Download Supplementary Information (doc 417K)

Mandoli et al. supplementary information
Supplementary material and methods
2
Supplementary references
7
Supplementary figures
8
1
Supplementary material and methods
Cell culture
ME-1 cells were cultured in RPMI 1640 supplemented with 10% FCS at 37 C. U937Tet-off CBF-MYH11 cells (Helbling et al., 2005) were cultured in RPMI 1640
supplemented with 10% FCS at 37 C. For conditional expression of CBF-MYH11,
U937 cells were washed 5 times in 50 mL phosphate-buffered saline (PBS) and seeded at
a density of 2 x 105 cells/ml in the absence of tetracycline. The increase of CBFMYH11 expression was detected by RT-PCR and by western blot.
inv(16) patient characteristics
Mononuclear CD34+ inv(16) AML blasts from peripheral blood of a de novo AML
patient was studied after informed consent was obtained in accordance with the
Declaration of Helsinki.
Cellular fractionation and western blotting
Nuclear and cytosolic fractions were harvested as described (Andrews and Faller, 1991).
Briefly cells were washed with cold PBS, resuspended in cold hypotonic lysis buffer and
incubated on ice for 10 minutes. Cytoplasmic fraction was yielded after centrifugation for
10 seconds. The pellet was suspended in hypertonic buffer, incubated on ice for 20 min,
centrifuged for 2 min at 4oC and supernatant was collected. Cytoplasmic and nuclear
fractions were mixed with sample buffer and separated on 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (Bio-Rad),
blocked in 5% nonfat dry milk in Tris (tris(hydroxymethyl)aminomethane)-buffered
saline with 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, and then incubated
with primary antibodies in TBS-T (with 5 % nonfat dry milk) overnight at 4°C. CBF,
MYH11 and RUNX1 were detected with rabbit polyclonal antibody against CBF
(1:1000), rabbit polyclonal antibody against MYH (1:1000) and rabbit polyclonal
antibody against RUNX1 (1:1000), respectively, followed by an IgG-HRP-conjugated
secondary antibody against rabbit (Dako). As a fractionation control GAPDH (sc-32233;
Santa Cruz), which is primarily cytoplasmic, was used. Proteins were visualized using
ECL (GE healthcare).
Chromatin immunoprecipitation (ChIP)
Cells were crosslinked with 1% formaldehyde for 20 min at room temperature, quenched
with 0.125 M glycine and washed with three buffers: (i) PBS, (ii) buffer of composition
0.25% Triton X 100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6 and (iii)
0.15 M NaCl, 10mM EDTA, 0.5 mM EGTA, 20mM HEPES pH 7.6. Cells were then
suspended in ChIP incubation buffer ( 0.15% SDS, 1% Triton X 100, 150 mM NaCl, 10
mM EDTA, 0.5 mM EGTA, 20mM HEPES pH 7.6) and sonicated using a Bioruptor
sonicator (Diagenode) for 20 min at high power, 30 sec ON, 30 seconds OFF. Sonicated
chromatin was centrifuged at maximum speed for 10 min and then incubated overnight at
4°C in incubation buffer supplemented with 0.1% BSA with protein A/G-Sepharose
beads (Santa Cruz) and 1µg of antibody. Beads were washed sequentially with four
different wash buffers at 4˚C: two times with a solution of composition 0.1% SDS, 0.1%
DOC, 1% Triton, 150 mM NaCl, TEE (10mM Tris pH 8, 0.1mM EDTA and 0.5mM
2
EGTA), one time with a similar buffer but now with 500 mM NaCl, one time with a
solution of composition 0.25 M LiCl, 0.5% DOC, 0.5% NP-40, TEE and two times with
TEE. Precipitated chromatin was eluted from the beads with 400 l of elution buffer (1%
SDS, 0.1 M NaHCO3) at room temperature for 20 minutes. Protein-DNA crosslinks were
reversed at 65°C for 4 hours in the presence of 200 mM NaCl, after which DNA was
isolated by Qiagen column. Antibodies and primers for qPCR can be found in
Supplementary table S1. For qPCR, relative occupancy was calculated as fold over
background, for which the second exon of the Myoglobin gene or the promoter of the
H2B gene was used.
