Dimethyl sulfoxide (DMSO), tetraethylthiuram disulfide (disulfiram), phenothiazine, and amiodarone HCl were purchased from Sigma Aldrich. Tegaserod maleate, di-N-desethylamiodarone, and desethylamiodarone were purchased from Santa Cruz Biotechnology. TMR HaloTag ligand was purchased from Promega. SuperSignal West Pico ELISA chemiluminescent substrate, high sensitivity streptavidin-HRP conjugate, and high capacity streptavidin-agarose beads were purchased from Pierce. StabilCoat buffer was purchased from Surmodics. AlphaScreen histidine detection kits (nickel chelate) and ½-area white 96-well plates were purchased from Perkin Elmer. Ni-NTA and glutathione sepharose 4B resins were purchased from GE Life Sciences.
Plasmids for the GST fusions of JARID1A PHD3 (1601–1660), AIRE PHD1 (293–354), ING2 PHD (201–281), BHC80 PHD (486–543), RAG2 PHD (414–487), and JMJD2A double tudor domain (895–1011) were kindly provided by D. Allis (Rockefeller Institute), G. Musco (Dulbecco Telethon Institute), T. Kutateladze (University of Colorado-Denver), Y. Shi (Harvard Medical School), W. Yang (US National Institutes of Health), and R.M. Xu (New York University), respectively. The (HQ)5-HaloTag N-terminal vector was kindly supplied by Dr. Mike Slater and Dr. Jim Hartnett of Promega Corporation (Madison, WI).
Histone H3 (1
) peptides were synthesized using solid phase synthesis on the Intavis ResPep SL robot (Intavis, Koeln, Germany). Standard Fmoc/tBu amino acid couplings were used and premodified amino acid building blocks Kme3
(Novabiochem) were incorporated into positions 4 and 12, respectively, depending on the peptide. Binding peptides contained EPEG-biotin
at position 12 and “unlabeled” competing peptides were unmodified at this position. All peptides (except for fluorescein-labeled species) contained a C-terminal tryptophan for concentration determination by absorbance at 280 nm. Peptide mass was confirmed by MALDI and crude products purified by preparative HPLC (Beckman-Coulter).
Histone H3 (1
) peptides were synthesized as above except for substitution of K(ivDde) (Novabiochem) at position 14. Following synthesis, this lysine was deprotected with 4% v/v hydrazine and labeled with 5-carboxyfluorescein (Novabiochem), followed by standard deprotection and purification.
Protein expression and purification
JARID1A PHD3 (1601–1660) was amplified from a GST plasmid and cloned into a bacterial expression vector encoding an N-terminal (HQ)5–HaloTag followed by a TEV site. Recombinant HQ5-HaloTag-JARID1A PHD3 was expressed in BL21DE3 pLysS cells under kanamycin and chloramphenicol resistance using a 3-hour ambient temperature induction period under 0.5 mM IPTG. Cell pellets were frozen at −80°C and thawed in 30 mM HEPES, 150 mM NaCl, pH 7.4 (HBS) supplemented with 0.5 mM TCEP, 0.01% v/v Triton X-100, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mg/mL lysozyme, followed by two cycles of freeze-thawing and light sonication to lyse cells (5 s 20% amplitude, 5 s off, 1 minute total per sonication cycle, ¼″ probe, Thermo Fisher). Following clarification by centrifugation, the lysate supernatant was bound to Ni-NTA resin in batch for 2 hours at 4°C, washed in batch with HBS and HBS containing 20 mM imidazole, pH 7.4, followed by batch elution with 0.3 M imidazole in HBS, pH 7.4. Elutions were dialyzed in 2 steps into HBS supplemented with 3 mM DTT and glycerol up to 10% v/v. Protein was concentrated in 30 kDa MWCO amicons (Millipore), supplemented with 0.5 mM TCEP, quantitated by Bradford, and stored at −80°C. (HQ)5-HaloTag-AIRE PHD1, RAG2 PHD, BHC80 PHD, and JMJD2A double tudor domain were cloned, expressed, and purified in a similar manner.
