MOLECULAR AND CELLULAR BIOLOGY, Feb. 2005, p. 1100–1112
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 3
Eukaryotic Translation Initiation Factor 4E Activity Is Modulated by
HOXA9 at Multiple Levels
Ivan Topisirovic,1† Alex Kentsis,1Jacqueline M. Perez,1Monica L. Guzman,2
Craig T. Jordan,2and Katherine L. B. Borden1*
Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York
University, New York,1and Department of Medicine, University of Rochester, Rochester,2New York
Received 12 July 2004/Returned for modification 19 August 2004/Accepted 5 November 2004
The eukaryotic translation initiation factor 4E (eIF4E) alters gene expression on multiple levels. In the
cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates
nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E’s ability to oncogenically
transform cells. We report here that the homeodomain protein, HOXA9, is a positive regulator of eIF4E.
HOXA9 stimulates eIF4E-dependent export of cyclin D1 and ornithine decarboxylase (ODC) mRNAs in the
nucleus, as well as increases the translation efficiency of ODC mRNA in the cytoplasm. These activities depend
on direct interactions of HOXA9 with eIF4E and are independent of the role of HOXA9 in transcription. At the
biochemical level, HOXA9 mediates these effects by competing with factors that repress eIF4E function, in
particular the proline-rich homeodomain PRH/Hex. This competitive mechanism of eIF4E regulation is
disrupted in a subset of leukemias, where HOXA9 displaces PRH from eIF4E, thereby contributing to eIF4E’s
dysregulation. In regard to these results and our previous finding that ?200 homeodomain proteins contain
eIF4E binding sites, we propose that homeodomain modulation of eIF4E activity is a novel means through
which this family of proteins implements their effects on growth and development.
Dysregulation of the eukaryotic translation initiation factor
4E (eIF4E) is linked to oncogenic transformation in cell cul-
ture and in vivo (28). eIF4E levels are upregulated in a subset
of acute myelogenous and chronic myelogenous leukemias, as
well as in non-Hodgkin B-cell lymphomas, breast cancer, and
head and neck squamous cell carcinoma (7, 23, 34, 35). Over-
expression of eIF4E leads to dysregulated cellular proliferation
and malignant transformation in immortalized cell lines (19–
21). In addition, eIF4E overexpression can contribute to leu-
kemogenesis by impeding granulocytic and monocytic differ-
entiation (34). eIF4E plays roles in both the nucleus and the
cytoplasm, and both of these functions contribute to its phys-
iological effects on cell growth and oncogenic transformation.
In the cytoplasm, eIF4E functions in the rate-limiting step of
cap-dependent translation initiation (28). Here, eIF4E directly
binds the methyl-7-guanosine (m7G) cap present on the 5? end
of mRNAs, thereby recruiting transcripts to the ribosome (28).
In order for translation to proceed, eIF4E must associate with
other factors of the eIF4F complex, including eIF4G, the scaf-
fold of this complex, eIF4A, and an RNA helicase, as well as
other factors such as the ribosome-bound eIF3 and the
poly(A)-binding protein (28). Surprisingly, overexpression of
eIF4E does not lead to increased production of all transcripts
(28). For example, overexpression of eIF4E leads to increased
translation of ornithine decarboxylase (ODC) and vascular
endothelial growth factor but not of actin or GAPDH (glycer-
aldehyde-3-phosphate dehydrogenase) (4, 9). Transcripts that
are more efficiently produced when eIF4E is overexpressed are
referred to as eIF4E-sensitive transcripts, and those that are
not are referred to as eIF4E insensitive (4, 9). This sensitivity
is thought to result from long and highly structured 5? untrans-
lated region (5?UTRs) (4, 9).
In the nucleus, eIF4E functions in nucleocytoplasmic
mRNA transport of a subset of transcripts (5, 27, 29). A sub-
stantial fraction (up to 68%) of eIF4E is found in multiprotein
nuclear structures referred to as eIF4E nuclear bodies (12, 17,
22, 29). The majority of these bodies colocalize with promy-
elocytic leukemia protein (5, 17). Here, eIF4E promotes the
selective transport of specific mRNAs, such as cyclin D1 and
ODC, from the nucleus to the cytoplasm without affecting
transport of housekeeping mRNAs such as GAPDH and actin
or altering the levels of these transcripts (17, 27, 32, 33). As in
the cytoplasm, eIF4E requires its m7G cap binding activity for
its mRNA transport function (5). The molecular mechanism
for how eIF4E-sensitive transcripts are transported and
whether eIF4E directly transports mRNAs or participates in a
process required for transport is not yet known. However, for
ease of terminology we will refer to this general phenomenon
as eIF4E-dependent mRNA transport. The underlying basis
for sensitivity to eIF4E at this level of regulation is due to the
presence of a 100-nucleotide eIF4E sensitivity element (4ESE)
in the 3?UTR of targeted transcripts (6). The mRNA transport
function of eIF4E contributes to its ability to transform cells
and to impede differentiation (5, 34). Interestingly, eIF4E-
dependent mRNA transport is upregulated in a distinct subset
of acute myeloid leukemia (AML) and blast crisis chronic
myeloid leukemia (bcCML) patient specimens (34). Thus,
eIF4E-dependent mRNA transport likely contributes to its
Given that eIF4E modulates gene expression at the levels of
* Corresponding author. Mailing address: Institute for Research in
Immunovirology and Cancer, University of Montreal, Montreal, Que-
bec H3T 1J4, Canada. Phone: (514) 343-6291. Fax: (514) 343-7379.
† Present address: Institute for Research in Immunovirology and
Cancer, University of Montreal, Montreal, Quebec, Canada.
translation and mRNA transport (27), its activity must be reg-
ulated at multiple levels to effectively keep its proliferative
properties under control. The best-described family of regula-
tors involve the translation functions of eIF4E and include the
eIF4E binding proteins (4EBPs) which contain conserved
eIF4E binding sites (28). This site is defined by YXXXXL?
(where X is any amino acid and ? is any hydrophobic amino
acid [see Fig. 1A]) (4, 9). These proteins use this site to interact
with the dorsal surface of eIF4E. In general, regulatory pro-
teins using this site do not substantially alter eIF4E’s cap bind-
ing activity. Instead, they act by sterically blocking association
with eIF4G, which also binds the dorsal surface, and thereby
Regulation of the nuclear fraction of eIF4E is only now
becoming clear. To date, two negative regulators have been
identified: the promyelocytic leukemia protein (PML) and the
FIG. 1. HOXA9 directly binds eIF4E with a conserved eIF4E binding site. (A) Sequence alignment of HOXA9 from a variety of species.
Residues that are part of the conserved eIF4E binding site are highlighted in yellow; the “?” symbol indicates residues that are hydrophobic.
Numbers indicate the position in the amino acid sequence. eIF4G, PRH, and Bicoid, two homeodomain-containing proteins that interact and alter
eIF4E activity, are included for comparison. The schematic below indicates the relative positions of the eIF4E binding site (indicated by the
arrowhead) and the homeodomain (HD) in HOXA9. Accession numbers sequences are as follows: p31269 (human), P09631 (mouse), q6pwd5
(striped bass), q9IA26 (horn shark), 042506 (puffer fish), NP002720 (human PRH), p09081 (Bicoid), and NP 937884 (eIF4G). (B to F) GST
pull-down analysis of HOXA9. HOXA9 or the Y11A HOXA9 mutant was immobilized on glutathione-agarose, and the ability to bind wild-type
or mutant forms of eIF4E, monitored by Western blotting (W.B.) and Coomassie blue staining (C.B.), indicates equal loading. In panel B, only
the bound fractions are shown. In panels D to F, the bound (i.e., the fraction associating with the beads) and unbound fractions are shown. In
addition, in panel D, the supernatant from the sixth wash (wash) of the beads is shown.
VOL. 25, 2005 HOXA9 STIMULATES eIF4E ACTIVITY1101
proline-rich homeodomain protein (PRH), also known as the
hematopoietically expressed homeodomain Hex (1, 5, 17, 31–
33). In cells that express all three proteins endogenously, the
majority of these three proteins colocalize to the same nuclear
structures (33). Both PML and PRH directly associate with the
dorsal surface of eIF4E, repress its mRNA transport function,
and subsequently repress its transformation activity (5, 31–33).
At the biochemical level, the RING domain of PML represses
eIF4E activity by inducing a conformational change in eIF4E,
which in turn reduces its affinity for the m7G cap of mRNA by
?100-fold (5, 14). Importantly, PML does not contain a con-
served eIF4E binding site, indicating that at least in the nu-
cleus, not all regulators need to contain this motif.
