Supplementary Information
A) Supplementary Figure Legends
Supplemental Figure 1 (a) MOZ interacts with MLL-N in vivo. Co-immunoprecipitation of
MLL-N with MOZ from HEK293T cell extracts. Whole-cell extracts were used for
immunoprecipitation with an anti-MOZ antibody, then immunoprobed with an anti-MLL-N
antibody. Input: unprecipitated extracts; ctrl: control IP. (b) Schematic representation of MOZ
and the deletion mutants. Domains are labeled as follows: NEMM, N-terminal part of Enok,
MOZ, or MORF; PHD, PHD zinc finger; ED, glutamate/aspartate-rich region; SM,
serine/methionine-rich region; and P, proline/glutamine stretch. The chromosomal
translocations involving MOZ with other HATs are also indicated (arrows).
Supplemental
Figure
2
Input
samples
corresponding
to
eluted
DNA
before
immunoprecipitation were amplified with HOXA5, HOXA7, or HOXA9 promoter specific
primers. Serial dilutions of the chromatin from HEK293T or CD34+ cells were used to
demonstrate linearity of the PCR reaction.
Supplemental Figure 3 Analysis of MLL and MOZ knockdown in CD34+ cells. (a) Left
panel: MLL siRNA treatment decreased the MLL mRNA level. CD34+ cells were transfected
with 500 pmol of Luc siRNA, MLL siRNA or MOZ siRNA. Total RNA obtained from
CD34+ cells was analyzed for MLL mRNA by real time RT-PCR with MLL-specific primers.
Data were normalized to the endogenous Hprt mRNA control. Data: Mean +/- SD of
triplicates. Four independent experiments were performed. Right panel: MLL siRNA
treatment reduced the MLL protein level. Extracts were prepared from cell treated as in right
1
panel for SDS-PAGE and immunoblotting with anti-MLL-C antibody (top). Ponceau R
staining was also performed (bottom). (b) Left panel: MOZ siRNA treatment decreased the
MOZ mRNA level. CD34+ cells were treated and total RNA was isolated as in (a) to
determine the MOZ mRNA level by real time RT-PCR with MOZ-specific primers. Data
were normalized to the endogenous Hprt mRNA control. Data: Mean +/- SD of triplicates.
Four independent experiments were performed. Right panel: MOZ siRNA treatment reduced
the MOZ protein level. Cell extracts were prepared and used for immunoblotting as in (a)
except that an anti-MOZ antibody was used (top). Ponceau R staining was also performed
(bottom).
Supplemental Figure 4 (a) The recruitment of MOZ is altered in absence of MLL and vice
versa. Purified CD34+ cells were transfected once with 500 pmol of Luc siRNA, MOZ
siRNA or MLL siRNA. Then, a ChIP analysis examining the recruitment of MOZ or MLL on
HOXA5, HOXA7, and HOXA9 promoters in CD34+ cells was carried out 24h later. Lanes
were loaded with products of PCR amplification using template prepared from either 1%
sheared chromatin (Input control) (lane 1) or immunoprecipitated chromatin using IgG (lane
2) or specific antibodies directed against MOZ (lane 3) or MLL (lane 4). (b) PCR analysis on
GAPDH promoter after ChIP experiments in siRNA transfected CD34+ cells. Lanes were
loaded with products of PCR amplification using template prepared from either 1% sheared
chromatin (Input control) or immunoprecipitated chromatin using IgG (Ctrl), specific
antibodies directed against MOZ, MLL, di-methylated H3K4, tri-methylated H3K4,
acetylated histone H3, or acetylated histone H4K16.
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B) Supplementary methods
Reagents
For ChIP and IP, a mouse monoclonal antibody against residues 856-870 of MOZ was
produced by Mustapha Oulad-Abdelghani (IGBMC, Illkirch, France). The anti-MOZ (N19)
antibody used in immunofluorescence assays was purchased from Santa Cruz Biotechnology
(CA, USA). The anti-MOZ antibody for Western blotting was provided by Abcam
(Cambridge, UK). The c-Myc (9E10) antibody was obtained from Santa Cruz Biotechnology
(CA, USA), the anti-FLAG M2 antibody from SIGMA (Saint-Louis, MO, USA), and the antiHA (3F10) antibody from Roche Applied Science (Mannheim, Germany). Antibodies
directed against MLL-C, WDR5, acetyl-histone H3K9K14, acetyl-histone H4K16,
dimethylated histone H3K4, and trimethylated histone H3K4 were provided by Upstate
biotechnology (Lake Placid, NY, USA). The anti-MLL-N antibody (N4.4) for Western
blotting was provided by Millipore. For FLAG immunoprecipitation assays, anti-FLAG M2
antibodies coupled to agarose beads were used (EZview Red ANTI-FLAG M2 Affinity Gel,
SIGMA). For control in IP and ChIP, IgG were provided by Santa Cruz. Biotinylated histone
H3 peptides (residues 1-21) (trimethylated on K4 or not) were purchased from Upstate
Biotechnology.
