Supplementary Appendix
Fuller description of Materials and Methods
Chromatin ImmunoPrecipitation (ChIP) coupled with acid elution method and ChIP coupled
with genome-wide promoter array analyses (ChIP-on-Chip)
Step 1. Preparation of bone marrow mononuclear cells for ChIP and ChIP-on-chip.
For ChIP-on-chip, at least 30 to 40 million mononuclear cells are necessary to obtain a sufficient
amount of ChIPed DNA for the NimbleGen genome-wide promoter array analysis, in which
three mock IgG controls and three experimental groups with specific antibodies are performed.
For a single individual ChIP experiment, 5 million mononuclear cells are needed.
The following is the procedure for preparing mononuclear cells from blood/bone marrow
aspirates.
1. 5 mL of blood/bone marrow aspirates were mixed with 5 mL of Phosphate Buffered
Saline (PBS) in a 15-mL conical tube.
2. The mixed bone marrow/blood was delicately overlaid onto 5.0 mL of Ficoll gradient
Histopaque-1077 (Sigma#1077-1, St. Louis, MO).
3. Cellular components were separated by centrifuge at 400 x g for 20 minutes at room
temperature and the mononuclear layer at the interface between the two solutions
was collected.
4. Ten ml of PBS were added to the mononuclear cells and then centrifuged at 500 x g
for 5 minutes.
5. The cell pellets were resuspended in PBS and the cells were counted to adjust the final
concentration of 5 million cells per mL in PBS.
Step 2. Cross-linking DNA/histone complexes.
1. Add 10% formaldehyde/PBS (pH = 7.5) directly to tissue culture media to a final
concentration of 1% of formaldehyde.
2. Stop the cross-linking reaction by adding 1.25 M glycine to a final concentration of
0.125 M glycine. Gently rock cells at room temp for 5 minutes.
3. Collect suspension cells and scrape adherent cells into a 50 ml conical tube.
Centrifuge the cells at 2,500 rpm, 40 C for 5 min.
4. Discard the supernatant and wash pellet once with cold PBS plus 1 x protease
inhibitors cocktail (from Roche). Centrifuge the cells at 2,500 rpm, 4 0 C for 5 min.
5. Discard the supernatant and re-suspend the cells with cold PBS plus 1 x protease
inhibitors cocktail (from Roche) adjusting the volume to 10 x 10 6 cells/1 ml.
6. Aliquot the cells in Eppendorf tubes (10 x 106 cells/1 ml/tube). Centrifuge the cells at
2,500 rpm, 40 C for 5 min.
7. Discard the supernatant. Snap-freeze the cell pellets in dry-ice and store the cell
pellets in -80 C.
Step 3. Chromatin Immunoprecipitation.
1. Thaw two tubes of the cell pellets in ice and re-suspend cell pellets in cell lysis buffer (10
x 106 cells/200 ul) plus 1 x protease inhibitors cocktail.
2. Centrifuge the cells at 5,000 rpm for 5 minutes at 40 C to pellet the nuclei.
3. Discard the supernatant and resuspend nuclei vigorously in nuclei lysis buffer plus 1 x
protease inhibitors cocktail (10 x 106 cells/100 ul nuclei lysis + 1 x protease inhibitors).
Incubate on ice for 10 minutes.
4. Transfer the nuclei lysates to a 4 ml sonication tube and add 1 ml chromatin
immunoprecipitation buffer with 0.4 % of Triton X-100 (IP buffer) plus 1 x protease
inhibitors cocktail. Resuspend the nuclei lysates vigorously with pipette.
5. Sonicate chromatin to an average length of about 300 – 500 bp while keeping samples
on ice (the time and number of pulses will vary depending on sonicator, cell type and
extent of crosslinking). The sonication condition is 4 x cycles, 30 sec/cycle, 40% output
and 2 sec/pulse.
6. Carefully remove the supernatant and transfer to a new tube. To reduce non-specific
background, pre-clean chromatin by adding 150 ul of Protein A Sepharose beads.
Incubate on a rotating platform at 40 C for 1.5 hr. Then spin down and remove the
protein-A beads.
