Protein pull-down experiments using compounds
GST fusion proteins were isolated from bacterial lysates using glutathione sepharose beads (GE Healthcare). In vitro compound pull-down assays were performed by pre-binding 5 μg of biotinylated UNC1215 to 2 μg of GST fusion protein in 500 μl of 1x binding buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2) giving a final compound concentration of 0.01 μg/μl (6.4 μM). Incubation was done overnight rocking at 4 °C. Next, 25 μL of streptavidin agarose beads (Millipore) were pre-washed with binding buffer and incubated with the compound-protein mix for 1 h rocking at 4 °C. After three washes with 500 μl binding buffer, 30 μl of 2x protein loading buffer was added to the beads and boiled. The samples were resolved on a SDS-PAGE and western blots were performed using anti-GST antibody.
Protein pull-down experiments using the H4K20me2 peptide
In vitro peptide pull-down assays were performed as described above by pre-binding 1 μg of biotinylated H4K20me2 peptide (MW 2.416 kDa) with 1 μg of GST fusion protein in 500 μL of 1x binding buffer.
L3MBTL3 protein was crystallized at a concentration of 10 mg/mL using the sitting drop vapor diffusion method at 18 °C. The reservoir solution contained 25% PEG 3350, 0.2 M NH4
Ac, 0.1 M Bis-Tris buffer at pH 5.5. Using a nylon loop, crystals were passed through reservoir solution additionally containing 20% glycerol, flash-frozen and stored in liquid nitrogen until data collection.41
Data collection/structure determination and refinement
A continuous sweep of 180 single-degree oscillation images was collected at beam line 19ID of the Advanced Photon Source at a wavelength of 0.9793 Å. Data were reduced using XDS, 42
The structure was solved with the program PHASER45
and a model related to Protein Data Bank (PDB)46
entry 3UT1 (to be published). Geometry restraints for the inhibitor were prepared with PRODRG.47
The atomic model was re-built, refined and validated using COOT,48
/autoBUSTER (Buster version 2.10.0. (Global Phasing Ltd, Cambridge, UK, 2011)) and the MOLPROBITY server,50
respectively. For data and model statistics, refer to Supplementary Table 4
Images were acquired using a Quorum Spinning Disk Confocal microscope equipped with 405, 491, 561, and 642 nm lasers. Image analysis and foci quantification were performed using Volocity 5.4.1. Foci were quantified using the % GFP intensity function.
For the localization experiments with GFP-3MBT, GFP-FLMBT, 3MBT-GFP, FLMBT-GFP, mCherry-3MBT, mCherry-FLMBT, GFP-FLMBTÄSAM, and the site-directed mutants, 100,000 HEK293 cells/1mL/well were seeded on coverslips in 12 well plates. 16–24 hours later, cells were transfected with the appropriate tagged construct using GeneJuice reagent, according to the manufacturer’s instructions, for an additional 48 hours. Prior to imaging, cells were treated with Hoechst 33342 dye for 30 min, washed with PBS, and incubated with the appropriate media.
For foci elimination experiments, the same transfection procedure was followed. In these experiments, cells were treated with the indicated concentrations of inhibitors for at least 24 hours before imaging.
For merocyanine-UNC1215 and GFP-FLMBT co-localization experiments, cells were seeded as described above and transfected with wildtype GFP-FLMBT, D274A GFP-FLMBT, or D381A GFP-FLMBT for 16–24 hours, followed by a 2 hour incubation with 2.5 μM of the merocyanine dye (mero76) or the merocyanine-UNC1215 conjugate/1 mL/well at 37 °C. After incubation with Hoechst 33342 dye and PBS washes, the media was changed to DMEM/F-12 without phenol red (11039) prior to imaging.
U2OS cells were similarly transfected with GFP fusion constructs. Cells were fixed with 2% formaldehyde in PBS for 10 minutes, permeabilized with 0.1% Triton X-100 PBS for 15 minutes, blocked with 5% goat serum, 3% BSA, 0.1% Tween in PBS for 1 hr and incubated with a BCLAF antibody (Abcam) overnight at 4 °C in the blocking buffer. A secondary Alexa488 antibody (Cell Signaling Technology) was used and coverslips were mounted with Fluoroshield DAPI (Sigma).
