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NEOPLASIA
Physical and functional link of the leukemia-associated factors AML1 and PML
Lan Anh Nguyen, Pier Paolo Pandolfi, Yukiko Aikawa, Yusuke Tagata, Misao Ohki, and Issay Kitabayashi
The AML1-CBF␤ transcription factor complex is the most frequent target of specific chromosome translocations in acute
myeloid leukemia (AML). The promyelocytic leukemia (PML) gene is also frequently involved in AML-associated translocation. Here we report that a specific
isoform PML I forms a complex with AML1.
PML I was able to recruit AML1 and coac-
tivator p300 in PML nuclear bodies and
enhance the AML1-mediated transcription in the presence of p300. A specific
C-terminal region of PML I and a Cterminal region of AML1 were found to be
required for both their association and
colocalization in the nuclear bodies. Overexpression of PML I stimulates myeloid
cells to differentiate. These results sug-
gest that PML I could act as a mediator for
AML1 and its coactivator p300/CBP to
assemble into functional complexes and,
consequently, activate AML1-dependent
transcription and myeloid cell differentiation. (Blood. 2005;105:292-300)
© 2005 by The American Society of Hematology
Introduction
Chromosome translocations are frequently detected in 50% to 70%
of human leukemia. The AML1 (CBFA2/PEBP2␣B/RUNX1) gene
is the most frequent target of leukemia-associated chromosome
translocations.1 The AML1 gene was first cloned at the breakpoint
of chromosome 21 in t(8;21) translocation, which is frequently
found in the M2 subtype of acute myeloid leukemia (AML).2 So
far, it has been reported that AML1 is disrupted by other translocations such as t(3;21),3,4 t(12;21),5,6 and t(16;21).7,8 AML1 also has
been found to be mutated in familial platelet disorder (FPD)
associated with a predisposition to leukemia9 and in sporadic cases
of AML and myelodysplastic syndrome (MDS) without chromosome translocations.10-12 AML1 protein forms a heterodimer with
CBF␤ and binds to the specific DNA sequence TGT/cGGT13,14 to
regulate the expression of a number of hematopoietic genes. Both
AML1 and CBF␤ are essential for the development of all lineages
of definitive hematopoiesis.15-19 AML1 is hypothesized to act as an
organizing factor that facilitates the assembly of transcriptional
activation complexes. In this regard, AML1 could synergize with
other transcription factors such as AP-1,20 C/EBPa,21 c-Myb,22
PU1,23 and Ets-124 to activate transcription. Furthermore, AML1
cooperates with coactivators such as YAP,25 ALY,26 Ear-2,27 p300/
CREB-binding protein (CBP),28 and MOZ.29 On the other hand,
AML1 has been reported to function as a transcriptional repressor
of some target genes via its interaction with corepressors like
TLE1,30,31 SIN3, and NCoR.32 Notably, the genes encoding some of
the AML1-interacting proteins are also the targets of leukemiaassociated chromosome translocations. In particular, CBF␤ is
disrupted in inv(16)33; p300 is disrupted in t(8;22)34,35 and t(11;
22)36; CBP is disrupted in t(8;16)37 and t(11;16)38-40; and MOZ is
disrupted in t(8;16),37 inv(8),41 and t(8;22).34,35 Thus, understanding
the contexts in which AML1 acts in cooperation with multiple
factors is necessary in order to elucidate the regulatory function
of AML1 in hematopoiesis and the mechanisms of leukemogenesis caused by chromosome translocations that disrupt AML1
and its cofactors.
The promyelocytic leukemia (PML) gene is involved in the
chromosome translocation t(15;17), which occurs in most cases of
the acute promyelocytic leukemia (APL) M3 subtype of AML.42-47
Targeted disruption of the PML locus demonstrated that PML could
control the differentiation of hematopoietic progenitors, cell proliferation, and tumorigenesis.48 PML coordinates tumor suppressive
functions such as induction of apoptosis, growth arrest, and cellular
senescence.49 PML concentrates in speckled subnuclear structures,
termed PML nuclear bodies (NBs)/ND10/PODs, together with
many proteins (Zhong et al50). The proteins that can colocalize with
PML into NBs include Sp100, p53, pRb, Daxx, and CBP, which
suggests that PML could modulate, at least in part from the NBs, a
variety of nuclear processes such as gene expression and genome
stabilization. PML can modulate transcription by recruiting transcription factors such as p53 to NBs. Recruitment of p53 to the
PML-NBs was shown to accompany Ras- and PML-induced
senescence and p53-dependent apoptosis.51-53 Although PML⫺/⫺
mice have been shown to have remarked reduction of granulocyte
and monocytes in their peripheral blood and bone marrow,48 the
current understanding of the function of PML is not enough to
explain the deficiency in myeloid hematopoiesis. Multiple isoforms
of PML are generated as a result of alternative splicing54 (Jensen et
al55). Whether these isoforms possess the specific functions remains largely unexplored.