Illumina high throughput sequencing
End repair was performed using the precipitated DNA of ~ 6 million cells (3-4 pooled
biological replicas) using Klenow and T4 PNK. A 3’ protruding A base was generated
using Taq polymerase and adapters were ligated. The DNA was loaded on gel and a band
corresponding to ~300 bp (ChIP fragment + adapters) was collected. The DNA was
isolated, amplified by PCR and used for cluster generation on the Illumina 1G or HiSeq
genome analyzer. The 35-45 bp tags were mapped to the human genome using the eland
or BWA program allowing 1 mismatch. For each base pair in the genome the number of
overlapping sequence reads was determined and averaged over a 10 bp window and
visualized in the UCSC genome browser (http://genome.ucsc.edu).
Re-ChIP
Re-Chip was performed as described (Martens et al., 2012). Briefly chromatin was first
incubated overnight at 4°C with first antibodies as for regular ChIPs. After standard
washing, elution was performed with 1% SDS (30 min, 37 °C). Eluates from at least
three ChIPs were combined, diluted with incubation buffer with protease inhibitors and
incubated overnight with secondary antibodies and protein-A beads at 4 °C. The
subsequent steps were performed as for regular ChIPs followed by qPCR.
Co-immunoprecipitation
Coimmunoprecipitation experiments were performed as before (Martens et al., 2002) in
assay buffer (0.1% NP-40, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5) containing a
mixture of protease inhibitors). ME-1 protein lysates were incubated overnight with
CBFβ-MYH11 or IgG antibodies and prot A/G beads (Santa Cruz), washed 4 times in
assay buffer and tested using western blotting for the presence of CBFβ-MYH11 using
different CBFβ antibodies (Diagenode, Santa Cruz) or with a TAF7 antibody (Santa
Cruz, sc-101167).
SILAC labeling and nuclear extracts preparation
ME-1 cells were SILAC labeled using RPMI (-Arg, -Lys) medium (Gibco/Invitrogen)
containing 10% dialyzed fetal bovine serum (Gibco/Invitrogen) and 1%
penicillin/streptomycin supplemented with either 13C615N4 L-arginine and 13C615N2 Llysine (Isotec) or non-labeled L-arginine and L-lysine (Sigma). Cells were cultured in
SILAC medium for at least 8 doublings to ensure full incorporation of the labeled amino
acids.
3
Nuclear extracts were prepared as previously described (Spruijt et al., 2013). Briefly,
cells were washed with PBS and incubated in hypotonic buffer (10 mM HEPES pH 7.9,
10 mM KCl, 0.1 mM MgCl2) for 15 minutes and homogenized using a type B (tight)
pestle in the presence of 0.15% NP-40 (Roche) and complete protease inhibitors (Roche).
The nuclei were pelleted by centrifugation, washed with PBS, resuspended in hypertonic
buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA pH 8,
10% glycerol, 0.1% NP-40, 1 mM DTT and complete protease inhibitors) and rotated for
1 h at 4°C to extract nuclear proteins. The nuclear extract was obtained by a final
centrifugation step at 20000g for 30 min at 4°C. The resulting supernatant/nuclear
extracts were frozen in liquid nitrogen and stored at −80°C.
DNA pull down
DNA pull down was performed as described previously (Spruijt et al., 2013) with some
modifications. Bait (containing the RUNX1 motif) and control (containing a scrambled
RUNX1 motif) oligonucleotides were generated by annealing sense and antisense
strands. Sense strand of both bait and control oligos were biotinylated at the 5’ end for
coupling to streptavidin beads. 75 ul of Dynabeads MyONE C1 (Invitrogen) streptavidin
magnetic beads were washed with DB buffer (20 mM Tris-HCl, pH 8.0, 2 M NaCl, 0.5
mM EDTA, 0.03% NP-40) and incubated with 10 ug of bait and control DNA separately
in a total volume of 0.35ml of DB buffer for 1h at RT on rotation wheel. After coupling,
the beads were washed two times with DB buffer and two times in PB buffer. A standard
setup in pull down consist of two experiments, forward and reverse. In the forward
experiment control oligo is incubated with light extract and bait oligo incubated with
heavy extract while in the reverse experiment, the control oligo is incubated with heavy
and the bait with light extract. 400ug of SILAC-labeled nuclear extract was added to
beads in a total volume of 600 ul PB buffer (150 mM NaCl, 50 mM Tris/HCl pH 8.0, 10
mM MgCl2, 0.5% NP-40, Complete Protease Inhibitor-EDTA [Roche]) supplemented
with 10ug of poly dIdC and incubated for 90 minutes at 4°C on a rotation wheel. Beads
were washed three times with PB buffer, resuspened in 2X NuPage loading buffer
containing 20 mM DTT and analyzed by western and mass spec. For mass spec analysis
pull down proteins from the forward experiment were mixed together (light control pull
down proteins mixed with heavy bait pull down) and similarly for the reverse (heavy
control pull down proteins mixed with light bait pull down proteins).