7. Save the supernatant and add 2 ml of IP buffer and mix well. Now the total volume
should be 3.2 ml. Save 200 ul as the input control.
8. Equally divide the samples into specific antibodies and an equal amount of IgG controls.
(The amount of antibodies needed for ChIP varies greatly. For Pu.1 antibody, 2 ug per 10
million cells is optimal.)
9. Incubate with constant rotation overnight at 4 C.
10. Add 70 ul of Salmon Sperm DNA protein A -50% slurry cells (see appendix). Salmon
sperm DNA must be omitted for ChIP-on-chip.
11. Incubate at a constant rotation for 2 hr at 4 C.
12. Pellet agarose by gentle centrifugation (1000 rpm at 4ºC, 4 min). Carefully remove the
supernatant that contains unbound, non-specific DNA. Wash the protein A
agarose/antibody/ complex for 3 minutes on a rotating platform with 1ml of each of the
buffers containing 1 x protease inhibitor cocktails (adding freshly) listed in the order as
given below:
1. Low Salt Immune Complex Wash Buffer , one wash
2. High Salt Immune Complex Wash Buffer, one wash
3. LiCl Immune Complex Wash Buffer, one wash
4. 1X TE , two washes.
13. Save the pellets and proceed to elution.
Step 4. Elution of Immunoprecipitated Chromatin. (This step is different from the traditional
ChIP protocol.)
1. Add 200 ul of the low pH elution, buffer the beads and gently re-suspend by pipetting
several times in ice. (Do not vortex!)
2. Micro-centrifuge at 1,000 rpm for 2 min and carefully transfer the supernatant fraction
(eluate) to another tube in ice.
3. Immediately add 20 ul of the high pH buffer to the 200 ul eluate to neutralize the pH of
the eluate (in ice).
4. Each ChIP should have a total volume of 220 ul.
5. Direct Q-PCR can be performed using 2-4 ul of the eluates.
Direct Q-PCR on the neutralized elutes:
1. Save 220 ul of 440 ul total elute for Q-PCR.
2. Use 2 - 4 ul of elute per Q-PCR reaction (25 ul). One can set the Q-PCR conditions
according to optimal annealing temp and DNA concentration vs. background noise. 12 ul of input should give a very strong signal.
For ChIP-on-chip experiments, additional steps are carried out as follows.
Step 5. Reversing cross-linked DNAs.
1. Add 2 ul of RNAse A (10mg/ml)/tube, incubate at 37 C for 30 min.
2. Add 2μl of 10mg/ml Proteinase K/tube and incubate for one hour at 45ºC.
3. Add 10 ul of 5 M NaCl to the elute (220 ul) and incubate at 65 C for 4 hr.
4. Add 2 ul of glycogen, 120ml of phenol:chloroform:isoamyl alcohol (25:24:1, Sigma P3803) to the extract DNAs.
5. Precipitate DNAs by adding 480 ul of 100% ethanol.
6. Dissolve the DNA in 50 ul of distill water.
Step 6. Blunting DNAs.
8. Add 10 ul of 10 x T4 polymerase buffer (NEB), 1 ul of 20 mM dNTP and 0.5 ul T4 DNA
polymerase (NEB cat. # 203L) and use dH2O to bring the volume to 100 ul.
9. Incubate at 16°C for 20 minutes.
10. Add 10 ul of 3 M NaOAc, 1 ul of Glycogen and 240 ul of 100% ethanol, store at –80°C
for 15-30 minutes, and then precipitate DNAs by centrifuge at 14,000 rpm for 15
minutes.
11. Wash the DNA pellets with 500 ul of 70% ethanol, air-dry and dissolve the DNAs in
50 ul of dH2O.
Step 7. Ligation.
1. Make double-stranded linker oligos oJW102/oJW103. The sequences are:
oJW102 (5’-GCGGTGACCCGGGAGATCTGAATTC-3’)
oJW103 (5’-GAATTCAGATC-3’)
2. Dissolve the two oligos (oJW102 and oJW103) in dH2O to a final concentration of
50 mM each.