In this study, we found that the 2 major leukemia-associated
proteins, AML1 and PML, can function in the same complex. A
specific PML isoform could interact with AML1 transcription
From the Molecular Oncology Division, Cancer Genomics Project, National
Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan;
and the Cancer Biology/Genetics Program and Department of Pathology,
Sloan-Kettering Institute Memorial Sloan-Kethering Cancer Center, New
York, NY
Promotion of Fundamental Studies in Health Sciences of the Organization for
Pharmaceutical Safety and Research of Japan.
Submitted March 29, 2004; accepted July 26, 2004. Prepublished online as
Blood First Edition Paper, August 26, 2004; DOI 10.1182/blood-2004-03-1185.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported in part by Grants-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology; and by the Program for
292
Reprints: Issay Kitabayashi, Molecular Oncology Division, National Cancer
Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; email: [email protected].
© 2005 by The American Society of Hematology
BLOOD, 1 JANUARY 2005 䡠 VOLUME 105, NUMBER 1
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BLOOD, 1 JANUARY 2005 䡠 VOLUME 105, NUMBER 1
factor, target AML1 into nuclear bodies together with its coactivator p300, enhance AML1-mediated transcription, and stimulate
differentiation of myeloid cells. Our findings suggest cooperation
of AML1 and PML in hematopoiesis.
Materials and methods
Cells and retrovirus
Murine myeloid progenitor L-G cells were cultured in RPMI medium
supplemented with 10% fetal calf serum (FCS), recombinant mouse
interleukin-3 (IL-3) (0.1 ng/mL) and 50 ␮M ␤-mercaptoethanol. To
monitor the growth and differentiation of myeloid cells in response to
granulocyte colony-stimulating factor (G-CSF), L-G cells were maintained
in the medium with G-CSF (5 ng/mL) instead of IL-3. Every day, viable
cells were counted using a Coulter counter (Beckman Coulter, Hialeah, FL).
To evaluate the cell differentiation, cells were stained with May-Gruenwald
and Giemsa solutions. BOSC23 and SaOS2 cells were maintained in
Dulbecco modified Eagle medium (DMEM) supplemented with 10% FCS.
To produce the retrovirus, BOSC23 cells were transfected with LNCXderived vectors by the calcium phosphate precipitation method, and the
culture supernatants were recuperated 48 hours after transfection. For
infection, L-G cells were incubated in the culture supernatant of BOSC23
transfectants with 4 ␮g/mL polybrene for 24 hours and then selected with
antibiotic G418 (1 mg/mL).
Plasmids
The AML1 expression vectors pLNCX-FLAG-AML1a and pLNCX-FLAGAML1b have been previously described.28
PML isoforms. The PML expression vector pCMV-HA-PML, which
was a generous gift from Dr A. Kakizuka (Kyoto University),44 carries the
PML transcript corresponding to human PML VI. To construct the
expression vector for other human PML isoforms I-V, we isolated cDNA
fragments encoding the specific 5⬘-terminal region of these isoforms from
the cDNA pool of HL60 cells by reverse transcriptase–polymerase chain
reaction (RT-PCR) using the up-primer 5⬘-ATCCAAGAAAGCCAGCCCAGAG-3⬘ (1208-1229) common to all, and the down primers
5⬘-TGGGAATGCTGTGATGGGTG-3⬘ (2749-2730), 5⬘-ACTAAATTAGAAAGGGGTGGGG-3⬘ (1975-1953), and 5-AGCACAGCTTGGGACTCAATGC-3⬘ (1865-1844), specific to PML I (M79462), PML IV (X63131),
and PML V (M79463), respectively, and the 5⬘-GGTGTTGGCTGCAAGGTTACAAG-3⬘ (2527-2505) that was shared by isoforms II (M79464)
and III (S50913). These fragments that represented the specific Ctermini of the individual isoforms were replaced with specific sequences
of PML VI on the expression vector pCMV-HA-PMLVI by using
appropriate enzymes. The sequences of these constructs were checked
by DNA sequencing. Subsequently, full-length cDNA of the PML
isoforms I-VI were subcloned into a retrovirus-derived LNCX vector by
using HindIII and NotI restriction enzymes.
PML I mutants. We introduced point mutations into PML I by the
method of site-specific mutagenesis using overlapping extension PCR. To
create unSUMOylated PML protein, which could not be SUMOylated, we
substituted 3 lysine residues at codon 65, 160, and 490, which were found
be modified by SUMO-156 into arginine (AAG65Lys-AGG65Arg, AAG160LysAGG160Arg, and AAG490Lys-AGG490Arg, bold letters indicate substituted
nucleotides). Deletion mutants of PML I were generated by using appropriate restriction enzymes.
mCCP1-luc reporter. Mouse mCCP1 promoter (⫺243 to ⫹27) was
isolated by PCR and subcloned between the NheI and HindIII sites of the
pGL3-Basic Vector (Promega, Madison, WI).