Given that PRH is a homeodomain protein, we were sur-
prised to find that it contained a conserved eIF4E binding site,
N-terminal to its homeodomain, which it uses for its direct
interaction with eIF4E (33). Using a bioinformatics approach,
we discovered that ?200 of the ?800 homeodomain-contain-
ing proteins in the Swiss-Prot database contain putative eIF4E
binding sites (33). Thus, it is possible that eIF4E is regulated in
tissue specific manners by association with a wide variety of
homeodomain proteins in a variety of contexts.
Loss of homeodomain protein regulation of eIF4E may con-
tribute to disease progression in ?40% of AML specimens
(34). Here, eIF4E-dependent cyclin D1 and ODC mRNA
transport is substantially upregulated (34). This apparently oc-
curs because of upregulation of eIF4E, enlargement of eIF4E
nuclear bodies, and downregulation of PRH, as well as the
displacement of PRH from the nucleus. Importantly, restora-
tion of a normal phenotype, including reassociation of PRH
with eIF4E and downregulation of eIF4E levels, leads to down-
regulation of eIF4E-dependent mRNA transport (34).
Here we investigate the possibility that eIF4E can be regu-
lated at multiple levels by the interplay of homeodomain pro-
teins. Further, we examine whether loss of this level of regu-
lation contributes to leukemogenesis. HOXA9 is another
homeodomain protein that also contains a putative eIF4E
binding site (34) and is implicated in leukemogenesis (30).
Because HOXA9 is upregulated in a variety of myeloid leuke-
mias and its overexpression in collaboration with Meis leads to
leukemogenesis in animal models (30), we investigated
whether HOXA9 does indeed bind eIF4E and thus whether it
modulates eIF4E function. We demonstrate that HOXA9, un-
like PRH, is a stimulator of eIF4E activity and suggest that this
stimulation could be important in its leukemogenic role. Our
studies indicate that HOXA9 modulates both the nuclear and
the cytoplasmic functions of eIF4E. Importantly, competition
between HOXA9 and PRH for eIF4E appears to be important
in maintaining normal cell growth control. These results pro-
vide an example of a novel mechanism for regulating eIF4E
function: competition between inhibitory and stimulatory ho-
meodomain proteins. Further, this subset of homeodomain
proteins are positioned to affect gene expression transcription-
ally and independently, at the level of mRNA transport and
translation, allowing them to act as potent modulators of gene
MATERIALS AND METHODS
Cell isolation and culture. AML blood cells and normal bone marrow cells
were isolated and processed as described previously (11, 13, 34). Primary AML
and CML cells were obtained from the peripheral blood of patients at the
Markey Cancer Center, University of Kentucky Medical Center. Normal bone
marrow was obtained as waste material after pathological analysis or surgical
marrow harvest or from the National Disease Research Interchange. All tissues
were obtained with the approval of the Institutional Review Board and appro-
priate informed consent.
Cell sorting and I?B expression. Adenovirus (Ad) vectors were constructed to
express either green fluorescent protein (GFP) alone or a combination of GFP
with the NF-?B inhibitor I?B as previously described (11). Isolated populations
were at least 95% pure. Control populations of normal granulocytes and mono-
cytes were obtained by labeling peripheral blood mononuclear cells with CD14-
phycoerythrin and CD15-fluorescein isothiocyanate (FITC) (Becton Dickinson).
Cells were sorted by using appropriate forward- versus side-scatter gates and
CD14?CD15?(monocytes) and CD14?CD15?(granulocytes) were isolated.
Overexpression studies of HOXA9. The bicistonic MSCV-Hoxa9-GFP con-
struct was kindly provided by Guy Sauvageau. In this construct, full-length
murine Hoxa9 cDNA lays downstream of the retroviral long terminal repeat,
followed by a pgk-GFP reporter cassette. PCR-based site-directed mutagenesis
(QuikChange; Stratagene) was used to generate Y11A HOXA9 mutant from the
MSCV-Hoxa9-GFP construct. The integrity of both constructs was verified by
automated DNA sequencing. Each plasmid was transiently transfected into the
Phoenix-Ampho packaging line (kindly provided by Gary Nolan), and retroviral
supernatants were used to infect human U937 cells from the American Type
Culture Collection. For experiments with Ad infection, purified populations of
GFP?cells were isolated by using a FACSVantage flow cytometer. Dead cells
were excluded by using propidium iodide, and sorted populations were at least
95% pure. U937 cells infected with retroviruses were sorted twice to obtain
GFP?populations that were at least 99% pure.
Western blot analysis and coimmunoprecipitation. Western blot analysis and
coimmunoprecipitation studies were as described previously (32). A total of 20
?g was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The following antibodies were used: mouse monoclonal anti-
eIF4E antibody (BD Transduction Laboratories), mouse monoclonal anti-cyclin
D1 antibody (BD Pharmingen), rabbit polyclonal anti-HOXA9 antibody (Up-
state), mouse monoclonal anti-?-actin antibody (Sigma), rabbit polyclonal anti-
ODC antibody (Biomol), and affinity-purified rabbit polyclonal anti-PRH anti-
body (33). All primary antibodies were used at 1:2,000, except rabbit polyclonal
anti-HOXA9 antibody that was used at 1:500. Note that experiments for speci-
ficity of the HOXA9 antibody indicate that the signal from the antibody is
specifically blocked if the HOXA9 antibody is preincubated with purified
HOXA9 protein. Horseradish peroxidase-conjugated secondary antibodies were
used at 1:20,000, and the signals were detected by chemiluminescence (Super
Signal West Pico; Pierce). Coimmunoprecipitations were carried out as de-
scribed previously (3, 17). Briefly, the appropriate antibody or immunoglobulin
G (IgG; Calbiochem) previously cross-linked to protein A-Sepharose beads were
added to precleared lysates and incubated overnight at 4°C. Beads were washed
five times with immunoprecipitation buffer, collected, and examined by Western
Cell fractionation and Northern blot analysis. Cell fractionation and Northern
blot analysis was performed as previously described (17, 32, 33). Total RNA and
RNA from cytoplasmic and nuclear fractions were isolated by the TRIzol
(Gibco) procedure according to instructions of the manufacturer and as de-
scribed previously (32). RNA from nuclear fractions was additionally treated
with RNase-free DNase I (Promega). A total of 5 ?g of total and fractionated
RNA was loaded on a 1% formaldehyde-agarose gel and subsequently trans-
ferred onto positively charged nylon membrane (Roche). Membranes were pre-
hybridized in ULTRAhyb buffer (Ambion) for 1 h at 45°C and probed with cyclin
D1 cDNA probe (10 pM), eIF4E cDNA probe (5 pM), GAPDH cDNA probe (5
pM) (Ambion), tRNALysantisense oligoprobe (30 pM), and U6 antisense oli-
goprobe 3? (30 pM) in the same buffer for 16 h at 45°C. cDNA probes were
generated as described previously (32), and signals were detected by using CDP
Star chemiluminescence (Ambion) as described by the manufacturer.
Indirect immunofluorescence and laser scanning confocal microscopy. Cells
were fixed and permeabilized as described previously (5, 32, 33) and incubated,
as indicated, with mouse monoclonal anti-eIF4E antibody (1:100; BD Transduc-
tion Laboratories), mouse monoclonal anti-PML antibody 5E10 (1:10), rabbit
polyclonal anti-HOXA9 antibody (1:100), or affinity-purified rabbit polyclonal
anti-PRH antibody (1:50) in blocking buffer for 2 h at room temperature. After
incubation with primary antibody, cells were washed three times in 1? phos-
phate-buffered saline (PBS; pH 7.2) and incubated with Texas Red-conjugated
donkey anti-rabbit antibody and Cy5-conjugated donkey anti-mouse antibody
(Jackson Immunoresearch Laboratories) for 45 min at room temperature. After
secondary antibody incubation, cells were washed three times in 1? PBS (pH
1102 TOPISIROVIC ET AL.MOL. CELL. BIOL.
7.4) and mounted in Vectashield with DAPI (4?,6?-diamidino-2-phenylindole;
Vector Laboratories, Inc.). For triple staining, cells were additionally fixed with
3.7% paraformaldehyde for 10 min at room temperature, washed, and then
incubated with 1:20 dilution of FITC-conjugated mouse monoclonal anti-eIF4E
antibody (BD Transduction Laboratories) at 4°C overnight. Fluorescence was
observed by using a ?100 objective lens on a Leica inverted scanning confocal
microscope with excitation at 488, 568, or 351/364 nm. All channels were de-
tected separately, and no cross talk between the channels was detected. Micro-
graphs represent single sections through the plane of cells with a thickness of
?300 nm. Experiments were repeated three times with more than 500 cells in
Polyribosome analysis, RNA isolation, and real-time PCR. Cell pellets (?500
mg) were homogenized in 1 ml of ice-cold lysis buffer (20 mM HEPES [7.5], 10
mM magnesium acetate, 100 mM potassium acetate, 1? EDTA-free complete
protease inhibitor cocktail [Roche], 400 U of SUPERasine/ml [Ambion]) and
incubated for 30 min on ice with occasional vortexing. Lysates were spun at 3,000
? g for 10 min at 4°C. Supernatants were transferred to clean tubes and spun at
12,000 ? g for 20 min at 4°C. Supernatants obtained in the previous step were
spun at 100,000 rpm (TLA 100.3 rotor; Optima TLX ultracentrifuge; Beckman)
for 1 h at 4°C to pellet the ribosomes. Ribosomal pellets were resuspended in 200
?l of ice-cold lysis buffer, loaded on the top of the 10 to 40% sucrose gradients
(buffered with the lysis buffer), and centrifuged at 55,000 rpm for 60 min by using
a TLS 55 rotor in Optima TLX ultracentrifuge (Beckman) at 4°C. Then, 200-?l
fractions were collected, and 1/10 of each fraction was used for the measure-
ments of the optical density at 254/280 (OD254/280). Two of ten were saved for
Western blot analysis. RNA was isolated from the remaining seven of ten of each
fraction by the TRIzol procedure according to the manufacturer’s instructions.