3. Assemble the double stranded linker oligos by adding 50 ul of 100 mM each oligo,
20 ul of 20 x ligation buffer (NEB # 202) and 80 ul of dH2O, heating the mixture up
to 80°C for 10 minutes and then slowly cooling down to room temperature.
4. Prepare the Ligase Mix by adding 10 ul of 10X ligase buffer 10ml, 1 ul of the 50 mM
annealed linkers, 1 ul of T4 DNA ligase (NEB #202L), and bring the volume to 100 ul
by dH2O.
5. Incubate overnight at 16°C.
6. Add 10 ul of 3M NaOAc and 1 ul of glycogen and 240 ul of 100% ethanol to
precipitate DNAs.
7. Wash with 500uL of 70% ethanol and air dry for 5 minutes.
8. Dissolve the DNAs in 50 ul of dH20.
Step 8. LM-PCR.
1. Make the PCR mixture by adding 10 ul of 10X Taq polymerase buffer (Qiagen), 8 ul
of 2.5mM dNTP, 2 ul of 50 uM oJW102 and 0.5 ul of Taq polymerase (Qiagen) to 50
ul the ligated DNA (50 ul) and bring the volume to 100 ul.
2. Amplify the DNAs following the steps 1. 55°C for 2’, 2. 72°C for 5’ 3. 95°C for 2’ 4.
95°C for 1’ 5. 60°C for 1’ 6. 72°C for 2’ 7. Go to step 4 for 25 times 8. 72°C for 5’ 9.
4°C forever.
3. Purify with QIAquick PCR purification kit. Elute in 50ml Qiagen elution buffer.
4. Measure the DNA concentration of each sample by using Nanodrop
Step 9. Labeling the DNAs.
Step 10. Hybridization to the NimbleGen arrays (3x720K)
Step 11. Two-color array scanning.
Step 12. Data Analysis.
Steps 9-12 are performed by NimbleGen according to the company’s protocol.
Appendixes:
Cell Lysis buffer
5 mM PIPES pH 8.0, 85 mM KCL, 0.5% NP40, 1 x protease inhibitor
cocktail (add before using)
Nuclei Lysis buffer
50 mM Tris-Cl pH 8.1, 10 mM EDTA, 1% SDS, 0.5% Empigen BB
(30%) (optional), 1 x protease inhibitor cocktail (add before using)
IP Dilution buffer
0.5% Triton X 100, 1.2 mM EDTA, 16.7 mM Tris-Cl pH 8.1, 167 mM
NaCl, 1 x protease inhibitor cocktail (add before using)
Low Salt Wash Buffer
0.5% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM
NaCl.
High Salt Wash Buffer
0.5% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM
NaCl.
LiCl Wash Buffer
0.25M LiCl, 0.5% IGEPAL CA630, 1mM EDTA, 10mM Tris, pH 8.1
TE buffer
10 mM Tris-Cl, pH 7.5, 1 mM EDTA (Make from 1 M stock of TrisCl (pH 7.5) and 500 mM stock of EDTA (pH 8.0).
Low pH elution buffer
0.125 M glycine pH=2.5
High pH neutralizing buffer
1 M Tris pH=9.0 (pH value must be accurately
Preparation of Salmon Sperm DNA protein A -50% slurry cells
0.75 ml protein A beads, 600 ug sonicated salmon sperm DNA, 1.5 mg BSA
Computational motif analysis and gene ontology Analysis
The human genome sequence (version hg18) was downloaded from the website
(http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/). A PERL script was written to
extract the DNA sequences corresponding to the H3K27me3 peaks. Overrepresented motifs
were identified by Gibbs-sampling. The expected occurrences for the PU.1 motif by random
chance were calculated by taking into account the GC content in the data. Specifically, the
probability that a particular base would occur in the motif was determined by counting the
number of occurrences of the base and dividing by N, the total number of bases in the DNA
sample. The PU.1 motif RRGGAASY has 8 bases and 16 variants. The probability (P i) that a
particular PU.1 variant would occur by chance is the product of probabilities of the individual
bases occurring, i.e., Pi = ∏ pij, where pij is the probability that the jth base in motif i would
occur. Because both strands of DNA were searched, the expected number of times a particular
PU.1 variant (i) occurs in a set of DNA sequences is: (Ei) = 2 PiN. The actual number of
occurrences (Ai) for a particular PU.1 variant (i) was determined by counting the number of
occurrences in both strands of DNA. The ratio Ai/Ei is the overrepresentation (Oi) of the ith
variant of the PU.1 motif. The average overrepresentation of the PU.1 motif was calculated by
16
Oa = [  Oi ]/ 16.