Identification of proteins in the AML1b complex
We purified AML1b complex and identified peptides in the AML1b
complex as previously described.29 Briefly, cell lysates were prepared from
L-G cell infectants that stably expressed AML1b tagged with FLAG peptide
in lysis buffer (250 mM NaCl, 20 mM sodium phosphate pH 7.0, 30 mM
INTERACTION OF AML1 WITH PML I
293
sodium pyrophosphate, 10 mM NaF, 0.1% NP-40, 5 mM dithiothreitol
(DTT), 1 mM phenylmethyl sulforyl fluoride [PMSF]) supplied with Complete
protease inhibitor (Roche, Mannheim, Germany), and then cleared by ultracentrifugation with a Hitachi RP50T2 rotor (Hitachi, Tokyo, Japan) at 4°C, 81 000 g
(30 000 rpm) for 30 minutes. The supernatant was incubated with anti-FLAG
antibody conjugated agarose beads (Sigma, St Louis, MO) and rotated at 4°C for
8 to 10 hours. After extensive wash with lysis buffer, the absorbed proteins
were eluted from agarose beads with FLAG peptide in lysis buffer (0.2
mg/mL) for 1 hour. The eluents were concentrated by using a filtration
device (Vivaspin, 10K-PES; Sartorius, Hannover, Germany) and separated
by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE). Proteins were stained with Coomassie brilliant blue, excised, destained
with 25 mM ammonium bicarbonate and 50% acetonitrile, dried, digested with
sequence grade modified trypsin (Promega) in 50 mM Tris [tris(hydroxymethyl)
aminomethane] pH7.6, extracted with 5% trifluoroacetic acid (TFA), 50%
acetonitrile, and subjected to liquid chromatography/mass spectrometry/
mass spectrometry (LC/MS/MS) analysis.
Immunoprecipitation, immunoblotting, and antibodies
Cell lysates were prepared in lysis buffer (250 mM NaCl, 20 mM sodium
phosphate pH 7.0, 30 mM sodium pyrophosphate, 10 mM NaF, 0.1%
Nonidet P-40 (NP-40), 5 mM DTT, 1 mM phenylmethyl sulfonyl fluoride
[PMSF]) supplied with complete protease inhibitor (Roche). After removal
of cell debris by ultracentrifugation with a Beckman TLA55 rotor
(Beckman Coulter, Marseilles, France) at 4°C and 72 000 g (40 000 rpm)
for 30 minutes, the supernatant was incubated with anti-FLAG antibody–
conjugated agarose beads (Sigma) and slightly rotated at 4°C for 8 to 10
hours. The absorbed beads were extensively washed with lysis buffer.
Precipitated proteins were eluted from the beads by 200 ␮L of lysis buffer
containing the FLAG peptide at a final concentration of 2 mg/mL. The
eluate was concentrated by using filtration devices (Vivaspin 10K-PES;
Sartorius) and dissolved with the same volume of 2 ⫻ SDS-PAGE sample
buffer (125 mM Tris-HCl, 4% SDS, 10% ␤-mercaptoethanol) and then
subjected to SDS-PAGE. When immunoprecipitation was not performed,
total protein lysates were prepared in 2 ⫻ SDS-PAGE sample buffer.
Antibodies were detected by chemiluminescence using enhanced chemiluminescence (ECL) plus Detection Reagents (Amersham Biosciences,
Buckinghamshire, United Kingdom). Primary antibodies were anti-FLAG
(M2) (Sigma), anti-HA (3F10) (Roche), anti-human AML rabbit polyclonal,28 anti-human PML rabbit polyclonal (PML001) and mouse monoclonal (1B9) (MBL), anti–mouse PML rabbit polyclonal,48 and anti-p300
rabbit polyclonal (N15) (Santa Cruz Biotechnology, Santa Cruz, CA)
antibodies were used in this study.
Immunofluorescence analysis
SaOS2 cells were plated at 1 ⫻ 105 per chamber on 4 chamber vessels (BD
Falcon, Bedford, MA), the day before transfection. Plasmid DNA was
transfected by using Lipofectamine2000 reagent (Invitrogen, Carlsbad, CA)
according to the supplier’s instructions. Four hours after transfection, the
medium was refreshed, and cells were incubated at 37°C for a further 36
hours for gene expression. Before staining, transfectants were washed with
phosphate-buffered saline (PBS). Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes and washed twice in PBS. Next, cells were
permeabilized with 0.1% Triton-X 100 in PBS for 3 minutes, washed 4
times with PBS and blocked with 10% FCS in PBS for 2 hours at room
temperature. Cells were incubated with primary antibodies at a final
concentration of 2 ␮g/mL in PBS containing 3% bovine serum albumin
(BSA) at 4°C for 10 hours. Primary antibodies were revealed by a 4-hour
incubation with appropriate fluorescence-conjugated secondary antibodies
(Santa Cruz Biotechnology) in a 100-fold dilution in PBS containing 3%
BSA. For double staining, cells were incubated with the 2 antibodies under
the same conditions. Slides were mounted in mounting liquid Vectashield
containing 0.4 mg/mL 4⬘,6-diamidino-2-phenylindole (DAPI) to visualize
nuclear DNA (Vector Laboratories, Burlingame, CA). The images under
fluorescence microscope (Olympus, Melville, NY) at magnification ⫻ 60
were captured with a CoolSNAP-HQ CCD camera (Roper Scientific,
Ottobrunn, Germany).
