Supplementary Methods
Methods
Cellular assays
To measure cellular proliferation, an ATP-Lite Luminescent Cell Viability Assay (CTG assay) was used
(Perkin Elmer; cat. #6016941). Cells (0.2 × 105 /well) were seeded into 96-well plates (Costar; cat. #3903)
in 100µL of growth media. Compounds were diluted in DMSO in a 3-fold dilution series and added to
the plates. Cell number relative to the DMSO treated samples was calculated to obtain IC50 values and
curves using a four parameter logistic function in XLFit. For the BrdU assay, 3 ml of cells (0.25 × 106/ml)
were seeded into 6 well plates. On day 6, cells were split to 1.5x106 cells/ml, 3 ml/well and incubated
for another 24 hr prior to the addition of BrdU. The assay was performed using a BrdU assay kit (BD
Biosciences; cat. #559619), and the samples were analyzed using a 96-well High Throughput Sampler
system on a Flow Cytometer (BD Biosciences FACS Canto II). Data were analyzed using FlowJo FACS data
analytical software.
Antibodies for western blot
Antibodies for H3 (#9715), H3K27Me2 (#9728), H3K27Me3 (#9756), H3K4Me2 (#9725), H3K36Me2
(#2901), EZH2 (#5246) and -actin (#4970) were from Cell Signaling Technologies; H3K27Me1 (#07-448),
H3K79Me2 (#04-835), H3K79Me3 (#07-952) were from Millipore; and EZH1 (#ab13665), H3K9Me2
(#ab1220), H3K36Me3 (#ab9050) were from Abcam.
Biochemical assays
Baculoviral expression plasmids for EZH2 Y111L and Y641N/Y661D were generated by site directed
mutagenesis using the Quickchange kit (QiAGEN). H3K27 peptide substrates [H2NATKAARKSAPATGGVKKPHRYRP-GG(K-Biotin)-OH] were purchased with unmethylated (H3[21-44]),
monomethylated {H3[21-44(H3K27-CH3)]} and dimethylated {H3[21-44(H3K27-CH3)2]} lysine 27 from
New England Peptide (Gardner, MA). d4-SAH was from Cayman Chemical Co. (Ann Arbor, MI). Enzyme
time course and titration were carried out in assay buffer (20 mM Tris, 0.5 mM DTT and 0.005% Triton X100, pH8.0). Reactions were performed at room temperature with 10 µM SAM and 30 µM peptides. The
ratio of SAH produced to SAH-d4 is reported as normalized activity. To determine steady-state kinetic
parameters, measurements were taken over five concentrations of H3[21-44] peptides and five
concentrations of SAM between 20 nM to 10 µM. Data was fitted globally for a random bi-bi sequential
mechanism as described (30).
Figure Legends for Supplementary Figures
Supplementary Figure 1
Examples of sequencing traces from K-P, K-R2#4 and K-R10#6. The mutated residues are indicated by
bold italics and the DNA base pair changes are indicated on the graph.
Supplementary Figure 2
K-R2#4 & 5 and K-R10#6 & 7 were resistant to compound 3 treatment. BrdU incorporation in DMSO
treated samples was normalized to 100%. Relative incorporation of BrdU of compound 3 (brown)
treated cells is shown (average from two independent experiments).
Supplemental Figure 3
Commassie blue staining of purified PRC2 complexes from sf9 cells. EZH2 was Flag-tagged and Flag-EZH2
co-migrated with SUZ12 in the protein gel. PRC2 subunits are indicated.
Supplementary Figure 4
Biochemical inhibition of PRC2 complexes by EI1. Percent inhibition of PRC2WT and PRC2Y111L activity by
EI1 at 1 µM and 20 µM [SAM] with Me0 (A) and Me1 (B) peptide. Percent inhibition of PRC2Y641N and
PRC2Y641N/Y661D activity by EI1 at 1 µM and 20 µM SAM with Me1 (C) and Me2 (D) peptide. Data were
generated in triplicate and were fitted in Eq. 1 (see method) using Graph pad prism. This IC50 shift
between 1M and 20M [SAM] was consistent with the SAM-competitive nature of the inhibitor.
Supplementary Figure 5
Biochemical inhibition of PRC2 complexes by EPZ-6438. Percent inhibition of PRC2WT and PRC2Y111L
activity by EPZ-6438 at 1 µM and 20 µM [SAM] with Me0 (A) and Me1 (B) peptide. Percent inhibition of
PRC2Y641N and PRC2Y641N/Y661D activity by EPZ-6438 at 1 µM and 20 µM SAM with Me1 (C) and Me2 (D)
peptide. Data were generated in triplicate and were fitted in Eq. 1 (see method) using Graph pad prism.
This IC50 shift between 1M and 20M [SAM] was consistent with the SAM-competitive nature of the
inhibitor.
Supplementary Figure 6
Biochemical inhibition of PRC2 complexes by compound 3. Percent inhibition of PRC2WT and PRC2Y111L
activity by compound 3 at 1 µM and 20 µM [SAM] with Me0 (A) and Me1 (B) peptide. Percent inhibition
of PRC2Y641N and PRC2Y641N/Y661D activity by compound 3 at 1 µM and 20 µM SAM with Me1 (C) and Me2
(D) peptide. Data were generated in triplicate and were fitted in Eq. 1 (see method) using Graph pad
prism. This IC50 shift between 1M and 20M [SAM] was consistent with the SAM-competitive nature of
the inhibitor.
Supplementary Figure 7
A)
Western blot of H3K27Me3, Me2, and Me1 in the indicated samples. Histone H3 served as a
loading control. K-R2#5 contained the Y111L mutation only, and K-R10#7 contained both Y111L and
Y661D mutations. EPZ-6438 treatment increased H3K27Me2 in resistant cells containing the Y111L
mutation alone. This blot shows similar results as Fig 4.
B)
Western blot of H3K4Me2, H3K9Me2, K36Me2, K36Me3, K79Me2, K79Me3 in the indicated
samples. Histone H3 served as a loading control. Samples were treated with indicated concentration of
EI1 for 3 days.
C)
Same as (B) except all samples were treated with EPZ-6438.
Supplementary Figure 8
Histone tail profiling by mass spectrometry. Each row corresponds to a histone H3 peptide with the
specific combination of marks shown on the right. Cells were treated with 2 or 10 M EI1 or EPZ-6438,
respectively, for 3 days. X axis shows change in the abundance of each peptide, expressed as the ratio
of (indicated sample)/DMSO control in log2 scale. Red lines indicate 2-fold changes. The mutation
status of EZH2 in the cell clones profiled is annotated.