His6-JARID1A PHD3 was cloned from the HQ-HaloTag vector into a pQE80L ampicillin-resistance vector and expressed in BL21DE3 pLysS cells. Expression and purification was performed as above except for the use of 3 kDa MWCO amicons (Millipore).
GST fusions to histone reader domains were expressed, lysed, and clarified as described for (HQ)5-HaloTag fusion proteins. The lysate supernatant was bound to glutathione sepharose 4B resin for 2 hours at 4°C, followed by washing in batch with HBS – 0.01% v/v Triton X-100 and elution with 25 mM glutathione in HBS. Elutions were dialyzed as above and protein concentrated in 10 kDa amicons.
HaloTag ligand 96-well plates
HaloTag plates were prepared at Promega by reacting an amine terminated long chain pegylated HaloTag ligand with amine reactive 96 well plates. HaloTag plates were made using white, black or clear base plates for chemiluminescence, fluorescence and absorbance readout respectively. These plates are available from Promega on special request (E.mail: cod/at/promega.com).
A sandwich immunoassay was used to determine the inter- and intra-plate sensitivity and reproducibility of the HaloTag plates. A dilution series of HaloTag-GST fusion protein (Promega) in PBS + 10 mg/mL BSA was made and added in triplicate to the HaloTag plate (50 μL/well). After a 1-hour incubation, the plate was washed and bound fusion protein detected by a sequential 1-hour incubation with anti HaloTag antibody and anti rabbit antibody conjugated to HRP. Colorimetric substrate for HRP was added and absorbance read on a plate reader at 450 nm. All binding steps occurred at ambient temperature. Microplates were sealed during binding steps to prevent evaporation. Results of these studies indicated an LOD of 10 ng/mL and excellent intra-plate reproducibility on clear HaloTag plates. Inter-plate reproducibility was determined using a similar assay on a clear, black and white plate (result not shown). For white and black plates, the substrate was transferred to a clear plate and absorbance read at 450 nm.
HaloTag assay for histone peptide binding
HaloTag fusion protein and peptide were diluted into StabilCoat buffer supplemented with 0.5 mM TCEP. High sensitivity streptavidin-HRP was diluted into SA-AP buffer (30 mM Tris, pH 7.6, 1 M NaCl, 20 mM K/PO4). All steps occurred at ambient temperature, with the microplate sealed to prevent evaporation.
The wells of a 96-well HaloLink microplate were rinsed with 0.2 mL 30 mM HEPES, 150 mM NaCl, pH 7.4, 0.01% v/v Triton X-100 (HBST). (HQ)5-HaloTag-JARID1A PHD3 (50 μL 40 μM) was added to each well and the plate shaken at 200–300 rpm for 2 hours at ambient temperature. Wells were aspirated and washed 3 times with 0.1 mL HBST. Biotinylated histone peptide (50 μL 250 nM) was added to each well and the plate shaken at 200–300 rpm for 1 hour. Wells were aspirated and washed 3 times with 0.1 mL HBST. Streptavidin-HRP (50 μL, 1:2,000 dilution) was added to each well and the plate shaken for 20 minutes at 300 rpm. Wells were aspirated and washed 6 times with 0.1 mL HBST. Immediately before detection, 0.1 mL chemiluminescent substrate was added to each well. Followed by 30 seconds shaking at slow speed, luminescence was measured (1 s integration time) on a Biotek Synergy™ H4 multimode plate reader. The initial concentration matrix assay was detected using a Tecan GENios Pro plate reader. These conditions were used for all (HQ)5-HaloTag-reader domain fusions except BHC80 PHD (1 μM biotinylated histone peptide).
