
JARID1A
IC50 18.9 µM
3000
DF%
DF%
2000
1000
0
JARID1C
6000%
IC50 12.4 µM
5000%
4000%
DF%
3000
Z’ factor
2000
1000
-7
-6
-5
-4
N-oxalylglycine [LOG(M)]
-3
-2
0
3000%
N-oxalylglycine (60 µM)
No inhibitor
2000%
1000%
0%
-8
-7
-6
-5
-4
-3
0
-2
N-oxalylglycine [LOG(M)]
HTRF JARID demethylation assays were validated by measuring activity of N-oxalylglycine (NOG) inhibitor. JARID1A
assay was performed using 10 μM α-KG and JARID1C assay was done using 2 μM α-KG. Serial dilutions of NOG were
pre-incubated for 5 min with each of the JARID1 enzymes. The enzymatic reaction was initiated by the addition of
100 nM biotinylated H3K4(1-21)me3 peptide substrate and cofactor mixture. These enzyme reactions were stopped
after 120 min or 30 min incubation at RT with the detection reagents.
5
10
15
20
25
30
35
40
45
50
Number of well
The robustness of the inhibition assay was checked performing a Z’ factor determination. Forty eight replicates
were run using NOG at 60 μM (IC80) and the following conditions: 100 nM H3K4(1-21)me3 substrate ; 10 ng/well
JARID1C ; 2 μM α-KG ; RT, 30min reaction.
The Z’ of 0.96 obtained at IC80 underlines the robustness of the assay and its suitability for HTS.
Conclusion
We have developed a selection of position and methylation level-specific
monoclonal antibodies that can be used to create HTRF screening and
profiling assays for a variety of epigenetic lysine-modifying enzymes. These
homogeneous, non-radioactive assays use simple two step protocols that require
no washing, transfer or separation procedures and directly measure position
and methylation-level specific substrates.
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This poster highlights the three main development phases and the performance
of demethylation HTRF assays using JARID enzymes. The optimized assay is
compatible with the use of iron(II), a cofactor necessary for JARID demethylation
to occur.
Copyright © 2013 Cisbio Bioassays, France. HTRF®, TRACE®, and the HTRF™ logo are trademarks belonging to Cisbio Bioassays.
Connect to www.cisbio.com to find a list of our regional distributors.
Like for all other EPIgeneous™ toolbox antibodies currently optimized for
methyltransferase and demethylase enzymes, the application developed with
the H3K4Me2 cryptate conjugate allows a very selective, sensitive and robust
detection of JARID1A and JARID1C activity.
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