Human cord blood CD34+ cells
Cells were layered over Ficoll-Paque (1.77 g/L) (Eurobio, Les Ulis, France), and the interface
containing mononuclear cells was harvested after centrifugation. Then, the cells were washed,
and CD34+ cells were purified using the CD34+ cells magnetic isolation kit and
AutoMACSTM separator according to the manufacturer’s instructions (Miltenyi Biotec,
Bergish Gladbach, Germany). The isolated cells were cultured during seven days in
3
StemSpan™ H3000, supplemented with 100 ng/mL rhFlt-3 ligand, 100 ng/mL rhSCF, 20
ng/mL rhIL-3 and 20 ng/mL rhIL-6 (StemCell Technologies, Vancouver, BC, Canada) before
RT-PCR assays, ChIP experiments or colony-forming assays.
Myeloid colony-forming assays
For myeloid culture, a total of 1×103 CD34+ cells or CD34+ siRNA-treated cells (48h after
transfection) were added to 1 ml of methyl-cellulose (1%) (MethoCult M3434; StemCell
Technologies) in IMDM with 30% fetal calf serum (Bio Whittaker), 2 mM L-glutamine
(Invitrogen), 1% BSA, MTG (100 µM), hSCF (50 ng/ml), hGM-CSF (10 ng/ml) (StemCell
Technologies) and hIL-3 (10 ng/ml) (StemCell Technologies). The culture plates were
incubated at 37°C and in a humidified atmosphere of 5% CO2 in air.
Real time RT-PCR
RNA was extracted from CD34+ cells using NucleoSpin®RNAII (Macherey-Nagel). cDNAs
from total RNA were synthesized using M-MLV Reverse Transcriptase (Promega). Real time
PCR was performed in triplicates using TaqMan probes from Applied Biosystems (Foster
City, CA) and analyzed in an Applied Biosystems 7500 Fast Real-Time PCR System. Values
for each PCR were normalized with Hprt or 2-microglobulin. The TaqMan® assays were the
following: Hs00430330_m1 (Hoxa5), Hs00600844_m1 (Hoxa7), Hs00365956_m1 (Hoxa9),
Hs00198899_m1 (Moz), Hs00610538_m1(Mll).
SDS-PAGE, Immunoprecipitation and Western blotting
HEK293T (transiently transfected as indicated above or not), K562, CD34+ cells were
harvested, washed with PBS, and Dounce-homogenized (10 strokes with a type B pestle) in a
cold immunoprecipitation-lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-HCl pH8,
4
protease inhibitor cocktail). Samples were incubated on ice for 30 min. After centrifugation, 1
mg of total proteins was then precleared with protein G agarose beads (Upstate). MOZ, MLLC or Myc-specific antibodies (or IgG as a control), associated with protein G agarose beads,
were used for immunoprecipitating proteins with gentle shaking at 4°C overnight. As control,
each
extract
was
also
immunoprecipitated
with
irrelevant
IgG.
For
FLAG
immunoprecipitations, protein extracts were immunoprecipitated with anti-FLAG M2 affinity
gel. Immunoprecipitation complexes were washed five times in ice-cold immunoprecipitationlysis buffer. Proteins were eluted by boiling in Laemmli buffer. Eluated proteins were then
separated by SDS-PAGE and electroblotted to nitrocellulose membranes. Equivalent loading
between lanes was confirmed by Ponceau R staining. Membranes were blocked in 1x PBS-T
(0.1%) and fat-free dry milk or BSA (5%) (blocking buffer) during one hour at room
temperature. Membranes were incubated with the primary antibodies diluted in the blocking
buffer at 4°C overnight. Membranes were washed three times in 1x PBS-T (0.1%) during 10
min each. Secondary antibodies conjugated with horseradish peroxidase were added, and the
membranes were incubated at room temperature during one hour. Membranes were washed
three times in 1x PBS-T (0.1%) during 10 min each. ECL Western blotting reagent kit
(Millipore, Billerica, MA, USA) was used for protein detection.