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BLOOD, 1 JANUARY 2005 䡠 VOLUME 105, NUMBER 1
Luciferase assay
Wild-type or PmL⫺/⫺ mouse embryo fibroblasts (MEF) cells48 were
transfected by the calcium phosphate precipitation method in 24-well
plates, and luciferase activity was assayed 48 hours later using a luminometer Lumat LB9507 (Berthold, Bad Wildbad, Germany) according to the
manufacturer’s protocol (Promega). Results of reporter assays represent the
average values for relative luciferase activity generated from 3 independent
experiments that were normalized using the activity of the enzyme from
pRL-CMV as an internal control.
Results
AML1b forms complex with a specific isoform PML I
We previously purified the AML1 complexes from a cell lysate of
L-G cells expressing a FLAG-tagged full-length AML1 (AML1b)
or a splicing variant lacking a C-terminal transcriptional activation
domain (AML1a)29 (Figure 1A). Mass spectrometry analysis
indicated that the 125-kDa protein in the AML1b complex contained the sequences that are found in murine promyelocytic
Figure 2. PML I isoform specifically interacts with AML1b. (A) Schematic
representation of the PML protein and genomic structure of various PML transcripts.
The PML protein contains the RBCC motif that consists of the RING finger domain
(RING), the 2 B boxes (B1 and B2), and the helical coiled-coil region. The proline rich
region (P) and serine-proline–rich region (S-P) have been thought to contain the
putative phosphorylation sites.44 SUMOylation sites at lysines 65, 160, and 49056 are
indicated. The PML gene consists of 9 exons (shaded boxes) with exon 7 divided into
exons 7a and 7b, and the introns being retained in several transcripts as a
consequence of alternative splicing (black boxes).54 The nomenclature for PML
isoforms proposed by Jensen55 was used in this study. The alternative usage of 3⬘
exons generates multiple transcripts I-VI. In the case of PML III and PML VI, the
encoding regions in which frame-shift occurred as a result of the retained intron are
indicated by the gray color. The total number of amino acids for each isoform is given
on the right side. (B) The interaction with AML1b is specific to the PML I isoform.
BOSC23 cells were cotransfected with HA-tagged AML1b and together either with
control vector (mock) or FLAG-tagged PML isoforms (I-VI). The expression of AML1
in the lysates of transfectants was detected by immunoblotting using anti-HA (3F10)
antibody (top). The lysates of transfectants were immunoprecipitated with anti-FLAG
(M2) antibody. The immunoprecipitates were analyzed by immunoblotting using
anti-FLAG (M2) (middle) and anti-HA (3F10) antibodies (bottom). (C) Identification of
regions of PML I required for interaction with AML1b. BOSC23 cells were cotransfected with FLAG-AML1b and either with control vector (mock) or constructs of PML I
as indicated. The expression of PML I in the lysates of transfectants were detected by
immunoblotting using anti-HA antibody (3F10) (top). The lysate of transfectants were
immunoprecipitated with anti-FLAG (M2) antibody and were analyzed by immunoblotting using anti-FLAG (M2) (middle) and anti-HA (3F10) antibodies (bottom).
Figure 1. PML I is a part of the AML1 complex. (A) Purification of the AML1b
complex. The complexes were purified from cell lysates prepared from L-G infectants
carrying empty vector (mock), stably expressing FLAG-tagged AML1a (AML1a, a
splicing variant lacking the C-terminal transcriptional regulatory domain), and
FLAG-tagged full-length AML1b proteins (AML1b). The complexes were immunoprecipitated on anti-FLAG antibody-conjugated agarose, and the bound materials were
eluted with the epitope peptide. Proteins were resolved by SDS-PAGE and visualized
by silver staining. The proteins were identified by mass analysis. (B) The amino acid
sequences of peptides derived from the 125-kDa protein specific to the fraction
purified from the FLAG-AML1b expressing cells. The protein was identified as PML
(accession number NM178087). (C) Detection of PML protein in the AML1 complex.
The purified AML1 complexes were analyzed by immunoblot with anti–mouse PML
antibody (rabbit polyclonal, Wang et al, 1998). (D) Interaction of endogenous AML1
and PML proteins. Cell lysates were prepared from 5 ⫻ 107 K562 cells and then were
immunoprecipitated with anti–human PML monoclonal antibody 1B9 (MBL) or control
IgG. The immunoprecipitates were analyzed by immunoblot with anti–mouse AML1
rabbit polyclonal antibody.
leukemia (PML) protein (Figure 1B). To confirm this interaction,
we performed immunoblot analysis using anti–mouse PML antibody and found that PML was certainly contained in the AML1b
fraction (Figure 1C). To test interaction of endogenous AML1 and
PML, cell lysates were prepared from K562 myeloid cells and then
immunoprecipitated with anti–human PML monoclonal antibody
(1B9). Immunoblot analysis using anti-human AML1 rabbit polyclonal antibody indicated that the immunoprecipitates obtained
with anti human PML monoclonal antibody (1B9) contained
AML1, whereas the immunoprecipitates with control IgG did not
contain AML1 (Figure 1D). These results suggested that the PML
protein might be a binding factor of AML1b.