Transfected cells were treated with inhibitors for 3–6 hours and analyzed on a Zeiss laser scanning confocal microscope LSM510 META Confocal using 488 nm laser bleaching and subsequent image acquisition every 2 seconds. The data was processed in Volocity 5.4.1 software, normalized to the signal in an unbleached area, and expressed as a percent of the initial signal. The exponential curve fitting and half-life calculations were done using GraphPad Prism4 software. The dissociation constants were determined as reported by Yguerabide et al.51
Affinity purification with biotin-UNC1215 conjugate
HEK293 cells were transiently transfected with Flag-FLMBT using GeneJuice reagent according to manufacturer’s instructions. Approximately 10 million cells were used per immunoprecipitation. Frozen cell pellets were lysed in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, and complete protease inhibitor cocktail (Roche). After vortexing and centrifugation at 13,200 × g, 4 °C, 5 min to pellet the DNA, the supernatant was diluted to 150 mM NaCl (while keeping other reagents’ concentrations constant) and was precleared with Streptavidin Mag Sepharose™ magnetic beads (GE Healthcare) for 1 hour at 4 °C. Streptavidin beads were incubated with 5 nmol biotin-UNC1215 (or incubated in the absence of biotin-UNC1215 as a negative control) in 300 μL IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail (Roche) for 30 min at room temperature. Precleared lysate was added to the biotin-UNC1215 prebound streptavidin beads, in the absence or presence of 5 nmol or 50 nmol cold, untagged UNC1215 overnight at 4 °C. Three washes were performed with IP buffer, followed by elution with buffer containing 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton, 3% SDS, 5 mM DTT, and 15 mM ME, analysis by SDS-PAGE, and immunoblotting.
HEK293 cells were transfected with Flag or GFP tagged L3MBTL3, treated with inhibitor (1 μM) for 18 hours, and lysed in 50 mM TrisHCl, 450 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors. The lysates were diluted to adjust the NaCl concentration to 200 mM, and then an anti-Flag antibody (Sigma) and protein A Dynabeads (Life Technologies) were added for incubation at 4 °C overnight. The beads were washed 3 times with the lysis buffer, eluted using 100 mM Na2H2PO4, 150 mM NaCl, 2 mM EDTA, 5 mM dithiothreitol, 1% Triton X-100, 3% SDS, 1 mM sodium orthovanadate, and 150 mM β-mercaptoethanol. SDS-PAGE separated proteins were transferred to PVDF membrane (Millipore) and immunoblotted using BCLAF1 (BTF1) antibody (Abcam).
Protein interactor screen and mass spectroscopy analysis
5 × 15 cm plates were used for one biological replica, with two biological replicas performed for each set of conditions. HEK293 cells transiently transfected with GFP-3MBT or GFP-FLMBT were lysed in high salt AFC buffer (10 mM Tris-HCL, pH 7.9, 420 mM NaCl, 0.1% NP-40) by freeze-thawing on a dry ice/ethanol mixture and in a 37 °C water bath. Freeze-thawing was repeated three times, after which cells were sonicated on ice (8 times, 0.3 s on, 0.7 s off), incubated with benzonase nuclease (to give a final concentration of 12.5–25 units/mL) for 30 min at 4°C, and centrifuged for 1 hr at 40,000 rpm. The cell lysates were then incubated with an anti-GFP antibody (Abcam) at 4°C overnight, protein G (Sigma) at 4 °C for 4 hours, and washed with low salt AFC buffer (10 mM Tris-HCl, pH 7.9, 100 mM NaCl, 0.1% NP-40), 5 × 1 mL each time. Bound proteins were eluted with 500 mM ammonium hydroxide, dried, resuspended in 50 mM ammonium bicarbonate, trypsin digested, and subjected to MS analysis. X tandem search algorithm was used to identify the peptides. The total spectral counts were normalized to the molecular weight of proteins and the complex was visualized using cytoscape.52
For cytoscape analysis, only those interactors were included that were not present in the vector alone purification, and had spectral counts equal to or higher than six.
Synthesis of UNC1021, UNC1079, UNC1215, and other related analogs are described in Supplementary Note 1
. Additional methods are presented in Supplementary Information
, including biochemical assays, selectivity assays, protein expression and purification, protein mutagenesis, and compound toxicity studies. Additional results are also included in Supplementary Information
Protein Data Bank: coordinates and structure factors for the co-crystal structure of the L3MBTL3-UNC1215 complex have been deposited with accession code 4FL6.