LC-MS/MS Analysis
Forward and reverse DNA pull-down were separated by SDS PAGE and subjected to ingel trypsin digestion as described (Vermeulen et al., 2007) Collected peptides were
desalted using StageTips (Rappsilber et al., 2007) and measured on an LTQ-Orbitrap
mass analyzer essentially as described (Vermeulen et al., 2007). Raw mass spectrometric
data were analyzed using the MaxQuant pipeline (Cox and Mann, 2008).
CBF-MYH11 knock down
FH1tUTG lentiviral construct for inducible shRNA expression was prepared as described
previously (Herold et al., 2008). Lentiviral particles were produced in Cos-7 cells and
subsequently ME-1 cells were infected with filtered viral supernatant. Positive cells were
sorted by FACS and induced for shRNA expression using doxycycline. After validating
4
CBFβ-MYH11 knockdown by qPCR and western blot analysis, strand specific RNA-seq
was performed.
Strand specific RNA sequencing
Total RNA was extracted from ME-1 cells with the RNeasy kit and on-column DNase
treatment (Qiagen) and the concentration was measured with a Qubit fluorometer
(Invitrogen). 250 ng of total RNA was treated by Ribo-Zero rRNA Removal Kit
(epicentre) to remove ribosomal RNAs according to manufacturer instructions. 16 µl of
purified RNA were fragmented by addition of 4 µl 5x fragmentation buffer (200 mM
Tris acetate pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate) and
incubated at 94°C for exactly 90 s. After ethanol precipitation, fragmented RNA was
mixed with 5 μg random hexamers, followed by incubation at 70 °C for 10 min and
chilling on ice. We synthesized first-strand cDNA with this RNA primer mix by adding 4
μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng of actinomycin
D, 200 U SuperScript III, followed by 2 h at 48 °C. First strand cDNA was purified by
Qiagen mini elute coloum to remove dNTPs and eluted in 34 μl elution buffer. Secondstrand cDNA was synthesized by adding 91.8 μl, 5 μg random hexamers, 4 μl of 5× firststrand buffer, 2 μl of 100 mM DTT, 4 μl of 10 mM dNTPs with dTTP replaced by dUTP,
30 μl of 5× second-strand buffer, 40 U of Escherichia coli DNA polymerase, 10 U of E.
coli DNA ligase and 2 U of E. coli RNase H, and incubated at 16 °C for 2 h followed by
incubation with 10 U T4 polymerase at 16 °C for 10 minutes. Double stranded cDNA
was purified by Qiagen mini elute column and used for Illumina sample prepping and
sequencing according to the Illumina protocol. We incubated 1 U USER (NEB) with 250
bp size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C
before PCR.
Bioinformatic analyses
Identification of CBFβ-MYH11 binding sites in ME-1 cells
Four antibodies (2 for each factor CBFβ and MYH11) were used to identify the binding
sites of the CBFβ-MYH11 fusion protein in ME-1 cells. Peak calling algorithm
MACS1.3.3 (Zhang et al., 2008) was used to detect the binding sites for these four
antibodies at a p-value cut off for peak detection of 10-6 (Supplementary Table S1). To
identify high confidence CBFβ-MYH11 binding sites, an overlap was taken of the
binding sites detected by MACS for all four antibodies.
Tag counting
Tags within a given region were counted and adjusted to represent the number of tags
within a 1 kb region. Subsequently the percentage of these tags as a measure of the total
number of sequenced tags of the sample was calculated and displayed as heat maps in
Figures 1F, 2E, 4D and S3.