IC50 values for the HaloTag assay, and all other peptide binding assays, were calculated by fitting relative binding/fraction bound versus log[Inhibitor] curves to the equation provided by GraphPad Prism curve fitting software:
where Fb is fraction bound/relative binding and [I] is inhibitor/competitor concentration. Z′ factor is calculated by the following equation:
where μ is the mean of the positive (p) and negative (n) controls, and σ is the standard deviation of the positive and negative controls (19
AlphaScreen histone binding assay
Protein, peptides, and AlphaScreen beads were diluted into StabilCoat buffer supplemented with 0.5 mM TCEP. First, 13.5 μL 100 nM (HQ)5-HaloTag-reader domain followed by 13.5 μL 150 nM biotinylated histone H3K4 +/− me3 peptide, was added to the wells of a half-area white 96-well plate. The final concentration of protein and peptide was 50 and 75 nM, respectively. The sealed plate was shaken at ambient temperature for 30 minutes at 220 rpm. AlphaScreen streptavidin donor and nickel chelate acceptor beads were added to a final concentration of 15 μg/mL (3 μL 150 μg/mL to protein/peptide mixture) and the sealed plate shaken for another 20 minutes at 220 rpm at ambient temperature. Fluorescence was measured on a Biotek Synergy H4 multimode plate reader following a 5 minute delay, using a 680/30 nm excitation/570/100 nm emission filter set with a 635 nm top mirror, sensitivity set to 190. Filter switching to wheel plugs after excitation and once again before emission introduced a necessary time delay for measurement.
Small molecule screening
Small molecule screening was performed at the University of Wisconsin Carbone Cancer Center Small Molecule Screening Facility. The HaloTag assay was performed as above except for the addition of 1 μL 10 mM compounds dissolved in DMSO from the NIH Clinical Collection I by a Matrix Hydra liquid handler (Thermo-Fisher) immediately after addition of biotinylated peptide solution, for a final concentration of 200 μM compound. Wash buffer was dispensed by the Matrix Hydra, and all other additions were performed by multichannel pipetting. Screening was performed over 2 independent experiments with each plate containing DMSO controls for H3K4me3 peptide binding, unmethylated H3 peptide binding and 7.5 μM unlabeled H3K4me3 peptide competition.
Secondary screens, concentration dependence, and counterscreen studies
The HaloTag assay and AlphaScreen assays were performed as above, except for the addition of 1 μL compound dissolved in DMSO to each well immediately after addition of biotinylated peptide. Compounds were assayed at final concentrations between 0 and 200 μM, 2–3% v/v DMSO.
Affinity pull downs
Streptavidin-agarose beads were equilibrated in HBST, followed by immobilization of biotinylated histone peptides in 1% BSA (w/v)-HBST for 30 minutes in batch. In all experiments except amiodarone HCl and phenothiazine, approximately 300 pmol of biotinylated peptide per 6.6 μL bed volume resin was immobilized. Approximately 50 pmol biotinylated peptide was immobilized for studies on amiodarone HCl and phenothiazine due to the poor solubility of these compounds in order to maximize the sensitivity of the assay. Following peptide immobilization, beads were washed three times with 0.5 mL HBST followed by centrifugation for 1 minute at 800 x g, and resuspended in 1% BSA-HBST to form a 50% slurry. (HQ)5-HaloTag-JARID1A PHD3 labeled with TMR HaloTag ligand (10 μM PHD3 labeled with 10 μM TMR ligand in 1% BSA-HBST for 15 minutes at ambient temperature, covered from light) was dispensed to 0.6-mL microcentrifuge tubes, followed by 13.2 μL peptide-linked beads (6.6 μL bed volume) for a final concentration of 100 nM TMR-(HQ)5-HaloTag-JARID1A PHD3 in 1% BSA-HBST. Compounds at varying concentrations (0–500 μM) were added, with a final concentration of 5% v/v DMSO in all samples. Reactions were protected from light and mixed for 45 minutes at ambient temperature, followed by centrifugation and three 0.5-mL HBST washes. Beads were spun down and all remaining liquid aspirated. Beads were boiled in 20 μL SDS-sample buffer for 5–10 minutes before separation by SDS-PAGE (12%, 200 V). Gels were scanned on a Typhoon FLA9000 using the TAMRA setting at 100 μm resolution.
