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NEOPLASIA
From the Department of Medicine, The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY;
and the Department of Biochemistry, Vanderbilt University School of
Medicine, Nashville, TN.
The AML-1/ETO fusion protein, created by the (8;21) translocation
in M2-type acute myelogenous leukemia (AML), is a dominant repressive
form of AML-1. This effect is due to the ability of the ETO portion of
the protein to recruit co-repressors to promoters of AML-1 target
genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia
creates the promyelocytic leukemia zinc finger PLZFt/RAR AML-1/ETO is a chimeric transcription factor
generated as a consequence of the (8;21) chromosomal translocation,
present in approximately 40% to 50% of patients with M2-type acute
myelogenous leukemia (AML).1 The proteins that are fused
in this event are AML-1 (also known as CBFA2, PEP2 Repression by AML-1/ETO is mediated by the ETO moiety and particularly
requires the zinc finger or MYND domain of ETO, which binds to
corepressors such as N-CoR and SMRT.4-6 ETO itself does not bind specific DNA sequences, and we recently showed that ETO is
itself a corepressor for the promyelocytic leukemia zinc finger (PLZF)
protein.19 Like ETO, PLZF is expressed in early
hematopoietic cells and binds to N-Cor, SMRT, Sin3a, and histone
deacetylase (HDAC).20-24 PLZF represses genes involved in
cell proliferation and inhibits the growth of myeloid and other
cells.25,26 PLZF is also disrupted by the
t(11;17)(q21;q23) in retinoid-resistant acute promyelocytic leukemia
(APL).27 This generates the PLZF/RAR Because ETO is a corepressor for PLZF, and both ETO and PLZF are
expressed in the early stages of myeloid differentiation, we
hypothesized that AML-1/ETO might interact with PLZF in t(8;21) leukemic cells. Similarly, because the PLZF/RAR DNA constructs and cell lines
Immunoprecipitations and immunoblotting
Cell fractionation and electrophoretic mobility shift assay Plates of 1 × 106 293T cells were transfected using Superfect (Qiagen) and 1.5 µg expression vectors for each of AML/ETO, wild-type PLZF, and control vectors. After 48 hours, the cells were harvested, nuclear extracts were prepared as described,32 and protein concentration of extracts were assayed using the DC Assay System (Bio-Rad, Hercules, CA). EMSA was performed with an [ -32P] dCTP-labeled double-stranded
oligonucleotide containing a high-affinity binding site for PLZF, as
described previously.33 Complementary oligonucleotides
(2.5 pmol) were annealed in TE with 100 mmol/L NaCl, and the
overhanging ends were filled in using Klenow and [-32P] dCTP. For 20 minutes on ice, 10 fmol probe was
incubated with 2 µg of each nuclear extract in 20 mmol/L HEPES, 1 mmol/L MgCl2, 10 µmol/L ZnCl2, 4% glycerol,
and 100 µg/mL 1 bovine serum albumin. For supershift
analysis, 100 ng PLZF mouse monoclonal antibody was pre-incubated with
nuclear extracts for 20 minutes on ice before the probe was added. The
DNA-protein complexes were separated by electrophoresis through a 4%
nondenaturing, 0.5 × TBE acrylamide gel, dried, and visualized by
exposure to Kodak XAR film (Eastman Kodak, Rochester, NY) and
analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Nuclear matrix extracts were prepared as described.34
Immunofluorescence 293T cells were plated onto glass coverslips before transfection with PLZF and AML-1/ETO; 100 ng each of AML-1/ETO and PLZF was co-transfected, an amount that was carefully titrated to reproduce the natural staining pattern of each protein. After blocking in 10% donkey serum for 30 minutes, the cells were exposed to a 1:50 dilution of mouse monoclonal PLZF antibody for 1 hour. This was followed by exposure to a 1:100 dilution of rabbit polyclonal ETO antibodies or rabbit AML-1 (anti-RHD) polyclonal antibodies (Oncogene Research Products) for 1 hour. Samples were then treated for 30 minutes with donkey antimouse antibody conjugated to fluorescein isothiocyanate (FITC), goat antirabbit antibody conjugated to Texas red (Jackson Immuno-Research), or both. Vectashield mounting medium with DAPI (Vector Laboratories, Burlington, CA) was then applied, and the slides were examined with a Leica-TCS-SP (UV) confocal microscope (Leica, Heidelberg, Germany). To eliminate the possibility of cross-channel bleed-through, the samples were scanned separately in the nonoverlapping portion of the spectrum of each fluorescent marker. The resultant images were then overlaid to determine co-localization. This experiment was repeated 4 times, and multiple fields were imaged. As negative controls, the cells were incubated with secondary antibody alone or in the presence of irrelevant mouse antibody (DAKO, Carpinteria, CA) at a 1:50 dilution, irrelevant rabbit antibody (R&D, Minneapolis, MN) at a 1:100 dilution, or both, followed by incubation with the appropriate secondary antibodies.Transcriptional assays To determine the transcriptional effects of the GAL4-fusion constructs, we used a reporter containing 5 GAL4-binding sites linked 5' to the herpes virus thymidine kinase (tk) promoter and the firefly luciferase gene.19 A reporter gene to assay the transcriptional effects of PLZF consisted of 4 copies of a high-affinity PLZF binding site found within the IL-3 receptor alpha chain (IL-3R) promoter linked 5' to the tk-luciferase
reporter.33 A tk-luciferase construct lacking
specific binding sites was used as a negative control for the above
reporters. 293T cells were plated at a density of
2 × 105 cells/well of 12-well or 4 × 105
cells/well of 6-well tissue culture dishes and transfected using Superfect (Qiagen). In 12-well dish transfections, 100 ng reporter and
5 ng internal control (tk-renilla luciferase) were used, and in 6-well dishes 200 ng reporter and 10 ng internal control were used.