Peak distribution analysis
To determine genomic locations of binding sites, the peak file was analyzed using a script
that annotates binding sites according to all RefSeq genes. With this script every binding
5
site is annotated either as promoter (-500 bp to the Transcription Start Site), exon, intron
or intergenic (everything else).
Motif analysis
To count motifs in CBFβ-MYH11 binding sites we derived the weight matrix of different
consensus binding sites for various proteins involved in hematopoiesis from Jaspar
(http://jaspar.genereg.net/). All CBFβ-MYH11 binding sites were subsequently examined
for the presence or absence of these motifs using a script that scans for homology of the
matrix within the DNA sequence underlying the CBFβ-MYH11 binding site
(pwmscan.py) (see also van Heeringen et al., 2011) using a threshold score of 0.9 (on a
scale from 0 to 1). Note that this analysis will detect the presence of a motif but will not
determine (statistical) enrichment for a particular motif.
Expression analysis
RNA-seq reads were uniquely mapped to the human reference genome and subsequently
used for bioinformatic analysis. RPKM (reads per kilobase of gene length per million
reads) (Mortazavi et al., 2008) values for RefSeq genes were computed using tag
counting scripts and used to analyze the expression level of genes in ME-1 cells. A t-test
was used to show statistical difference between expression of groups of genes. CD
markers were extracts from the HCDM website (www.hcdm.org).
Scripts used in this study
Task
Peak calling
Tag counting
Motif counting
Motif scoring
Peak annotation
Intensity plot
Expression level
Name script
MACS
peakstats.py
pwmscan.py
pwm_scores.py
genomic_distribution.py
makeColorProfiles.pl
RNAseq2RefSeq.pl
Used to generate figures
1D, 3C, 4C, S1C, S1D
2E, 4A, 5B, S3
3A
3A
1G, H
1F, 4D
5A, 6D, 6G-I
All scripts used in this study are available upon request.
For clustering and heatmap generation TMEV (http://www.tm4.org/mev/) was used and
for functional annotation GSEA (http://www.broadinstitute.org/gsea/).
6
Supplementary references
1.
Andrews NC, Faller DV. A rapid micropreparation technique for extraction of
DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res.
1991;19:2499.
2.
Cox J, Mann M. MaxQuant enables high peptide identification rates,
individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
Nat Biotechnol. 2008;26:1367-1372.
3.
Helbling D, Mueller BU, Timchenko NA, et al. CBFB-SMMHC is correlated
with increased calreticulin expression and suppresses the granulocytic differentiation
factor CEBPA in AML with inv(16). Blood. 2005;106:1369-1375.
4.
Herold MJ, van den Brandt J, Seibler J, Reichardt HM. Inducible and reversible
gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats.
Proc Natl Acad Sci U S A. 2008;105:18507-18512.
5.
Martens JH, Mandoli A, Simmer F, et al. ERG and FLI1 binding sites demarcate
targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia.
Blood. 2012;120:4038-4048.
6.
Martens JH, Verlaan M, Kalkhoven E, Dorsman JC, Zantema A. Scaffold/matrix
attachment region elements interact with a p300-scaffold attachment factor A complex
and are bound by acetylated nucleosomes. Mol Cell Biol. 2002;22:2598-2606.
7.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and
quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008;5:621-628.
8.
Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment,
pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc.
2007;2:1896-1906.
9.
Spruijt CG, Gnerlich F, Smits AH, et al. Dynamic readers for 5(hydroxy)methylcytosine and its oxidized derivatives. Cell. 2013;152:1146-1159.
10.
van Heeringen SJ, Veenstra GJ. GimmeMotifs: a de novo motif prediction
pipeline for ChIP-sequencing experiments. Bioinformatics. 2011;27:270-271.
11.
van Heeringen SJ, Veenstra GJ. GimmeMotifs: a de novo motif prediction
pipeline for ChIP-sequencing experiments. Bioinformatics. 2011;27:270-271.
12.
Vermeulen M, Mulder KW, Denissov S, et al. Selective anchoring of TFIID to
nucleosomes by trimethylation of histone H3 lysine 4. Cell. 2007;131:58-69.
13.
Zhang Y, Liu T, Meyer CA, et al. Model-based analysis of ChIP-Seq (MACS).
Genome Biol. 2008;9:R137.
7
8
9
10
11