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BLOOD, 1 JANUARY 2005 䡠 VOLUME 105, NUMBER 1
INTERACTION OF AML1 WITH PML I
295
Figure 3. PML I interacting domain is located at the C-terminal of AML1. (A) Schematic diagram of the structure of AML1 deletion mutants. The Runt, activation, and
inhibition domains are indicated as Runt, AD, and ID, respectively. Nuclear import signal (NLS) and nuclear matrix targeting signal (NMTS) are shown. (B) Identification of
regions of AML1b required for interaction with PML I. BOSC23 cells were cotransfected with PML I and control vector (mock) or AML1 constructions as indicated. The
expression of PML I in the lysates of transfectants was detected by immunoblotting using anti-HA (3F10) antibody (top). The lysates of transfectants were immunoprecipitated
with anti-FLAG (M2) antibody. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG (M2) (middle) and anti-HA (3F10) antibodies (bottom). (C) The Runt
domain is dispensable for the interaction of AML 1 with PML I. BOSC23 cells were cotransfected with PML I and full-length AML1b or deletion mutants of AML1 partially lacking
Runt domain as indicated. The expression of PML I (top) and AML1 (middle) proteins in the cell lysates was detected with anti-HA antibody and anti-FLAG antibody,
respectively. The immunoprecipitates with anti-FLAG antibody were analyzed using anti-HA antibody (lower).
Multiple isoforms of PML proteins are generated by alternative
usage of 3⬘ exons.55 To verify the possible association between
AML1b and PML, the FLAG-tagged major isoforms PML I-VI
(Figure 2A) were cotransfected with HA-AML1b into BOSC23
cells. Cell lysates were immunoprecipitated with anti-FLAG
antibody. Immunoblot analysis of the immunoprecipitate (IP)
samples showed that only PML I could interact with AML1b
(Figure 2B). These results indicated that PML I is one of the
components in the multiple protein complex assembled by AML1b.
as CBF␤, did not affect the binding to PML I (Figure 3C). Thus, the
PML I interacting site is located between amino acids 362 and 402
within the C-terminal region of AML1b. This region was demonstrated to be indispensable for the function of AML1 in transcription regulation, myeloid cell differentiation, and hematopoiesis,28,58,59 and it overlaps with the nuclear matrix targeting signal
(NMTS)60 (Figure 3A). Interestingly, 2 other members of the
RUNX family, AML2 and AML3, could bind to PML I (Figure
AML1b binding domain is located within the specific
C-terminus of PML I
Given that PML I differs from other isoforms in its C-terminus, we
focused on this region to determine the AML1 binding site. Two
kinds of C-terminal deletion mutants (mutant d851 and d592) were
constructed. Since the leucine zipper in some proteins has been
demonstrated to be essential for protein-protein interaction,57 we
constructed a mutant lacking the region (dL-Z) in order to
investigate whether this domain is required for the interaction of
PML I with AML1b. IP–Western blot analysis indicated that
deletion of the region from the C-terminal end to amino acid 851
did not affect the PML-AML1b interaction (Figure 2C). Further
truncation to residue 592 resulted in the loss of association with
AML1b. In addition, without the leucine zipper, mutant dL-Z still
bound to AML1b. Thus, the AML1b interacting domain was found
expanding from codon 592 to codon 851, which resides within the
specific C-terminal region of PML I.
PML-interacting domain overlaps the nuclear matrix signal
of AML1b
To determine the domains in AML1 that are required for interaction
with PML I, we performed IP-Western analysis using a series of
deletion mutants of AML1b (Figure 3A). Removal of 78 amino
acids of AML1b from the C-terminal end did not affect the
interaction with PML I (Figure 3B). However, further deletion up
to codon 362 resulted in loss of the interaction. Truncation of amino
acids 178-241 or 178-291 did not eliminate the interaction with
PML I. Deletion of the Runt domain, which has been demonstrated
to be essential for the association of AML1 with other proteins such
Figure 4. PML could interact with 2 other members of RUNX family AML2 and
AML3. (A) BOSC23 cells were cotransfected with PML I and AMLs as indicated. The
expression of AML proteins (upper) and PML I (middle) in the lysate of transfectants
was analyzed by immunoblotting and detected with anti-AML antibody28 mixed with
anti-AML3 antibody (Upstate Biotechnology, Charlottesville, VA) and anti-FLAG (M2)
antibody, respectively. The lysate of transfectants were immunoprecipitated with
anti-FLAG (M2) antibody, analyzed by immunoblotting, and probed with a mixture of
anti-AML1 antibodies (lower). (B) Comparison of amino acid sequences for the
C-terminal of AML1, AML2, and AML3. Identical amino acids are indicated in red. The
boxed region corresponds to the PML I interacting domain of AML1.