The combinations and quantities of expression plasmids are noted in the
figure legends. To determine the transcriptional effects of
RARPML/RAR, and PLZF/RAR, we used a RARE-tk-Luc reporter construct harboring a DR5 retinoid response element (gift of
Dr Len Freedman, Memorial-Sloan Kettering Cancer Center, New York, NY).
These experiments were performed in charcoal-stripped serum (Life
Technologies, Rockville, MD). To assess transcriptional activity, dual
luciferase assays were performed (Promega, Madison, WI), using an MLX
Microtiter Plate Luminometer (Dynex Technologies, Chantilly, VA). All
these experiments were performed in duplicate 3 to 6 times, with
results averaged and normalized to the internal control.
Immunoblotting, performed as above, confirmed the expression of
proteins. To assess effects of the proteins on cell growth, 5 separate
sets of 293T cell cultures at a density of
2.8 × 105/well (of a 6-well dish) were transfected with
pCDNA, pCDNA + AML-1/ETO, PLZF or PLZF + AML-1/ETO as above. The
cells were allowed to grow in parallel to the reporter assay cells, as
described, and were harvested at identical times for counting.
AML-1/ETO interacts with PLZF Because AML-1/ETO retains the domain (between ETO amino acids 220 and 330) required for interaction with PLZF,19 we transiently expressed both AML-1/ETO and PLZF in 293T cells to determine whether they formed a complex. Lysates from these cells were subjected to immunoprecipitation with antibodies against PLZF, the AML-1 runt domain, or the ETO C-terminal MYND domain. Lysates precipitated with AML-1 or ETO antibodies readily revealed the presence of PLZF (Figure 1A). In a reciprocal manner, immunoblots of lysates precipitated with PLZF antibody revealed the presence of AML-1/ETO, as detected by both the AML-1 and ETO antibodies (Figure 1B-C). 293T cells do not express AML-1 or ETO at detectable levels, as shown by the fact that immunoprecipitations performed with each antibody followed by immunoblotting with the same antibody did not show any endogenous protein. Thus, PLZF is interacting with the transfected chimeric protein. We then performed immunofluorescence studies in 293T cells to determine whether AML-1/ETO and PLZF colocalize in cell nuclei. PLZF normally localizes in a speckled nuclear pattern, and AML-1/ETO localizes in a combined speckled and diffuse nuclear pattern.30,35 Initially, we titrated the dose of each plasmid to achieve a staining pattern similar to that of the endogenous protein. Dual staining for both proteins with FITC and Texas red-conjugated secondary antibodies demonstrated that the speckled fraction of AML-1/ETO largely colocalizes with PLZF (Figure 1D). No staining was seen when nonspecific isotype antibodies were used as negative controls (data not shown). From these studies we concluded that PLZF and AML-1/ETO could associate in vivo in human cells.
Endogenous AML-1/ETO and PLZF interact in t(8;21) leukemia cells PLZF is normally expressed in early myeloid cells and represses promoters of genes involved in both differentiation and cell proliferation, such as cyclin A2 and the IL-3R
chain.25,33 Because the M2 leukemic phenotype corresponds
to a myeloblastic morphology, we hypothesized that PLZF would be
expressed in these cells. To answer this question we used the SKNO-1
cell line, derived from a young patient with t(8;21) M2
AML.31 These cells expressed PLZF at levels detectable by
direct immunoblotting (Figure 2A, lane
1). We then subjected lysates from these cells to immunoprecipitation using the PLZF, AML-1, and ETO antibodies as above. AML-1/ETO was
immunoprecipitated by antibodies to PLZF (Figure 2B-C). PLZF was also
detectable in both the AML-1 and ETO immunoprecipitates (Figure 2A).