RNA from each fraction was quantified by spectrophotometry, and 40 ng was
converted into cDNA by using the Sensiscript Reverse Transcription kit (Qia-
gen). Real-time PCR was carried out in triplicate with the QuantiTect SYBR
Green real-time PCR kit (Qiagen) in an Opticon thermal cycler (MJR) under the
following conditions: 95°C for 15 min, followed by 40 cycles of 94°C for 30 s, 57°C
for 30 s, and 72°C for 30 s. The following gene-specific primers were used:
GAPDH forward (5?-ACCACAGTCCATGCCATCAC-3?), GAPDH reverse
(5?-TCCACCACCCTGTTGCTGTA-3?), cyclin D1 forward (5?-CAGCGAGCA
GCAGAGTCCGC-3?), cyclin D1 reverse (5?-ACAGGAGCTGGTGTTCCATG
GC-3?), ODC forward (5?-GCATCAGCTTTCACGCTTG-3?), and ODC re-
transcription-PCR was performed by using a One-Step RT-PCR kit (Qiagen)
according to the manufacturer’s instructions. Reactions were carried out for 27
cycles under the same conditions as for real-time PCR.
Purification of eIF4E, HOXA9, and PRH and difference CD spectroscopy.
eIF4E was purified for pull-downs as described earlier (14). PRH was purified as
described earlier (33). eIF4E for cap binding and fluorescence studies was pu-
rified as a fusion protein of the B1 domain of protein G (eIF4E-GB [kindly
provided by Gerhard Wagner]). Expression of HOXA9–glutathione S-trans-
ferase (GST) was induced in BL21(RIL) codon plus cells at an OD600of 0.8 with
0.8 mM IPTG for 16 h at 18°C. Sedimented cells were suspended in 0.5 M
NaCl–50 mM Na-Tris (pH 7.5)–1 ?M tris(2-carboxyethyl)phosphine supple-
mented with protease inhibitors and 50 U of micrococcal nuclease, 5 mg of
RNase A, and 100 U of DNase I (per pellet of 1-liter culture) and then lysed by
20 rounds of sonication on ice by using a duty cycle. Lysates were cleared by
centrifugation and incubated with FastFlow glutathione-Sepharose at room tem-
perature for 20 min. Bound beads were exhaustively washed with 0.3 M NaCl–10
mM Na-Tris (pH 7.5), including a high-stringency 0.7 M NaCl–10 mM Na-Tris
(pH 7.5) wash, all of which were done at 4°C. Bound protein was eluted by
incubation with 0.3 M NaCl–50 mM Na-Tris (pH 7.5)–50 mM reduced glutathi-
one at room temperature for 20 min and dialyzed against 0.3 M NaCl–10 mM
Na-Tris (pH 7.5)–1 ?M ZnCl2at 4°C. Purity was assessed by SDS-PAGE, and
the concentration was calculated as ε280? 77,520 M?1cm?1. Measurement of
binding affinity using difference circular dichroism (CD) was performed as de-
scribed previously (14). Briefly, far-UV CD spectra were recorded by using
Jasco-810 spectropolarimeter with a 0.847-cm tandem cuvette (Hellma). Binding
partners were diluted in 0.3 M NaCl–50 mM Tris (pH 7.5)–5 mM glutathione at
25°C. Partners in two chambers of the cuvette were mixed by inversion and
allowed to equilibrate for 30 min. Five spectra for each condition before and
after binding were collected by using a 1-nm bandwidth and a 1-nm resolution
and then averaged. Relative ellipticity was converted to molar ellipticity (14).
The effects due to twofold dilution during mixing of the two tandem cuvette
sections were monitored by omitting one of the binding partners. Differences in
molar ellipticity at 222 nm for variable binding partner ratios were normalized,
where a value of 1.0 corresponds to the maximal CD change at saturating partner
concentrations. Binding isotherms were fit to a heuristic expression, assuming
stoichiometry of a single binding site as described previously (14). Cap affinity
methods are given elsewhere (10).
HOXA9 directly binds eIF4E. Our previous studies indi-
cated that ?200 homeodomain proteins contain potential
eIF4E binding sites (34). One such homeodomain protein,
HOXA9, contains a putative eIF4E binding site (Fig. 1A) and
is upregulated in many myeloid leukemias (2, 8, 16, 18). Similar
to PRH, this site is found N-terminal to the homeodomain
(Fig. 1A). Importantly, this binding site is found in HOXA9
from a wide variety of species ranging from fish to humans
(Fig. 1A). In order to determine whether HOXA9 directly
binds eIF4E, we carried out GST pull-down experiments with
proteins purified to homogeneity. Murine HOXA9 directly
binds to eIF4E (Fig. 1B) with a similar affinity as observed for
other eIF4E interacting proteins PML, Bicoid, and PRH (Fig.
1B and C) (5, 14, 24, 25). Mutation of the conserved tyrosine
to alanine (Y11A) in the eIF4E binding site of HOXA9 results
in a significant reduction in binding (Fig. 1D). Proteins that
contain conserved eIF4E binding sites typically associate with
the dorsal surface of eIF4E (28). Consistently, mutation of
W73 on the dorsal surface of eIF4E inhibits association with
HOXA9 (Fig. 1E). Similarly, PRH, PML, and Bicoid also
require W73 (Fig. 1E) (5, 14, 24, 25). Mutation of the cap-
binding site (W56A) does not impede association with
HOXA9, PML, Bicoid, or PRH (Fig. 1F) (34). Thus, HOXA9
utilizes its conserved eIF4E binding site to directly interact
with the dorsal surface of eIF4E.
HOXA9 localizes to eIF4E bodies only in a subset of leuke-
mia specimens. To establish the physiological importance of
the HOXA9 eIF4E interaction, we examined whether these
proteins colocalized in primary human blood cells (where they
are endogenously expressed) using immunofluorescence and
confocal microscopy and separately using immunoprecipita-
tion studies (Fig. 2). Incubation of the polyclonal anti-HOXA9
antibody with recombinant HOXA9 protein, which served as a
specific antigen, resulted in the disappearance of the signal in
both immunofluorescence and Western studies, indicating that
this antibody specifically recognizes HOXA9 protein (http://www
?16§ion?8 [referred to below as “our website”]). Speci-
mens were taken from normal donors or from a variety of
leukemic subtypes. Our previous studies indicated that the
PRH eIF4E interaction was disrupted in a distinct subset of
myeloid leukemias (34). In particular, normal bone marrow,
French-American-British Classification (FAB) subtype AML
specimens M1, M2, and ALL specimens all had normal levels
of eIF4E and PRH proteins and normal cyclin D1 mRNA
transport (34). However, in M4/M5 AML and bcCML speci-
mens, which account for ?40% of myelogenous leukemias,
eIF4E levels were substantially upregulated, PRH levels were
downregulated and PRH was displaced from the nucleus. This
was correlated with dysregulation of eIF4E and upregulation
of eIF4E-dependent cyclin D1 mRNA transport (34).