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coexpressed with PML I, AML1b was localized to small dotlike
structures (Figure 5C). These structures coincided with PML I
NBs, as revealed by the merged image, implying that AML1b and
PML I colocalized in NBs. In contrast, AML1a remained diffuse in
the nuclei (Figure 5B). These results suggest that binding to PML I
is essential for the accumulation of AML1b into nuclear bodies,
since AML1a does not contain a binding domain for PML I. To
further clarify this possibility, we investigated the subnuclear
distribution of AML1b when coexpressed with PML I deletion
mutants d851 or d592. As we expected, coexpressed AML1b was
sequestered to NBs containing deletion mutant d851 (Figure 5D),
but not to the one formed by mutant d592 (Figure 5E). Additionally, we could not find colocalization between AML1b and PML IV
(Figure 5F), which is one of the PML isoforms unable to associate
with AML1b. Lacking SUMO1 modification, PML I 3R forms
several aggregates. AML1 b was found together with PML I 3R in
these structures (Figure 5G).
PML I stimulates cooperation of AML1 and p300
Figure 5. PML I recruits AML1b into nuclear bodies. (A) SaOS2 cells were
transfected with LNCX-FLAG-AML1a, LNCX-FLAG-AML1b, or LNCX-HA-PML I.
(B,C) AML1b, not AML1a, is recruited into NBs. SaOS2 cells were cotransfected with
LNCX-FLAG-AML1a (B) or LNCX-FLAG-AML1b (C) together with LNCX-HA-PMLI.
(D,E) Localization of AML1b and PML I mutants. SaOS2 cells were cotransfected with
LNCX-FLAG-AML1b together with either LNCX-HA-PMLI d851 (D) or LNCX-HAPMLI d592 (E). (F) AML1b was not colocalized with PML IV into NBs. SaOS2 cells
were cotransfected with LNCX-FLAG-AML1b and LNCX-HA-PML IV. (G) AML1b is
presented in the aggregates formed by PML I 3R. SaOS2 cells were cotransfected
with LNCX-FLAG-AML1b and LNCX-HA-PML I 3R. In all experiments, AML1 was
stained with anti–human AML rabbit polyclonal antibody, as shown by the green color,
and PML was stained with anti-HA antibody (3F10), as shown by the orange color.
Merging of the 2 colors results in a yellow signal, indicating the colocalization of 2
proteins. Nuclear staining with DAPI is shown by the blue color.
4A). The comparison performed to examine for the presence of
identical amino acids revealed the region in AML1, which we
identified to be essential for the interaction with PML I, is highly
conserved in AML2 and AML3 (Figure 4B).
PML I targets AML1b into nuclear bodies
The association with PML was demonstrated to be essential for the
recruitment of pRb, CBP, Daxx, and p53 into NBs,61-64 prompting
us to examine whether AML1b, when binds to PML I, could
accumulate into NBs. In order to determine the subnuclear
localization of AML1 proteins in relation to PML, double immunofluorescent staining of transiently coexpressed AML1 and PML
was performed. AML1 proteins were coexpressed with HA-tagged
PML proteins in human osteosarcoma (SaOS2) cells, and location
of the expressed proteins were detected by anti-AML1 or anti-HA
antibodies, respectively. Without cotransfection with PML I, both
AML1a and AML1b were nuclear diffuse (Figure 5A). Being
The effect of PML proteins on AML1-dependent transcription was
investigated using the reporter gene under the control of the mCCP1
promoter, which contains 2 AML1-binding sites.65 Figure 6A shows that
among PML isoforms I-VI, only PML I significantly activates AML1dependent transcription activity of the mCCP1 promoter. The stimulation by PML I was strongly enhanced when p300, which could function
as a coactivator of AML1, was transfected together with PML I (Figure
6B). These results indicated that PML I could facilitate functional
cooperation of AML1b and p300 in transcription activation. We
therefore investigated the localization of AML1b and p300 in relation to
PML I by immunostaining. When p300 and PML I were cotransfected
into SaOS2 cells, they colocalized in PML-NBs (Figure 6Ci). Without
PML cotransfection, both p300 and AML1b were nuclear diffuse
(Figure 6Cii). In contrast, coexpression of PML I facilitated the
accumulation of these proteins together in small dotlike structures
(Figure 6Ciii). Thus, PML I could recruit both AML1 and its cofactor
p300 to the same nuclear bodies. By this way, the functional cooperation
of AML1 and p300 proteins might be enhanced.
PML I stimulates G-CSF–induced differentiation of myeloid cells
L-G is an IL-3–dependent myeloid precursor cell line that can be
differentiated into mature neutrophils in response to G-CSF.66
AML1b strongly stimulates the G-CSF–induced differentiation of
L-G cells.28,67 Therefore, we investigated whether PML I could
elicit any effect on the proliferation and differentiation of L-G cells.