Immunofluorescence microscopy
Cells were incubated 1h at RT with the anti-MOZ (dilution 1:100), anti-MLL-C (dilution
1:100), anti-WDR5 (dilution 1:100) or no antibody as a control. Then, cells were incubated
during 1 h with antibodies specific for mouse or goat immunoglobulin subclasses conjugated
to fluorochromes (anti-goat-Alexa 488 or anti-mouse-Alexa 568) (dilution 1:1000). Nuclei
were counterstained with DAPI. For cytospun cells, images were captured with a Cell
Observer Station (Zeiss) with a 63x oil objective, then deconvulated with the deconvolution
5
module of the Axiovision software. Adherent cells were analyzed using a confocal laser
scanning microscope (Leica TCS SP2). Images were processed with Adobe Photoshop
(adjustment of brightness and contrast).
Reporter gene assays
All transfections contained 1 µg of reporter plasmid (pHoxA7-luciferase or pMIP1luciferase), as well as 100 ng of TK- -galactosidase plasmid as an internal control to
normalize transfection efficiency and MOZ or MLL expression vectors.
ChIP procedure and ChIP primers
After pre-clearing with salmon sperm DNA/protein A/G agarose beads, the samples
underwent immunoprecipitation with antibodies specific for MOZ, MLL-C, di-, trimethylated H3 K4, acetyl H3 K9 K14, acetyl-histone H4 K16, or IgG (Control ChIP) at 4°C
overnight. Beads were washed, protein/DNA complexes eluted, then cross-links reversed by
heating at 65°C overnight. After RNA and protein digestions, DNA was purified on a spin
column (NucleoSpin Extract II, Macherey-Nagel, Düren, Germany). Input corresponds to
total sonicated DNA. DNA was amplified by PCR. PCR amplification was performed using
Taq polymerase (Promega) and ChIP primers.
The ChIP primers used to amplify regions of the promoter locus of HOXA5 were (5′CTCCACCCAACTCCCCTATT-3' and 5′-CGGTCGTTTGTGCGTCTAT-3'), HOXA7 were
(5′-CAGGGCTCACTAGCAGGAGT-3’ and 5′-GGCAAGAGGCTCAAATATGC-3’), and
HOXA9
were
(5′-GGGGAGACGGGAGAGTACAG-3’
and
5′-
CGTCCAGCAGAACAATAACG-3’) (Invitrogen), and the negative region (NEG: region
located
between
the
GAPDH
gene
and
the
CNAP1
gene)
were
(5′-
ATGGTTGCCACTGGGGATCT-3’ and 5′-TGCCAAAGCCTAGGGGAAGA-3’) (Active
6
Motif, Rixensart, Belgium). The ChIP primers used to amplify a region of the promoter locus
of GAPDH were provided by EZ ChIP (Upstate biotechnology).
Real-time qPCR analysis was analyzed in an Applied Biosystems 7500 Fast Real-Time PCR
System. For real-time qPCR analysis, the ChIP primers used to amplify regions of the
promoter
locus
of
HOXA5
were
(5′-CTCCACCCAACTCCCCTATT-3'
and
5′-
CGGTCGTTTGTGCGTCTAT-3'), HOXA7 were (5′-CCTGTGAGGACTGCTGAGATTG-3’
and
5′-CCCCCAGATTTACACCAAACC-3’),
GGGGAGACGGGAGAGTACAG-3’
and
and
HOXA9
were
(5′-
5′-CGTCCAGCAGAACAATAACG-3’)
(Invitrogen).
siRNA knockdown
siRNAs, synthesized by Invitrogen, target human MLL (Sense: 5’-AGUGGUUCCUGAGAA
UGGAUUUGAA-3’) and MOZ (Sense: 5’-UUAAUCUGCACUUCAGAGCCUCAGG-3’).
A luciferase siRNA was used as a negative control (Sense: 5’-CUUACGCUGAGUACUUCG
Att-3’).
Histone tail peptide-binding assays
Cell extracts or in vitro translated products were then precleared with neutravidin-coated
agarose beads (Pierce, Rockford, IL, USA). Biotinylated histone H3 tail peptides
(trimethylated on K4 or not) were incubated overnight either with the cell extracts or the in
vitro translated product, at 4°C. Then, the biotinylated histone H3 tail peptides were collected
by incubation with neutravidin-coated agarose beads. The beads were washed ten times in the
IP lysis buffer. Bound proteins were eluted by boiling in the SDS loading buffer, separated by
SDS-PAGE, and analyzed by immunoblotting with anti-Myc or anti-WDR5 antibodies.
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