Thus, we examined the distribution of HOXA9 in these
specimens to determine whether its interaction could contrib-
ute to a subset of leukemias. In normal and M1/M2 AML
specimens, PML, PRH, and eIF4E colocalize as expected (34;
VOL. 25, 2005HOXA9 STIMULATES eIF4E ACTIVITY1103
FIG. 2. HOXA9 associates with eIF4E and colocalizes with eIF4E nuclear bodies in the subset of myeloid leukemia specimens that show
disruption of eIF4E-PRH interaction and facilitated eIF4E-dependent mRNA transport. (A) Confocal micrographs of cells obtained from the
bone marrow of patients with the indicated FAB subtypes of AML. Cells were stained with anti-eIF4E-FITC conjugated antibody (green),
anti-HOXA9 antibody (red), and anti-PML monoclonal antibody 5E10 (blue). The HOXA9-eIF4E overlay is shown in yellow (indicated on
subpanel J by the “?” symbol), the eIF4E-PML overlay is shown in light blue (indicated on subpanel E by the “#” symbol), and the triple
HOXA9-eIF4E-PML overlay is shown in white (indicated on subpanel J by the “?” symbol). The objective lens magnification was ?100, and the
images were further magnified twofold except for subpanels K to O, which were magnified threefold. An example of HOXA9 aggregate that is
distinct from eIF4E nuclear body is shown in subpanel E (in red, indicated by an asterisk). (B) Whole-cell extracts, from the indicated specimens,
were immunoprecipitated with anti-eIF4E antibody and analyzed by Western blotting with anti-HOXA9 antibody (IP eIF4E/WB HOXA9).
Conversely, extracts obtained from the same set of cells were immunoprecipitated with anti-HOXA9 antibody and probed with anti-eIF4E
antibody (IP HOXA9/WB eIF4E). A total of 80% of the immunoprecipitated fraction (P) and 20% of the unbound fraction (S) were subjected
to SDS-PAGE on 12 or 15% polyacrylamide gels. (C) Western blot analysis of whole-cell extracts obtained from the indicated specimens. Western
blots were probed with anti-eIF4E, anti-cyclinD1, anti-PRH, or anti-HOXA9 antibody. In addition, ?-actin is shown as a control for protein
data not shown). In these same M1 and M2 subtype specimens,
HOXA9 also formed nuclear dot structures that were indistin-
guishable from normal specimens, i.e., diffuse cytoplasmic and
nucleoplasmic staining (Fig. 2A, subpanels A to E and K to O,
and Fig. 3A, subpanels A to E). These bodies (red dots in
panels E and O) did not have any apparent spatial relationship
to those containing PML, PRH, and eIF4E. Occasionally,
HOXA9 partially overlapped with an eIF4E nuclear body, but
this event appeared to be random.
Interestingly, examination of FAB M4/M5 specimens, which
have dysregulated eIF4E, revealed that eIF4E and HOXA9
proteins almost completely colocalized in the abnormally en-
larged eIF4E nuclear bodies characteristic of these leukemias
(Fig. 2A, subpanels F to J and P to Y). In addition, both
proteins are present throughout the cytoplasm. Because of the
diffuse localization in the cytoplasm, confocal microscopy is
not best suited for establishing interaction, and thus we carried
out immunoprecipitation studies. Consistently, immunopre-
cipitation studies of specimens from these leukemias reveal
that eIF4E and HOXA9 interact, whereas in normal specimens
only a small fraction of these proteins interact consistent with
the confocal data (Fig. 2B). In M1/M2 specimens with overex-
pressed HOXA9 but normal levels of eIF4E, an interaction
was observed between HOXA9 and eIF4E; however, a much
smaller fraction of eIF4E immunoprecipitated with HOXA9
than in the M4/M5 AML specimens (Fig. 2B and C). Since the
confocal data indicate that HOXA9 nuclear bodies are not
colocalizing with eIF4E nuclear bodies in the M1/M2 or nor-
FIG. 3. I?B-SR induced loss of NF-?B activity in CD34?cells derived from bcCML patients leads to the normalization of HOXA9 protein
levels and correlates with the restoration of nuclear architecture. (A) Confocal micrographs of the CD34?cells derived from the bone marrow of
apparently healthy individuals (BM) and from bcCML patients. bcCML CD34?cells were either transduced with empty vector (?I?B-SR), or with
I?B-SR (?I?B-SR). Cells were stained with DAPI (blue), anti-eIF4E antibody (green), and anti-HOXA9 antibody (red). The HOXA9-eIF4E
overlay is shown in yellow. The objective lens magnification was ?100, and the images were further magnified by twofold. Note that there is
substantial colocalization of the HOXA9 protein with the eIF4E bodies in the CD34?cells derived from the bcCML patients (subpanel I, shown
in yellow, indicated by a “?” symbol). Expression of I?B-SR leads to the loss of colocalization of HOXA9 protein with eIF4E nuclear bodies in
the aforementioned cells, with results resembling the staining pattern observed in CD34?cells derived from the healthy individual (subpanels N
and D; the eIF4E nuclear body, shown in green, and HOXA9, shown in red, are labeled with a “?” symbol and an asterisk, respectively).
(B) HOXA9 Western blot analysis of whole-cell extracts isolated from the same specimens. ?-Actin is shown as a control for protein loading.
VOL. 25, 2005 HOXA9 STIMULATES eIF4E ACTIVITY1105
mal specimens (Fig. 2A), the interactions detected by immu-
noprecipitation likely reflect interactions within the popula-
tions of eIF4E and HOXA9 which are found diffusely
throughout the cytoplasm and/or nucleus. Importantly, unlike
the M4/M5 specimens, PRH is still present at the eIF4E nu-
clear bodies in the M1/M2 or normal specimens, a finding
consistent with the observation that eIF4E-dependent cyclin
D1 transport is not upregulated in these specimens (34). Note
that the cyclin D1 levels are upregulated transcriptionally in
M1/M2 specimens (34).
In the M4/M5 specimens, PRH is, in general, absent from
eIF4E nuclear bodies (34), whereas enlarged HOXA9 nuclear
bodies colocalize with the enlarged eIF4E nuclear bodies (Fig.
2A, subpanels F to J and U to Y). This is due to two factors, the
substantial downregulation of PRH in these leukemias and the
fact that PRH is found almost completely in the cytoplasm of
these specimens (34). We propose that HOXA9 overexpres-
sion in conjunction with low PRH and high eIF4E levels con-
tributes to the upregulation of eIF4E-dependent cyclin D1
mRNA transport previously observed in the M4/M5 AML and
bcCML specimens (34).
HOXA9 nuclear activity and localization are mediated by
NF-?B. The subcellular localization of eIF4E and PRH in
M4/M5 AML leukemia specimens returns to the pattern ob-
served in normal specimens when the cells express a dominant-
negative inhibitor of NF?B, I?B-super repressor (I?B-SR)
(34). Specifically, expression of this inhibitor leads to down-
regulation of eIF4E, upregulation of PRH and return of PRH
to the eIF4E nuclear bodies. Importantly, the return of PRH to
eIF4E nuclear bodies correlates with downregulation of
eIF4E-dependent cyclin D1 mRNA transport (34). Thus, we
utilized the same system to determine whether return of nor-
mal cyclin D1 mRNA transport was correlated with displace-
ment of HOXA9 from the eIF4E body in the M4/M5 AML and
bcCML specimens (Fig. 3). We examined the effects of NF-?B
activity by using an Ad vector encoding I?B-SR that mediates
strong repression of NF-?B activity within 6 to 12 h (11). AML
or bcCML cells were transduced with bicistronic vectors coding
for Ad-GFP or Ad-I?B-SR-GFP, and CD34?/GFP?cells were
isolated by fluorescence-activated cell sorting (Fig. 3) (our
website). In AML or CML CD34?cells overexpressing GFP
from the control virus, the subcellular distribution of eIF4E,
PML, PRH, and HOXA9 proteins (as analyzed by confocal
microscopy) is indistinguishable from their respective distribu-
tions in the untransduced patient cells (Fig. 3) (34).
In cells expressing I?B-SR, we observe a substantial alter-
ation in eIF4E nuclear body architecture, as expected (Fig. 3A,
subpanels K to O) (our website) where eIF4E nuclear bodies
are smaller and there is less eIF4E in the nucleus. Importantly,
I?B-SR expression leads to downregulation of HOXA9 pro-
tein levels (Fig. 3B) (our website) and to its reorganization into
smaller bodies that are distinct from eIF4E nuclear bodies
(Fig. 3A) (our website). Thus, HoxA9 nuclear bodies (seen as
red dots) no longer colocalize with eIF4E nuclear bodies
(green dots). This leads to a reduction in cyclin D1 mRNA
transport and thus a reduction in levels of cyclin D1 protein in
the identical specimens (see Fig. 3 in reference 34). These
HOXA9 bodies are similar to those we observed in normal
bone marrow controls (Fig. 3A, subpanels A to E). Identical
results are observed whether experiments were done in an
M4/M5 AML or bcCML background. Concurrently, we ob-
served the return of PRH to the eIF4E nuclear body in these
identical specimens (34).