To this end, we infected L-G cells with a retrovirus encoding PML I
and followed up the growth and morphological changes of the
infectants cultured in the medium containing G-CSF (5 ng/mL)
(Figure 7). As expected, the overexpression of PML I wild-type
inhibited the cell proliferation (Figure 7B) and conferred upon the
L-G cells advantages for maturation into neutrophils. At day 6,
most PML I wild-type infectants differentiated into mature neutrophils, whereas control cells did not (Figures 7C, E). The cells
expressing the 3R mutant that lacks all SUMOylation sites
proliferated exponentially (Figure 7B) and remained in its immature/
intermediate status until day 8, when control cells differentiated
into neutrophils (Figures 7D, F). Altogether, these results indicate
that PML could be involved in the regulation of myeloid differentiation and that SUMO-1 modification also are essential for the
function of PML I.
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BLOOD, 1 JANUARY 2005 䡠 VOLUME 105, NUMBER 1
INTERACTION OF AML1 WITH PML I
297
Figure 6. PML I stimulates cooperation of AML1 and
p300. (A) PML I specifically enhanced AML1-induced
transcription. The effect of PML proteins on AML1dependent transcription was investigated using the reporter gene under the control of mCCP1 promoter.
PmL⫺/⫺ MEF cells were spread in 24-well plates and
transfected with 50 ng of reporter gene, 200 ng of
pLNCX-AML1b, 30 ng of empty vector or PML isoforms
as indicated, and 2 ng of pRL-CMV. (B) PML I stimulates
the cooperated transcription activity of AML 1 and p300.
PmL⫺/⫺ MEF cells were spread in 24-well plates and
transfected with 50 ng of CCP1-luc, 200 ng of pLNCXAML1b (lanes 4-9), 250 ng of pCMV-p300 (lanes 6-9),
and either 30 ng (lanes 2, 5, 8) or 100 ng (lanes 3, 6, 9) of
pLNCX-PML I and 2 ng of pRL-CMV. Luciferase activity
was assayed as described in the Experimental Procedure. (C) PML I recruits AML1b and p300 into nuclear
bodies. SaOS2 cells were cotransfected with pCMV-p300
(i-iii) together with LNCX-FLAG-AML1b (ii, iii) and LNCXHA-PMLI (i, iii). p300 stained with anti-p300 rabbit polyclonal antibody (N15) is shown by the green color, and
PML (i) stained with anti-HA antibody (3F10) and AML1
(ii, iii) stained with anti-FLAG antibody (M2) are shown by
orange color. Merging of the 2 colors results in a yellow
signal, indicating the colocalization of 2 proteins. Nuclear
staining with DAPI is shown by the blue color.
Discussion
AML1 forms a complex with specific PML I isoform
We previously purified AML1 complexes from murine progenitor L-G cells that stably expressed AML1a or AML1b proteins.
Multiple proteins were copurified with AML1b, but they were
absent from both the mock-purified fraction and AML1a complex, suggesting that they could interact specifically with
AML1b. Mass spectrometry revealed the presence of PML
protein in the purified AML1b complex. It is known that
alternative usage of 3⬘ exons of the PML locus results in
generation of several isoforms that have a specific C-terminus.
Our results showed that only PML I is contained in the AML1b
complex. Analysis of peptides derived from the PML protein in
the AML1b complex detected 4 fragments, 2 of which are
contained in the common region and 2 others that are in the
specific C-terminal region of PML I. No specific sequence for
other isoforms was detected. The molecular mass of the PML
protein in the purified AML1b complex is about 125 kDa, which
corresponds to that of PML I, the largest isoform among
identified isoforms of PML. Using IP-Western analysis, we
found that among the major isoforms PML I-VI, only PML I
could be coprecipitated with AML1b. The specific association of
PML I with AML1b suggests that their interaction might be
mediated through the C-terminal region of PML I. Indeed,
deletion analysis mapped the binding domain for AML1b to this
region of PML I. Taken together, we concluded that the specific
isoform PML I is a part of a multiple complex of AML1b. It has
been reported that p53 could specifically interact with PML IV
isoform.64 This and our findings suggest that each of the PML
isoforms might play important roles in regulation of factorspecific transcription.
Localization of AML1 in PML nuclear bodies
The PML nuclear body represents a specialization of the
protein-based architecture and exists as an entity that is
independent of either DNA or RNA.68 An increasing number of
transcription factors and coactivators that have been found to
reside together with PML in nuclear bodies give support for the
role of this structure in transcription regulation. In this study, we
demonstrated that AML1b is colocalized with PML I in nuclear
bodies. This accumulation required the interaction between
AML1b and PML I, since the respective binding domains in
both AML1b and PML I are essential for recruitment of AML1b
into the nuclear bodies. We previously found that p300 histone
acetyltransferases can function as coactivators of AML1b.28
Here we showed that PML I can recruit both AML1b and p300
coactivator to the same nuclear bodies and enhance their
cooperation in the activation of transcription.
The PML nuclear body is a stationary structure that associates
with the nuclear matrix. It has been reported that AML1 localizes
within the nucleus to punctuate foci that associate with the nuclear
matrix.58,60 Targeting AML1 to these sites involves at least 2
trafficking signals, one of which mediates nuclear import–nuclear
localization signal (NLS) and the other promotes association with
nuclear matrix–nuclear matrix targeting signal (NMTS).60 In our
study, we found that NMTS of AML1 is required for both
interaction with PML and localization in nuclear matrix-associated
PML bodies, suggesting that PML I could modulate distribution of
AML1 in the nuclear matrix.