i 1
To perform gene ontology analysis, the human gene ontology (GO) database was downloaded
from the Gene Ontology Consortium website
(http://www.geneontology.org/GO.downloads.ontology.shtml). The GeneTide database was
downloaded from http://genecards.weizmann.ac.il/genetide-bin/batch_query.cgi. GeneTide
allows cross references between the gene ids in the H3K27me3 data and the gene names in the
GO database.
Whole exome sequencing and Sanger sequencing
Genomic DNA was extracted from bone marrow cells using Qiagen DNeasy blood & Tissue Kit
(Cat. No. 69504, Valencia, CA) and following the manufacturer’s protocol. Fragmented genomic
DNA was tested for size distribution and concentration with the use of an Agilent 2100
Bioanalyzer (Agilent Technologies). Illumina libraries were generated from qualified fragmented
genomic DNA with the use of NEBNext reagents (New England Biolabs). The resulting libraries
were subjected to exome enrichment with the use of NimbleGen SeqCap EZ Human Exome
Library Version 2.0 (Roche NimbleGen), according to the manufacturer's instructions. Enriched
libraries were assessed by quantitative PCR for size distribution and DNA concentration by an
Agilent 2100 Bioanalyzer. The samples were then sequenced on an Illumina HiSeq2000. A
rational bioanalytic algorithm was applied to identify candidate alterations/mutations. Multiple
steps were performed to reduce the false-positive rate in the reported results. First, whole
exome assembly was non-redundantly mapped with the reference genome hg19. Next, an
analytic algorithm was used to call all the positions that varied from a reference genome. Each
potential mutation was compared against databases of known SNPs.
For Sanger DNA sequencing on the PU.1 regulatory regions, the genomic DNA isolated by using
the Qiagen DNeasy blood & Tissue Kit from the CN-RCMD specimens was further amplified by
using the primers and the condition described previously by Bonadies et al (Blood. 2010 Jan
14;115(2):331-4). Sanger sequencing was performed by at the sequencing core of University of
Michigan by following the standard protocol.
Citations for Figures and Tables:
Figure S1. PU.1 expression without drug treatment in various cell lines. A. PU.1
expression in OCI-M2 line; B. PU.1 expression in THP1 cells; C. PU.1 expression
in SC cells.
Figure S2. H3K27me3 inhibitors promoted myeloid differentiation by reducing
H3K27me3 at PU.1 and its target genes in NB4 cells, a promyelocytic cell line. (A)
DZNep inhibits the growth of NB4 cells in a dose-dependent manner. (B) All-trans retinoic Acid
(ATRA), the most effective drug for APL, inhibit the growth of NB4 cells. (C) ChIP experiments
show H3K27me3 in the DZNep-treated NB4 cells at PU.1 promoters. (D) H3K27me3 in the
DZNep-treated NB4 cells at the promoter of CD18, a direct PU.1 target. (E) Flow cytometric
study on PU.1 expression in the NB4 cells treated with 0.25 M DZNep for 6 days. Red, isotype
control; blue, no drug treatment (DMSO only); green, plus drug. (F) Flow cytometric study on
PU.1 expression in the NB4 cells treated with 3 M ATRA for 6 days. (G) Flow cytometric study
on CD18 expression, a differentiation marker for NB4 cells. The cells were treated with 0.25 M
DZNep for 6 days. The DZNep-treated cell viability was 88% at harvest. (H) Flow cytometric
study on CD18 expression in the NB4 cells treated with 3 M ATRA for 6 days. The ATRA-treated
cell viability was 92% at harvest.