HOXA9 overexpression leads to disruption of the PRH-
eIF4E interaction. The direct interaction with eIF4E and the
observation that HOXA9 associates with eIF4E in a subset of
leukemia specimens led us to investigate whether HOXA9 was
positioned to modulate eIF4E functions by modulating its in-
teractions with PRH (Fig. 4). First, we determined whether
HOXA9 overexpression alone was sufficient to alter the eIF4E
and PRH association observed in control cells. U937 cells were
transfected with a bicistronic MSCV vector encoding GFP,
along with HOXA9 or the Y11A HOXA9 mutant deficient in
eIF4E binding. GFP-positive cells were isolated by using flu-
orescence-activated cell sorting. In vector control cells, the
majority of PML, PRH, and eIF4E colocalized as observed
previously (33, 34). Endogenous HOXA9 bodies do not asso-
ciate with eIF4E nuclear bodies, similar to the studies above in
the normal primary specimens (our website). HOXA9 overex-
pression resulted in two major changes (Fig. 4). First, it leads
to a reduction in the association of PRH with eIF4E nuclear
structures (our website). Immunoprecipitation studies indicate
that when HOXA9 is overexpressed, PRH no longer physically
associates with eIF4E (Fig. 4D). Second, immunoprecipitation
studies indicate that overexpressed HOXA9 interacts with
eIF4E, in both the nuclear and cytoplasmic compartments
(Fig. 4). Consistently, HOXA9 now colocalized with eIF4E
nuclear bodies (our website). The subcellular distribution of
PML and eIF4E did not appear to be altered by HOXA9
overexpression (data not shown); thus, HOXA9 only alters
association of PRH with eIF4E. Importantly, the Y11A
HOXA9 mutant, which cannot bind eIF4E, does not associate
with eIF4E nuclear bodies and thus does not alter the distri-
bution of PRH, as shown by immunoprecipitation experiments
(Fig. 4A and D) and independently by confocal microscopy
experiments (our website).
HOXA9 promotes eIF4E-dependent mRNA transport. We
hypothesized that HOXA9 modulates the mRNA transport
activity of eIF4E. Western analysis of cyclin D1 protein levels
indicate that overexpression of HOXA9 leads to increased
cyclin D1 protein levels, whereas cyclin D1 levels are only
modestly increased in cells expressing the Y11A mutant (Fig.
4B). Importantly, overexpression of HOXA9 did not alter
PML or eIF4E protein levels, and PRH appeared to be slightly
reduced in both wild-type and mutant-overexpressing cells
(Fig. 4B and data not shown). To determine whether upregu-
lation of cyclin D1 is related to upregulation of eIF4E’s mRNA
transport function, we examined cyclin D1 mRNA levels (Fig.
4C). Consistent with previous studies, HOXA9 upregulates
cyclin D1 mRNA levels. Note that the two bands present in the
cyclin D1 Northern blots are both cyclin D1, with different
poly(A) tail lengths. Further, there is no alteration in the
production of GAPDH mRNA, an eIF4E-insensitive mRNA.
We examined whether eIF4E-dependent cyclin D1 mRNA
transport was modulated by HOXA9 (Fig. 4C, right panel).
Here, GFP?cells expressing either HOXA9, the Y11A mu-
tant, or vector were fractionated into nuclear and cytoplasmic
compartments, and the subcellular distribution of cyclin D1
mRNA was monitored. U6snRNA and tRNALysserve as con-
trols for the quality of the fractionation. Comparison of
1106 TOPISIROVIC ET AL.MOL. CELL. BIOL.
FIG. 4. HOXA9 requires integrity of its eIF4E-binding to associate with eIF4E and to facilitate nuclear export of ODC and cyclin D1
transcripts. (A) The same cells were fractionated into cytoplasmic (c) and nuclear (n) fractions. The protein extracts from each fraction were
immunoprecipitated with anti-eIF4E (IPeIF4E), IgG (IPIgG), or anti-HOXA9 (IPHOXA9) antibody. A total of 50% of the immunoprecipitated
fraction was subjected to SDS–15% PAGE, and the consequent Western blot was probed with anti-eIF4E antibody (W.B. eIF4E). (B) Western
blot analysis of whole-cell protein extracts of U937 cells overexpressing the indicated constructs. Western blots were probed with anti-eIF4E,
anti-cyclin D1, anti-PRH, anti-ODC, or anti-HOXA9 antibody. In addition, ?-actin is shown as a control for protein loading. The normalized
relative intensities of the bands were as follows: 1.00 ? 0.14 (vector), 2.22 ? 0.22 (HOXA9Y11A mutant), and 4.80 ? 0.10 (HOXA9 wild type)
for cyclin D1 and 1.00 ? 0.13 (vector), 1.00 ? 0.14 (HOXA9Y11A mutant), and 3.21 ? 0.16 (HOXA9 wild type) for ODC. The results are from
average values ? the standard deviations from three independent experiments. The quantification was performed with ImageQuant Software
(Molecular Dynamics). Area quantitation report values obtained for cyclin D1 or ODC were normalized against the corresponding area
quantitation report values obtained for ?-actin, and the value obtained for control, empty-vector-transduced cells was set at 1. (C) Total cellular
RNA (left panel) or RNA obtained from nuclear (n) or cytoplasmic (c) fractions (right panel) of these cells analyzed by Northern analysis (N.B.).
tRNALysand U6snRNA were used as markers for the cytoplasmic and nuclear fractions, respectively. GAPDH is shown as a control for RNA
loading. (D) HOXA9 competes with PRH for eIF4E binding. Whole-cell protein extracts were immunoprecipitated with anti-eIF4E (IPeIF4E) or
anti-HOXA9 (IPHOXA9) antibody. A total of 50% of the immunoprecipitated fraction was analyzed by SDS-PAGE on 12 or 15% gels, and the
resulting Western blot was probed with anti-HOXA9, anti-eIF4E, or anti-PRH antibody.
VOL. 25, 2005 HOXA9 STIMULATES eIF4E ACTIVITY1107
HOXA9 with controls indicates that HOXA9 overexpression
was correlated with substantially increased cyclin D1 mRNA
levels in the cytoplasmic fraction, indicating increased cyclin
D1 mRNA transport. In contrast, the subcellular distribution
of an eIF4E-insensitive mRNA, GAPDH, was not altered.
Thus, the specificity of the mRNA transport effects of HOXA9
is similar to those observed for eIF4E. Taken together, these
findings indicate that HOXA9 increases cyclin D1 expression
both through upregulation of its mRNA levels and by promot-
ing its mRNA transport.
To ensure that the HOXA9-dependent increase in cyclin D1
mRNA transport was mediated through its interaction with
eIF4E, parallel studies were carried out with the Y11A
HOXA9 mutant, which binds eIF4E with substantially reduced
affinity (Fig. 1). Analysis of total mRNA indicates that cyclin
D1 mRNA levels are increased to the same extent as wild-type
HOXA9, a finding consistent with a transcriptional upregula-
tion of cyclin D1 (Fig. 4C). However, the Y11A mutant did not
alter the subcellular localization of cyclin D1 mRNA relative to
vector controls (Fig. 4C). Thus, the ability of HOXA9 to dis-
place PRH from nuclear bodies and to increase cyclin D1
mRNA transport requires its interaction with eIF4E.
To establish whether HOXA9 was a general inhibitor of
eIF4E-dependent mRNA transport or specifically inhibited cy-
clin D1, we monitored the levels and subcellular distribution of
ODC, another transcript sensitive to eIF4E at the mRNA
transport level (Fig. 4C, right panel). HOXA9 overexpression,
but not expression of the Y11A mutant, led to increased trans-
port of ODC transcripts correlated with increased ODC pro-
tein levels. Importantly, neither HOXA9 nor the mutant al-
tered levels of ODC mRNA (Fig. 4C, left panel). Thus,
HOXA9 stimulates eIF4E-dependent mRNA transport of
both ODC and cyclin D1 mRNAs. This activity is independent
of the transcriptional functions of HOXA9.
HOXA9 modulates translation of eIF4E-sensitive tran-
scripts. Since HOXA9 directly binds eIF4E and modulates the
nuclear function of eIF4E, we extended our studies to assess
whether the cytoplasmic fraction of HOXA9 modulates trans-
lation of eIF4E-sensitive transcripts (Fig. 5). Previous studies
indicated that overexpression of eIF4E leads to increased poly-
somal loading of ODC but not of cyclin D1 or GAPDH tran-
scripts (27). Thus, we monitored polysomal loading of these
transcripts in HOXA9- or Y11A mutant-overexpressing cells
or vector controls. Appropriately transduced and GFP?sorted
cells were separated into different ribosomal fractions by cen-
trifugation on sucrose gradients, and ribosomal profiles were
determined for ODC, cyclin D1, and GAPDH mRNA by per-
forming quantitative real-time PCR. The results are repre-
sented as average cycle threshold values (CT) ? the standard
deviations for each ribosomal fraction. Parallel experiments
using semiquantitative PCR methods confirmed these results
(our website). Importantly, for any given set of cells, we used
the same ribosomal fractions to monitor all three transcripts so
differences in polysomal loadings are not the result of differ-
ences in fractionations between experiments.