Neither protein was present when the appropriate mouse and rabbit
control antibodies were used for immunoprecipitation (Figure 2A-C).
Thus, endogenous AML-1/ETO interacts with endogenous PLZF in t(8;21)
leukemia cells.
AML-1/ETO antagonizes PLZF-mediated transcriptional repression To determine the effect of AML-1/ETO on the transcriptional properties of PLZF, we transiently transfected both proteins along with a reporter containing PLZF binding sites from the IL-3R chain
promoter. Repression of this reporter by PLZF is well established and
is potentiated by ETO.19,33 We found that AML-1/ETO
strikingly inhibited transcriptional repression by PLZF. This occurred
when PLZF was expressed in the context of both pSG5 and pCDNA3.1+
expression vectors and was a specific effect of AML-1/ETO given that it
did not occur in the presence of the empty pCMV expression vector (Figure 3A and data not shown). AML-1/ETO
did not activate transcription from the reporter construct in the
absence of PLZF, suggesting that the block of repression was related to
the interaction of these 2 proteins (Figure 3B). AML-1/ETO blocks
activation by AML-1 even when expressed at a lower level than
AML-1.11,12 Similarly, AML-1/ETO antagonized repression by
PLZF antagonism even when AML-1/ETO was cotransfected at an 8-fold
lower concentration than PLZF. This effect was maintained over a broad
range of doses of AML-1/ETO (Figure 3C). This was not caused by toxic
effects because 293T cells transfected in conditions identical to those
of the cells above grew to levels similar to those of mock-transfected cells and had similar levels of viability, as determined by trypan blue
exclusion (Figure 3D and data not shown). Finally, the effect was
specific for PLZF because transcriptional repression by the unrelated
corepressor KRIP136 was not inhibited by the co-expression of AML-1/ETO (Figure 3E).
We next determined which of the 2 repression domains of PLZF
could be inhibited by AML-1/ETO. For this purpose we fused amino acids
1 to 400 of PLZF to the GAL4 DNA-binding domain
(GAL4-PLZF1-400) and cotransfected it with a reporter
plasmid containing 5 GAL4-binding sites. This fragment includes both
the N-terminal BTB/POZ repression domain (amino acids 1-130) and the
second repression domain (RD2) between amino acids 200 to 300. AML-1/ETO was also able to inhibit repression by this construct, with a
slightly lower efficiency than full-length PLZF, but also at a 1:8
ratio of AML-1/ETO to PLZF (Figure 4A and
data not shown). The reporter was not activated by AML-1/ETO,
indicating that the effect was related to the interaction between the 2 proteins (Figure 4B). We then analyzed this effect on the isolated
repression domains of PLZF, each of which is required for PLZF function
(see reference37 and see below). Wild-type ETO is able to
enhance repression mediated by both domains, though it only binds to
RD2.19 Both domains yielded powerful repression, with
GAL4-RD2 repressing the luciferase reporter 70-fold and GAL4-BTB/POZ repressing it 14-fold (Figure 4C-D). AML-1/ETO inhibited the constructs in a fashion similar to that of full-length PLZF, suggesting that transcriptional disruption occurred through effects on both repression domains (Figure 4C-D).
The C-terminal corepressor binding domain of AML-1/ETO is required to block PLZF repression Both the AML-1 and the ETO proteins can bind corepressors. However, AML-1 requires residues that are not present in the AML-1/ETO fusion protein to bind to the mSin3A and groucho corepressors.17,18 In contrast, AML-1/ETO contains all the domains required to associate with Sin3A, N-CoR, SMRT, and HDACs.4-6 Of these, the MYND domain is required to enhance repression by PLZF.19 We determined whether the same domain was required for the action of AML-1/ETO. PLZF and the isolated repression domains of PLZF were co-expressed along with AML-1/ETO or mutant AML-1/ETO constructs lacking either the HHR domain (AML-1/ETO HHR) or both the HHR and MYND domains
(AML-1/ETOHHR/MYND). In all cases, the MYND
corepressor-binding domain was absolutely required for AML-1/ETO to
block transcriptional repression by full-length PLZF or the PLZF
repression domains (Figure 5). We verified that AML-1/ETO HHR/MYND still associates with
PLZF by coimmunoprecipitations in transiently transfected 293T cells
(Figure 5).