A subset of AML1 was shown to colocalize with RNA
polymerase II at the site of ongoing transcription.69 Given that
the C-terminus of AML1 mediates association with the nuclear
matrix and its interaction with transcriptional cofactors such as
p300/CBP28 and MOZ,29 AML1 might assemble into macromolecular complexes with coregulatory proteins at nuclear matrixassociated sites. Recently, observation of living cells showed
that AML1 continuously and rapidly shuttles into and out of the
dynamic yet spatially stable foci.70 The C-terminal region of
AML1 that mediates association with other cofactors and the
nuclear matrix appears to reduce the mobility of this protein.
Thus, dynamic association of AML1 to stationary subnuclear
foci might be essential for AML1 to assemble complexes with
regulatory cofactors. Several proteins have different potentials
to localize within PML nuclear bodies. PML and Sp100 are
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BLOOD, 1 JANUARY 2005 䡠 VOLUME 105, NUMBER 1
NGUYEN et al
relatively immobile in the nucleoplasm and reside within the
PML nuclear bodies, which suggests that they play a structural
role in the integrity of these domains. In contrast, CBP, a
transcriptional cofactor, moves relatively rapidly into and out of
the PML nuclear bodies.68 From these data, taken together, we
propose that stationary PML nuclear bodies serve as a scaffold
for the formation of the reversible complex between AML1 and
cofactors such as p300/CBP. This model might be applied to
complexes of the other PML-interacting transcription factors
such as p53, AP1, and SP1 since these transcription factors also
use p300/CBP as coactivators.
Functional implication of AML1-PML I interaction
Figure 7. PML I inhibits the proliferation and stimulates the differentiation of
murine myeloid progenitor cells in response to G-CSF. (A) Expression of PML
protein in L-G infectants. Cell lysates were prepared in SDS-PAGE sample buffer and
then analyzed by Western blot using anti-HA antibody. Subfraction of PML protein
modified by SUMO-1 is indicated. (B) The L-G infectants were cultured in the
presence of 5 ng/mL G-CSF. The relative number of viable cells is shown. (C,D)
Morphology of L-G infectants exposed to G-CSF for 6 days (C) and 8 days (D) were
tested by staining with May-Gruenwald and Giemsa solutions. (E,F) Percentage of
L-G infectants differentiated by G-CSF at day 6 (E) and day 8 (F). Immature includes
myeloblasts and promyelocytes; intermediate, myelocytes and metamyelocytes; and
mature, band cells and segmented neutrophil cells.
In this study, we mapped the PML I binding site to the portion
between residues 363 and 402 in the C-terminal of AML1b.
Previously, this domain and the p300 binding site (residues 295
to 362) were both demonstrated to be essential for the stimulatory effect of AML1b on G-CSF–induced differentiation of the
myeloid progenitor L-G cells.28 In this study, we showed that
PML I could stimulate the L-G cells to mature into neutrophils
in response to G-CSF (Figure 7). However, this effect of PML I
could not be observed in the cell expressing AML1-MTG8 (data
not shown), a dominant negative form of AML1, which
represses AML1-dependent transcription.71 This suggests that
the ability of PML I to regulate cell differentiation might be
mediated via functional AML1 proteins. To further clarify this
possibility, the effect of PML I on differentiation of cells, in
which gene targeting or RNAi suppresses the expression of
AML1 proteins, needs to be investigated.
Mice lacking either AML1 or its heterodimeric partner CBF␤
are deficient in definitive hematopoiesis.15 PmL⫺/⫺ mice also
have an impaired capacity for terminal maturation of their
myeloid cells.48 The comparison of the phenotype of these mice
suggests that PML is not always essential for the function of
AML1, but it is involved in the development of myeloid cells
that is regulated by AML1. Both AML1 and PML are frequent
targets of chromosome abnormalities that are found in acute
myeloid leukemias. AML1 interacting proteins, which have
been identified so far (including CBF␤, CBP/p300, MOZ, and
PML), all are the target of leukemia-associated chromosome
translocations. The fact that about 50% of patients with acute
leukemia harbor chromosome abnormalities or mutations that
disrupt AML1 and/or its binding factors suggests that instability
of AML1 complex might be a common mechanism shared by
distinct myeloid malignancies and underscores the importance
of this complex in hematopoiesis and leukemogenesis.
Acknowledgments
We thank Dr A. Kakizuka for human PML VI cDNA, Dr T. Okuda
for AML2 and AML3 cDNAs, Dr S. Nakamura and Dr H. Ichikawa
for mCCP1-luciferase construct, and Dr David Baltimore for
BOSC23 cells.
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2005 105: 292-300
doi:10.1182/blood-2004-03-1185 originally published online
August 26, 2004
Physical and functional link of the leukemia-associated factors AML1
and PML
Lan Anh Nguyen, Pier Paolo Pandolfi, Yukiko Aikawa, Yusuke Tagata, Misao Ohki and Issay
Kitabayashi
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