Clearly, overexpression of HOXA9 leads to a substantial
change in the polysomal profile of ODC transcripts (Fig. 5).
Here, more transcripts were found in the heavier polysomal
fractions (fractions 10 and 11) compared to vector controls and
cells expressing the Y11A mutant, which were found with small
polysomes (fractions 7 and 9). Loading of transcripts onto
heavier polysomes indicates increased translational efficiency.
This correlates with increased translation of ODC (Fig. 4B).
Importantly, the polysomal loading profile of GAPDH was not
affected by expression of HOXA9 or the Y11A mutant com-
pared to vector controls. For the case of cyclin D1 mRNA,
ribosomal distribution was not qualitatively affected by en-
forced expression of either HOXA9 or the Y11A mutant. Note
that the total amount of polysomal cyclin D1 (as reflected by
lower CTvalues) was elevated in the cells overexpressing wild-
type form of HOXA9 compared to control cells and cells
overexpressing HOXA9Y11A, a finding consistent with the
upregulation of transport of cyclin D1 transcripts to the cyto-
plasm, which was detected in the HOXA9-overexpressing cells.
We further analyzed the polysomal fractions of these cells
(our website). Analysis of the polysomal profiles by monitoring
OD254or by Western analysis of ribosomal proteins indicated
the expected distributions of eIF4E and ribosomal proteins in
these fractions (our website). Overexpression of HOXA9 or
the Y11A mutant did not alter the distribution of these ribo-
somal proteins, indicating that increased polysomal loading is
not due to differences in the overall polysomal profile in
HOXA9, overexpressed HOXA9, and Y11A mutants were not
found in the polysomal fractions (our website). Note that
eIF4E was found both in fraction 1, with HOXA9, and inde-
pendently on the polysomes (our website). Thus, eIF4E is
available for association with eIF4G consistent with the ob-
served increased protein production (our website).
HOXA9 alleviates eIF4E repression through competition.
We sought to understand the biochemical underpinnings of the
ability of HOXA9 to promote eIF4E’s activity. HOXA9,
through its direct interaction with eIF4E, is positioned to mod-
ulate eIF4E’s activity through a number of mechanisms. First,
we examined the possibility that HOXA9 altered the affinity of
eIF4E for the m7G cap (our website). The affinity of m7GpppG
cap was assessed by monitoring fluorescence quenching of two
tryptophans in the cap binding site (W56 and W102) as a
function of m7GpppG cap analogue concentration as described
previously (14, 15). HOXA9 and, for comparison, PRH and
PML RING were purified to homogeneity from bacteria. For
these studies, we used the eIF4E-GB fusion protein in which
GB acts as a solubility enhancement tag (10). The eIF4E-GB
construct bound the cap analogue with a Kdof 130 ? 21 nM,
a finding consistent with previous reports (see reference 36 and
references therein). The addition of HOXA9 led to a slight
increase in cap affinity (Kd? 87 ? 34 nM). For comparison,
the addition of a PML RING led to a 100-fold reduction in
m7GpppG binding (Kd? 12 ? 3 ?M), as expected (14, 15).
Importantly, PRH, like HOXA9, had little effect on cap bind-
ing relative to eIF4E alone (Kd? 89 ? 14 nM). It appears that
neither homeodomain protein greatly alters the affinity of
eIF4E for the m7GpppG cap, and thus their respective effects
on eIF4E must be mediated through another mechanism.
Another possible mechanism by which HOXA9 promotes
eIF4E functions is through displacement of an inhibitor. We
examined the possibility that eIF4E activity could be modu-
lated via competition between HOXA9 and PRH (Fig. 6).
Note that, like HOXA9, PRH binds eIF4E in both subcellular
compartments (33). If these proteins compete for binding in
1108TOPISIROVIC ET AL.MOL. CELL. BIOL.
vivo, this should be reflected in their binding affinities for
eIF4E in vitro. Since PRH and HOXA9 both bind the dorsal
surface of eIF4E, requiring W73, one would expect that any
given eIF4E molecule can only bind either HOXA9 or PRH,
but not both, at any given time. To determine the dissociation
constant of HOXA9, and separately PRH, for eIF4E, we uti-
lized CD spectroscopy as we did previously (14, 15). When
either HOXA9 or PRH bind to eIF4E, the proteins undergo a
conformational change, as observed by CD spectroscopy. The
extent of this change as a function of eIF4E concentration is
shown (Fig. 6). The apparent Kds are 0.81 ? 0.17 ?M for
eIF4E-HOXA9 and 0.34 ? 0.035 ?M for eIF4E-PRH, indi-
cating that PRH binds to eIF4E slightly more tightly. However,
given the closeness of these Kdvalues, one would expect that
binding of eIF4E would be ultimately dominated by whichever
protein, HOXA9 or PRH, was in excess or at higher local
concentrations. Given these Kdvalues and ignoring other un-
known factors, cells expressing equimolar amounts of HOXA9
and PRH would have a ratio of ?0.4:1 of HOXA9-eIF4E to
PRH-eIF4E complexes. Under conditions in which HOXA9
levels are 10- or 100-fold increased, this ratio would change to
?4:1 or ?40:1, respectively. These ratios would be dramati-
cally affected by co-overexpression of eIF4E and HOXA9, as
observed in M4/M5 AML and bcCML, and even more affected
because PRH is downregulated while other inhibitor discussed,
PML, does not change its levels or localization (33). These
results strongly suggest that at the levels of overexpression seen
in HOXA9-overexpressing cells or in those of primary patient
specimens, HOXA9 effectively outcompetes PRH for eIF4E
binding and thereby relieves PRH-mediated repression and
modulates eIF4E activity.
We describe here a novel function for HOXA9 that depends
on its association with eIF4E. HOXA9 upregulates the trans-
port of cyclin D1 mRNA, and the levels of cyclin D1 tran-
scripts. In contrast, HOXA9 upregulates ODC mRNA trans-
port and translation but does not upregulate the levels of ODC
mRNA. Together, these findings suggest that HOXA9 has two
classes of functions, its role in transcription and a distinct role
in promoting both the nuclear and the cytoplasmic functions of
FIG. 5. HOXA9 facilitates translational initiation of ODC, but not of cyclinD1 and GAPDH mRNA. Polysomes from U937 cells transduced
with empty vector (MSCV), U937 cells overexpressing wild-type HOXA9 (MSCV/HOXA9wt) and U937 cells overexpressing HOXA9 mutant that
does not bind eIF4E (MSCV/HOXA9Y11A) were prepared and resolved by sedimentation on continuous 10 to 40% Sepharose gradients.
Ribosomal profiles of GAPDH, cyclin D1 and ODC transcripts were determined by quantitative real-time PCR analysis. To graphically present
the ribosomal profiles of these transcripts, average CTvalues obtained from two independent experiments both carried out in triplicate were plotted
against the number of the fraction. CTvalues for the control U937 cells (MSCV) are shown in blue, CTvalues for cells overexpressing wild-type
HOXA9 (HOXA9) are shown in red, and CTvalues for the Y11A mutant are shown in green. Bars represent the standard deviation.
VOL. 25, 2005HOXA9 STIMULATES eIF4E ACTIVITY1109
eIF4E. Our studies with the Y11A HOXA9 mutant demon-
strate that these functions depend on whether HOXA9 inter-
acts directly with eIF4E. Importantly, the transcriptional and
eIF4E-dependent properties of HOXA9 are independent of
each other, e.g., the Y11A mutant does not bind eIF4E but still
upregulates the levels of cyclin D1 mRNA. The parallel activ-
ities of HOXA9 and eIF4E strongly suggest that eIF4E is what
determines which transcripts are being regulated by HOXA9
and whether regulation occurs at the level of mRNA transport,
translation, or both. However, this does not rule out the pos-
sibility that HOXA9 directly interacts with these mRNAs
(through possible interactions with its homeodomain) and that
HOXA9 may only effect a subset of eIF4E-sensitive tran-
scripts. If true, this would infer that HOXA9 preferentially
interacts with a specific subset of eIF4E-sensitive transcripts.
In the nucleus, eIF4E promotes the transport of ODC, cyclin
D1, and other mRNAs. Many of these mRNAs contain 4ESE
in their 3?UTRs which, together with the m7G cap, are re-
quired for eIF4E to promote their export to the cytoplasm (6).