Corepressors do not rescue PLZF from inhibition by AML-1/ETO The above results suggested that one possible mechanism of AML-1/ETO interference of PLZF action could be through competition for corepressors. To test this model, we cotransfected N-CoR, SMRT, or ETO along with PLZF and AML-1/ETO. When full-length PLZF was coexpressed along with N-CoR, SMRT, or ETO, transcriptional repression was enhanced (Figure 6A). However, when AML-1/ETO was added at substoichiometric levels to any of these PLZF/corepressor combinations, repression was still blocked. A similar phenomenon was observed when SMRT or ETO was transfected in combination with GAL4-PLZF1-400, GAL4-BTB/POZ, and GAL4-RD2. Thus, a simple competition for co-repressors is unlikely to fully explain the effects of AML-1/ETO on PLZF.
AML-1/ETO disrupts the nuclear matrix compartmentalization of PLZF and reduces its ability to bind to DNA We next performed cell fractionation on 293T cells transiently transfected with the low levels of PLZF that reproduce the natural immunofluorescence pattern of the endogenous protein. Almost all the PLZF was present in the nuclear fraction (Figure 7A). Intriguingly, a large amount of the protein was in the nuclear matrix fraction. This is consistent with the finding that PLZF can be found in nuclear bodies, which have been shown to be associated with the matrix38,39 (Figure 7A). When PLZF and AML-1/ETO were transfected together, there was almost no PLZF localization to the nuclear matrix, though the nuclear localization of PLZF was not affected (Figure 7A). Immunoblotting of whole cell extract showed that PLZF was expressed to similar levels in both the PLZF and the PLZF + AML-1/ETO cells (Figure 7A). Thus, sequestration from the nuclear matrix compartment might contribute to the effect of AML-1/ETO on transcriptional repression by PLZF.
We next analyzed whether AML-1/ETO could affect DNA binding by PLZF. We
previously showed that PLZF forms a high-molecular-weight complex when
allowed to bind with an oligonucleotide containing PLZF-binding sites
from the IL-3R PLZF/RAR product of the (11;17)(q23;q21) translocation from the retinoic acid (RA)-resistant variant of APL. PLZF/RAR recruits N-CoR, SMRT, and HDACs to RAR target genes and fails to release these proteins in response to retinoic acid.22-24 ETO, which is normally expressed in
myeloblasts, binds the second repression domain of PLZF, retained in
PLZF/RAR, and could participate in RA-independent PLZF/RAR
transcriptional repression. To examine this possibility, we transiently
expressed PLZF/RAR and ETO in 293T cells and performed
immunoprecipitations with PLZF, RAR, or ETO rabbit polyclonal
antibody. Immunoblots performed using the PLZF or RAR antibodies
indicated that PLZF/RAR was present in all 3 of the precipitates
(Figure 8A-B). Direct immunoblots of the
cell lysates also confirmed expression of both proteins (Figure 8A-B).
A reciprocal experiment was performed in which cell lysates were
precipitated with the PLZF, RAR, or ETO antibodies and then
immunoblotted for ETO. In this case ETO was observed in all 3 precipitates but not in precipitates obtained with mouse and rabbit
control antibodies (Figure 8C). In contrast, we were unable to detect
an interaction between ETO and the PML/RAR fusion protein of RA
sensitive "classic" APL (data not shown).
ETO enhances PLZF/RAR and compared this to the effect of ETO on RAR and
PML/RAR. 293T Cells were transiently transfected with empty vector,
RAR, PML/RAR, or PLZF/RAR with or without ETO in
charcoal-stripped serum. Consistent with previous reports, PML/RAR
and PLZF/RAR had a modest repressive effect on their own on the RARE
reporter in the absence of RA (Figure 9).
However, the addition of ETO significantly enhanced repression of the
RARE reporter by PLZF/RAR but did not enhance repression by RAR
or PML/RAR. Thus, ETO is a corepressor for PLZF/RAR and for PLZF.
The investigation of common pathways of leukemogenesis led us to study the interaction between proteins involved in M2 AML and retinoid-resistant APL. We previously reported that the ETO protein, involved in the (8;21) translocation of M2-AML is a corepressor for PLZF.19 This effect was dependent on the MYND corepressor/HDAC-binding domain of ETO and was blocked by HDAC inhibitors.19 We expected that AML-1/ETO would interact with PLZF because the PLZF-binding site located at ETO residues 220 to 330 is retained in the fusion protein. Consistent with this, the 2 proteins interacted when expressed in transfected cells and endogenously in a cell line derived from a patient with t(8;21) AML. We modeled the interaction between PLZF and AML-1/ETO in transfected
cells and found that the fusion protein strongly blocked repression by
PLZF or the isolated repression domains of PLZF. This suggests a novel
function for AML-1/ETO as de-repressor of a transcriptional repressor.