Fluorescence polarization binding studies
Increasing concentrations of compounds were added to solutions containing 1 μM GST-JARID1A PHD3 and 3 nM H3(1
in 30 mM HEPES, 150 mM NaCl, pH 7.4, 0.01% v/v Triton X-100. Each condition was measured in triplicate in a black 384-well plate on a Biotek Synergy H4 multimode plate reader. Counterscreen studies utilized 4 μM GST-AIRE PHD1, 4 μM GST-JMJD2A DTD, 18 μM GST-UHRF1 TTD, 25 μM GST-RAG2 PHD, 60 μM GST-BHC80 PHD. For AIRE PHD1 and BHC80 PHD, 3 nM H3(1
peptide was utilized. For UHRF1 TTD, 3 nM H3(1
was utilized. The concentrations of protein for each GST fusion represent 90% fraction bound. From polarization values, fraction bound was calculated by
is the observed polarization at a given concentration of compound, Pf
is the polarization of free peptide probe, and Pb
is the polarization of maximally bound peptide probe (20
). Dissociation constants (Kd) were calculated using the equation:
where Fb is fraction bound ((21
Zinc ejection studies (PAR assay)
Zinc released was monitored through the colorimetric reagent, 4-(2-pyridylazo) resorcinol (PAR). In a final volume of 300 μL, 5 μM His6-JARID1A PHD3 or (HQ)5-HaloTag-reader domain was treated with increasing concentrations of disulfiram, methyl methanethiosulfonate, tegaserod maleate, or amiodarone HCl in a solution of 10 mM PAR in 30 mM HEPES, 150 mM NaCl, pH 7.4, 5% v/v DMSO (includes DMSO from compounds). 90 μL of each solution was added in triplicate to a clear 96-well plate. The plate was shaken for 10 minutes; alternatively, 1 DMSO-only sample was boiled during this time to monitor zinc released due to structural destabilization. Absorbance at 500 nm was read on a Biotek Synergy H4 multimode plate reader. DMSO-only absorbance values were subtracted from each sample.
Protein structures were downloaded from RCSB PDB (2KGI, JARID1A PHD3; 2GFA, JMJD2A DTD; 2G6Q, ING2 PHD). Small molecule ligands were prepared using Sybyl (Tripos). Ligands were docked “blindly” onto protein receptors using the AutoGrid function within AutoDock 4, the entire histone-binding domain or lobe (in the case of JMJD2A) was accommodated within the grid.
Site directed mutagenesis of JARID1A PHD3 and Schild analysis
Aspartate residues within a GST fusion to JARID1A PHD3 at positions 1624 and 1629 were each mutated to alanine and asparagine, and tryptophan 1625 to alanine, using QuikChange Site-Directed Mutagenesis II reagents. Dissociation constants for each mutant were determined by fluorescence polarization, where increasing concentrations of GST fusion were titrated against 3 nM H3(1
peptide, unmodified or trimethylated at lysine 4. EC50 values for each clone were determined in a similar manner, using fixed concentrations of either di-N-desethylamiodarone or tegaserod maleate (22
). In these analyses, increasing concentrations of GST fusions to JARID1A PHD3 clones were titrated against solutions containing a fixed concentration of inhibitor and H3K4me3 probe. The fold change in EC50 with a fixed concentration of inhibitor relative to the EC50 in the absence of inhibitor is referred to as the dose ratio (22
). A dose ratio greater than one suggests that the inhibitor is able to interact with JARID1A PHD3, causing a shift in apparent Kd
for the H3K4me3 peptide. In contrast, a dose ratio equal to one indicates that the inhibitor does not interact with JARID1A PHD3 and consequently does not influence its ability to interact with the H3K4me3 probe.