Mandoli et al. supplementary information

Supplementary material and methods2

Supplementary references7

Supplementary figures8

Supplementary material and methods

Cell culture

ME-1 cells were cultured in RPMI 1640 supplemented with 10% FCS at 37 C. U937-Tet-offCBF-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 ade novo AMLpatient was studied after informed consent was obtainedin 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 sulfate-polyacrylamide 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 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 200mM 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 (

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 mMNaCl, 50 mM Tris-HCl (pH 7.5) containing a mixture of proteaseinhibitors). 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 either13C615N4L-arginine and13C615N2L-lysine (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.

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 with10 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 600ul 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 in-gel 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 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 wasmixed 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, 1μl 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. Second-strand cDNA was synthesized by adding 91.8 μl, 5 μg random hexamers, 4 μl of 5× first-strand 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 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 ( 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 (

Scripts used in this study

Task / Name script / Used to generate figures
Peak calling / MACS / 1D, 3C, 4C, S1C, S1D
Tag counting / peakstats.py / 2E, 4A, 5B, S3
Motif counting / pwmscan.py / 3A
Motif scoring / pwm_scores.py / 3A
Peak annotation / genomic_distribution.py / 1G, H
Intensity plot / makeColorProfiles.pl / 1F, 4D
Expression level / RNAseq2RefSeq.pl / 5A, 6D, 6G-I

All scripts used in this study are available upon request.

For clustering and heatmap generation TMEV ( was used and for functional annotation GSEA (

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.

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