AML-1/ETO functions include the blockade of AML-1 transcriptional
activation in a dominant-negative fashion11,40 due to the
inappropriate recruitment of corepressors to AML-1 target
sequences,4-6 the active repression of basal transcription from the MDR-1 promoter independent of its dominant-negative effects on
AML-1,14 the dominant-negative inhibition of
transcriptional activation by C/EBP The mechanism through which AML-1/ETO inhibits PLZF function is complex. The simplest explanation is that AML-1/ETO antagonized PLZF by competing for corepressors. Consistent with this explanation, AML-1/ETO requires the MYND corepressor/HDAC-binding motif to antagonize PLZF. However, the addition of corepressors at doses that enhance repression by PLZF alone could not enhance repression by PLZF in the presence of low levels of transfected AML-1/ETO. The fact that AML-1/ETO inhibits both repression domains of PLZF is also consistent with a model in which AML-1/ETO interferes with corepressor/PLZF function through noncompetitive mechanisms, possibly through an aberrant complex formation. This association might be stable and would preclude the formation of effective repression complexes, supported by the strong co-precipitation and co-localization of AML-1/ETO and PLZF when co-expressed in 293T cells and the co-precipitation of a significant amount of AML-1/ETO with PLZF in SKNO cells. Part of the inhibition may be related to the ability of AML-1/ETO to almost completely exclude PLZF from the nuclear matrix compartment, an effect that correlates well with the suppression of PLZF function. It has been suggested that PLZF associates with the nuclear matrix.38 Proper nuclear matrix targeting may be essential for the function of certain transcriptional factors, such as AML-1.43 AML-1/ETO lacks the nuclear matrix signal sequence of AML-1 and localizes to a distinct matrix subcompartment.44 These results suggest a novel functional requirement for discrete nuclear matrix compartmentalization in PLZF repression, a possibility we are exploring further. Finally, PLZF binding to DNA was partially inhibited by AML-1/ETO. However, AML-1/ETO also blocked repression by GAL4-PLZF, GAL4-BTB/POZ, and GAL4-RD2, though it did not inhibit GAL4-KRIP1, suggesting that the effect on PLZF binding may be specific to PLZF. It should be noted that PLZF represses transcription through the action of HDACs, whereas the KRIP protein relies on a different mechanism, potentially through interaction with nonhistone, chromatin-associated proteins.45 Hence, it remains possible that AML-1/ETO may inhibit the function of other proteins related to PLZF in structure or function, such as FAZF/TZFP/ROG46-48 or LRF.49 In conclusion, we propose that AML-1/ETO inhibits PLZF through several mechanisms. These include the formation of an aberrant protein complex with nuclear corepressors and HDACs (unfavorable for transcriptional repression), sequestration of PLZF away from the nuclear matrix, and inhibition of the ability of PLZF to bind to DNA. There may be significant biologic consequences to the ability of
AML-1/ETO to inhibit transcriptional repression by PLZF and other
proteins. PLZF is a powerful growth suppressor in myeloid and other
cells.26 This may occur in part by the inhibition of cell
cycle regulators such as cyclin A2, delaying entry into and transit
through the S phase.25,26 In APL associated with t(11;17)
(q21;q23), the RAR To examine whether ETO could play a central role in multiple forms of
leukemia, we determined its ability to interact with the PLZF/RAR In conclusion, we report novel modes of action for AML-1/ETO on the
PLZF growth suppressor and for the ETO corepressor on the PLZF/RAR
We thank Drs Arthur Zelent and Samuel Waxman for continued support and Dr Kathy Borden for helpful advice.
Submitted April 17, 2000; accepted August 10, 2000.
Supported by National Institutes of Health grants R01 CA59936 (J.D.L.) and R01 CA64140 (S.W.H.) and by American Cancer Society Award DHP 160 (J.D.L.). J.D.L. is a Scholar of the Leukemia and Lymphoma Society. A.M. is supported by NIH grant K08 CA73762. Confocal laser scanning microscopy was performed at the MSSM-CLSM core facility, supported with funding from NIH shared instrumentation grant 1S10 RR0 9145-01 and National Science Foundation Major Research Instrumentation grant DBI-9724504.
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.
Reprints: Jonathan D. Licht, Dept of Medicine, Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, Box 1130, 1 Gustave Levy Pl, New York, NY 10029; e-mail: Jonathan.licht{at}mssm.edu.
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Abstract
Introduction