Negative regulators of this process were identified previously,
e.g., PML and PRH (5, 14, 33). PRH is similar to HOXA9 in
that it is a homeodomain containing protein with an N-termi-
nal eIF4E binding site (33). Unlike HOXA9, PRH and PML
impede eIF4E-dependent mRNA transport (5, 33). PML,
PRH, and HOXA9 bind the dorsal surface of eIF4E with
similar submicromolar affinity, suggesting that HOXA9 com-
petes with these negative regulators of eIF4E in order to alle-
viate repression. It is also possible that, through other regions
of the HOXA9 protein, HOXA9 recruits factors that enhance
eIF4E-dependent transport, perhaps by stabilizing active
transport complexes in addition to removing inhibitors.
In the cytoplasm, eIF4E functions in the rate-limiting step of
translation initiation. Interestingly, eIF4E overexpression in-
creases translational efficiency of a subset of eIF4E sensitive
transcripts including ODC but not cyclin D1 (27). The se-
quence features in the cytoplasm that impart sensitivity are not
yet known but are thought to involve the structural complexity
of the 5?UTR (28) and are distinct from the 4ESE, which plays
a role in the nuclear mRNA export functions of eIF4E (6). Our
data demonstrate that HOXA9 overexpression actually pro-
motes translational efficiency of ODC. It seems likely that
HOXA9 acts prior to the assembly of the eIF4F complex. In
particular, HOXA9 binds the same surface of eIF4E that is
bound by eIF4G. The eIF4E-eIF4G interaction is required for
formation of a productive translation initiation complex.
HOXA9 has a Kdfor eIF4E of ?1 ?M (Fig. 6), whereas the
eIF4E-eIF4G Kdis ?1 nM (10). Thus, eIF4G should easily
displace HOXA9, allowing for translation of ODC and other
eIF4E-sensitive transcripts. Importantly, HOXA9 is absent
from the polysomes, and thus there is no HOXA9-eIF4E in-
teraction in the polysomal fractions (our website). Also, both
the Y11A mutant (which is deficient in this activity) and the
wild-type proteins have the same subcytoplasmic distribution
(our website), strongly suggesting that the HOXA9-eIF4G ex-
change occurs prior to loading of ODC onto polysomes, a
finding consistent with the increased translation we observed.
Elucidating further details of this mechanism will be an area of
active future work.
FIG. 6. Affinity of HOXA9 and PRH for eIF4E are calculated by monitoring conformational changes upon binding as observed by using CD
spectroscopy. Far UV CD spectra were collected, and normalized molar ellipticity ([?]) at 222 nM was monitored as a function of eIF4E-GB
concentration with 15 nM GST-HOXA9 or 15 nM GST-PRH. Buffer alone shows no effects, as expected. PRH and HOXA9 bound eIF4E with
submicromolar affinities. HOXA9 (dashed line)- and PRH (dash-dotted line)-binding isotherms represent fits to a heuristic single-site binding
1110TOPISIROVIC ET AL.MOL. CELL. BIOL.
Previous studies indicated that another homeodomain pro-
tein, Bicoid, suppresses translation of caudal mRNA through
its N-terminal eIF4E binding site (25). The mechanism of
repression is quite different from that of stimulation of trans-
lation by HOXA9. The homeodomain of Bicoid binds an ele-
ment in the 3?UTR of caudal mRNA, known as a Bicoid
response element (BRE), and the eIF4E binding site of Bicoid
binds the dorsal surface of eIF4E at the same time (24). In this
case, mRNA specificity is determined by Bicoid and not eIF4E.
Translational repression of caudal mRNA presumably occurs
through blocking the eIF4E-eIF4G interaction (Kd? ?1 nM)
(24). The data in Fig. 1 and 6 would suggest that the Kdfor
Bicoid is similar to that of PRH and HOXA9. Thus, the asso-
ciation of Bicoid with both the BRE in caudal mRNA and the
eIF4E appears to be important for forming an effective repres-
sion complex. Thus, this mechanism is different from the stim-
ulatory mechanism of HOXA9, for which the choice of mR-
NAs is determined by eIF4E, and the weaker eIF4E-HOXA9
(Kd? ?1 ?M) association readily allows exchange with eIF4G
and thus translation. In this way, homeodomain proteins mod-
ulate eIF4E function in a variety of ways.
HOXA9 only affects eIF4E in cells that endogenously ex-
press the HOXA9 protein. For instance, HOXA9 overexpres-
sion in NIH 3T3 cells does not lead to upregulation of cyclin
D1 (data not shown). Neither does HOXA9 transcriptionally
upregulate cyclin D1 in NIH 3T3 cells (data not shown). These
findings suggest that HOXA9 requires tissue-specific cofactors.
Similarly, the findings that HOXA9 overexpression does not
alter eIF4E function in M1/M2 leukemia specimens and that
overexpression of HOXA9 alone in U937s is sufficient for its
stimulation of eIF4E activity suggest that HOXA9 acts in a
context-dependent manner, for which the precise context is
defined not only by cell type but by other multifactorial factors
that result in differing gene expression programs and thus
different complements of eIF4E regulatory factors.
Our finding that 200 homeodomain proteins contain eIF4E
binding sites suggest that there could be a plethora of eIF4E
regulators (33). Here, we suggest that such regulators could
work by competing for eIF4E binding and thereby modulate
development and differentiation, as our in vitro work strongly
suggests for HOXA9 and PRH (Fig. 6). For instance, the
presence of HOXA9 in the eIF4E nuclear body in HOXA9-
overexpressing cells is correlated with concomitant displace-
ment of PRH. This mechanism seems to be physiologically
relevant in M4/M5 AML and bcCML primary patient speci-
mens, for which the HOXA9 and eIF4E levels are high (the
present study) and PRH is displaced to the cytoplasm (34). In
this case, eIF4E functions are upregulated and correlate with
increased proliferation and impeded differentiation in human
cell lines (34). Importantly, treatment with the NF-?B repres-
sor I?B-SR restores PRH to eIF4E nuclear bodies, results in
the relocalization of HOXA9 to a distinct part of the nucleus,
and downregulates eIF4E-mediated growth and developmen-
tal arrest (34; the present study).
In summary, our findings demonstrate that the activity of
eIF4E is regulated through interactions with certain homeodo-
main proteins. These results are particularly interesting in light
of new findings suggesting that homeodomain proteins transit
between cells to effect gene expression (26). Given that so
many of these proteins contain the eIF4E binding motif, it
seems likely that eIF4E is regulated through competition of a
variety of homeodomain proteins, some inhibitory and some
stimulatory, and that these effect outcomes critical to normal
cellular proliferation, differentiation, and development. Fur-
thermore, we propose that changes in homeodomain protein
expression and/or localization may orchestrate alterations in
eIF4E-mediated gene expression. Such changes are indepen-
dent of the normal transcriptional activities commonly associ-
ated with homeodomain proteins and allow modulation of
gene expression at multiple levels, positioning these proteins as
potent regulators of cellular proliferation and differentiation.
We are grateful for the gifts of antibodies, constructs, and cell lines
kindly provided by Guy Sauvageau, L. de Jong, Gerhard Wagner, and
Nahum Sonenberg. We thank Jonathan Licht for use of the Opticon
thermal cycler. We are grateful for technical assistance from Melanie
McConnell, Vladimir Jankovic, and Biljana Culjkovic and for critical
reading of the manuscript and helpful discussions with Martin Wied-
mann and Guy Sauvageau.
Confocal laser scanning microscopy was performed at the MSSM-
LCSM core facility, supported by funding from the NIH (1 S10 RR0
9145-01) and the NSF (DBI-9724504). K.L.B.B. and C.T.J are scholars
of the Leukemia and Lymphoma Society. K.L.B.B. holds a Canada
Research Chair. Financial support was provided by the NIH (CA
98571 and CA90446).
1. Borden, K. L. 2002. Pondering the promyelocytic leukemia protein (PML)
puzzle: possible functions for PML nuclear bodies. Mol. Cell. Biol. 22:5259–
2. Calvo, K. R., D. B. Sykes, M. Pasillas, and M. P. Kamps. 2000. Hoxa9
immortalizes a granulocyte-macrophage colony-stimulating factor-depen-
dent promyelocyte capable of biphenotypic differentiation to neutrophils or
macrophages, independent of enforced Meis expression. Mol. Cell. Biol.
3. Carlile, G. W., W. G. Tatton, and K. L. Borden. 1998. Demonstration of a
RNA-dependent nuclear interaction between the promyelocytic leukaemia
protein and glyceraldehyde-3-phosphate dehydrogenase. Biochem. J. 335:
4. Clemens, M. J., and U. A. Bommer. 1999. Translational control: the cancer
connection. Int. J. Biochem. Cell Biol. 31:1–23.
5. Cohen, N., M. Sharma, A. Kentsis, J. M. Perez, S. Strudwick, and K. L.
Borden. 2001. PML RING suppresses oncogenic transformation by reducing
the affinity of eIF4E for mRNA. EMBO J. 20:4547–4559.
6. Culjkovic, B., I. Topisriovic, L. Skrabanek, M. Ruiz-Gutierrez, and K. L. B.
Borden. eIF4E selectively associates with nuclear mRNAs with a novel
3?UTR structure to modulate their nucleo-cytoplasmic mRNA transport.
Submitted for publication.
7. De Benedetti, A., and A. L. Harris. 1999. eIF4E expression in tumors: its
possible role in progression and malignancies. Int. J. Biochem. Cell Biol.
8. Golub, T. R., D. K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J. P.
Mesirov, H. Coller, M. L. Loh, J. R. Downing, M. A. Caligiuri, C. D. Bloom-
field, and E. S. Lander. 1999. Molecular classification of cancer: class dis-
covery and class prediction by gene expression monitoring. Science 286:531–
9. Graff, J. R., and S. G. Zimmer. 2003. Translational control and metastatic
progression: enhanced activity of the mRNA cap-binding protein eIF-4E
selectively enhances translation of metastasis-related mRNAs. Clin. Exp.
10. Gross, J. D., N. J. Moerke, T. von der Haar, A. A. Lugovskoy, A. B. Sachs,
J. E. McCarthy, and G. Wagner. 2003. Ribosome loading onto the mRNA
cap is driven by conformational coupling between eIF4G and eIF4E. Cell
11. Guzman, M. L., C. F. Swiderski, D. S. Howard, B. A. Grimes, R. M. Rossi,
S. J. Szilvassy, and C. T. Jordan. 2002. Preferential induction of apoptosis
for primary human leukemic stem cells. Proc. Natl. Acad. Sci. USA 99:
12. Iborra, F. J., D. A. Jackson, and P. R. Cook. 2001. Coupled transcription and
translation within nuclei of mammalian cells. Science 293:1139–1142.
13. Jordan, C. T., D. Upchurch, S. J. Szilvassy, M. L. Guzman, D. S. Howard,
A. L. Pettigrew, T. Meyerrose, R. Rossi, B. Grimes, D. A. Rizzieri, S. M.
Luger, and G. L. Phillips. 2000. The interleukin-3 receptor alpha chain is a
VOL. 25, 2005HOXA9 STIMULATES eIF4E ACTIVITY 1111
unique marker for human acute myelogenous leukemia stem cells. Leukemia Download full-text
14. Kentsis, A., E. C. Dwyer, J. M. Perez, M. Sharma, A. Chen, Z. Q. Pan, and
K. L. Borden. 2001. The RING domains of the promyelocytic leukemia
protein PML and the arenaviral protein z repress translation by directly
inhibiting translation initiation factor eIF4E. J. Mol. Biol. 312:609–623.
15. Kentsis, A., R. E. Gordon, and K. L. Borden. 2002. Control of biochemical
reactions through supramolecular RING domain self-assembly. Proc. Natl.
Acad. Sci. USA 99:15404–15409.
16. Kroon, E., J. Krosl, U. Thorsteinsdottir, S. Baban, A. M. Buchberg, and G.
Sauvageau. 1998. Hoxa9 transforms primary bone marrow cells through
specific collaboration with Meis1a but not Pbx1b. EMBO J. 17:3714–3725.
17. Lai, H. K., and K. L. Borden. 2000. The promyelocytic leukemia (PML)
protein suppresses cyclin D1 protein production by altering the nuclear
cytoplasmic distribution of cyclin D1 mRNA. Oncogene 19:1623–1634.
18. Lawrence, H. J., S. Rozenfeld, C. Cruz, K. Matsukuma, A. Kwong, L.
Komuves, A. M. Buchberg, and C. Largman. 1999. Frequent coexpression of
the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias.
19. Lazaris-Karatzas, A., K. S. Montine, and N. Sonenberg. 1990. Malignant
transformation by a eukaryotic initiation factor subunit that binds to mRNA
5? cap. Nature 345:544–547.
20. Lazaris-Karatzas, A., M. R. Smith, R. M. Frederickson, M. L. Jaramillo,
Y. L. Liu, H. F. Kung, and N. Sonenberg. 1992. Ras mediates translation
initiation factor 4E-induced malignant transformation. Genes Dev. 6:1631–
21. Lazaris-Karatzas, A., and N. Sonenberg. 1992. The mRNA 5? cap-binding
protein, eIF-4E, cooperates with v-myc or E1A in the transformation of
primary rodent fibroblasts. Mol. Cell. Biol. 12:1234–1238.
22. Lejbkowicz, F., C. Goyer, A. Darveau, S. Neron, R. Lemieux, and N. Sonen-
berg. 1992. A fraction of the mRNA 5? cap-binding protein, eukaryotic
initiation factor 4E, localizes to the nucleus. Proc. Natl. Acad. Sci. USA
23. Nathan, C. A., P. Carter, L. Liu, B. D. Li, F. Abreo, A. Tudor, S. G. Zimmer,
and A. De Benedetti. 1997. Elevated expression of eIF4E and FGF-2 iso-
forms during vascularization of breast carcinomas. Oncogene 15:1087–1094.
24. Niessing, D., S. Blanke, and H. Jackle. 2002. Bicoid associates with the
5?-cap-bound complex of caudal mRNA and represses translation. Genes
25. Niessing, D., N. Dostatni, H. Jackle, and R. Rivera-Pomar. 1999. Sequence
interval within the PEST motif of Bicoid is important for translational re-
pression of caudal mRNA in the anterior region of the Drosophila embryo.
EMBO J. 18:1966–1973.
26. Prochiantz, A., and A. Joliot. 2003. Can transcription factors function as
cell-cell signaling molecules? Nat. Rev. Mol. Cell. Biol. 4:814–819.
27. Rousseau, D., R. Kaspar, I. Rosenwald, L. Gehrke, and N. Sonenberg. 1996.
Translation initiation of ornithine decarboxylase and nucleocytoplasmic
transport of cyclin D1 mRNA are increased in cells overexpressing eukary-
otic initiation factor 4E. Proc. Natl. Acad. Sci. USA 93:1065–1070.
28. Sonenberg, N., and A. C. Gingras. 1998. The mRNA 5? cap-binding protein
eIF4E and control of cell growth. Curr. Opin. Cell Biol. 10:268–275.
29. Strudwick, S., and K. L. Borden. 2002. The emerging roles of translation
factor eIF4E in the nucleus. Differentiation 70:10–22.
30. Thorsteinsdottir, U., E. Kroon, L. Jerome, F. Blasi, and G. Sauvageau. 2001.
Defining roles for HOX and MEIS1 genes in induction of acute myeloid
leukemia. Mol. Cell. Biol. 21:224–234.
31. Topcu, Z., D. L. Mack, R. A. Hromas, and K. L. Borden. 1999. The promy-
elocytic leukemia protein PML interacts with the proline-rich homeodomain
protein PRH: a RING may link hematopoiesis and growth control. Onco-
32. Topisirovic, I., A. D. Capili, and K. L. Borden. 2002. Gamma interferon and
cadmium treatments modulate eukaryotic initiation factor 4E-dependent
mRNA transport of cyclin D1 in a PML-dependent manner. Mol. Cell. Biol.
33. Topisirovic, I., B. Culjkovic, N. Cohen, J. M. Perez, L. Skrabanek, and K. L.
Borden. 2003. The proline-rich homeodomain protein, PRH, is a tissue-
specific inhibitor of eIF4E-dependent cyclin D1 mRNA transport and
growth. EMBO J. 22:689–703.
34. Topisirovic, I., M. L. Guzman, M. J. McConnell, J. D. Licht, B. Culjkovic,
S. J. Neering, C. T. Jordan, and K. L. Borden. 2003. Aberrant eukaryotic
translation initiation factor 4E-dependent mRNA transport impedes hema-
topoietic differentiation and contributes to leukemogenesis. Mol. Cell. Biol.
35. Wang, S., I. B. Rosenwald, M. J. Hutzler, G. A. Pihan, L. Savas, J. J. Chen,
and B. A. Woda. 1999. Expression of the eukaryotic translation initiation
factors 4E and 2alpha in non-Hodgkin’s lymphomas. Am. J. Pathol. 155:247–
36. Zuberek, J., J. Jemielity, A. Jablonowska, J. Stepinski, M. Dadlez, R. Sto-
larski, and E. Darzynkiewicz. 2004. Influence of electric charge variation at
residues 209 and 159 on the interaction of eIF4E with the mRNA 5? termi-
nus. Biochemistry 43:5370–5379.
1112 TOPISIROVIC ET AL.MOL. CELL. BIOL.