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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 741-747
NUP98 Is Fused to PMX1 Homeobox Gene in Human Acute
Myelogenous Leukemia With Chromosome Translocation t(1;11)(q23;p15)
By
Takuro Nakamura,
Yukari Yamazaki,
Yoshiaki Hatano, and
Ikuo Miura
From PRESTO, Japan Science and Technology Corp, Tokyo, Japan; The
Cancer Institute, Japanese Foundation for Cancer Research, Tokyo,
Japan; and the Third Department of Internal Medicine, Akita University
School of Medicine, Akita, Japan.
 |
ABSTRACT |
The nucleoporin gene NUP98 was found fused to the
HOXA9, HOXD13, or DDX10 genes in human acute
myelogenous leukemia (AML) with chromosome translocations
t(7;11)(p15;p15), t(2;11)(q35;p15), or inv(11)(p15;q22), respectively.
We report here the fusion between the NUP98 gene and another
homeobox gene PMX1 in a case of human AML with a
t(1;11)(q23;p15) translocation. The chimeric NUP98-PMX1 transcript was detected; however, there was no reciprocal
PMX1-NUP98 fusion transcript. Like the NUP98-HOXA9
fusion, NUP98 and PMX1 were fused in frame
and the N-terminal GLFG-rich docking region of the NUP98 and
the PMX1 homeodomain were conserved in the NUP98-PMX1 fusion,
suggesting that PMX1 homeodomain expression is upregulated and that the
fusion protein may act as an oncogenic transcription factor. The fusion
to NUP98 results in the addition of the strong transcriptional
activation domain located in the N-terminal region of NUP98 to PMX1.
These findings suggest that constitutive expression and alteration of
the transcriptional activity of the PMX1 homeodomain protein may be
critical for myeloid leukemogenesis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HOMEOBOX GENES represent a class of
transcription factors that share a conserved DNA binding motif called
the homeodomain. This domain defines the DNA binding specificity of the
protein and plays an important role in transcriptional regulation of
downstream target genes.1 Homeobox proteins control
embryonic development in organisms and function in adult tissue,
including the hematopoietic system,2,3 in which expression
of class I HOX genes has been well characterized. The
HOXA and HOXB genes in the 3' region of the
clusters are expressed in most primitive bone marrow cells and are
downregulated as CD34+ hematopoietic progenitors
differentiate toward erythroid and myeloid phenotypes. Genes in the
5' regions are expressed at nearly equal levels in
CD34+ subpopulations.4 There is also good
evidence that several non-class I homeobox genes, such as
Hox11,5 Hex,6 and
Oct-2,7 are important in maintaining normal
myeloid, erythroid, or lymphoid populations. These findings indicate
that a broad range of homeobox genes are required for proper
differentiation and function of hematopoietic lineages.
The important role of homeobox genes in the hematopoietic system is
also indicated by the fact that several homeobox genes are involved in
leukemogenesis.2,3,8 In the mouse, a retrovirus-like intracisternal A particle is integrated in the 5' end of the
Hoxb8 gene in the myelo-monocytic cell line
WEHI-3B.9,10 Recently, cooperative activation of
Hoxa7/a9 and another homeobox gene Meis1 has been
identified in retrovirus-induced BXH-2 mouse myeloid leukemia.11 Cooperative leukemogenic action of
Hoxa9 and Meis1 was further demonstrated by the
induction of leukemia using retroviral transfer of the genes to mouse
primary bone marrow cells.12 In humans, overexpression of
the HOX11 gene occurs in rare cases of human T-cell acute
lymphoblastic leukemia (ALL) that have the chromosomal translocations
t(10;14)(q24;q11) or t(7;10)(q35;q24) in which the HOX11 gene
is activated due to juxtaposition of promoter elements from the T-cell
receptors.13-16 In these cases, upregulation of the
homeobox genes is thought to cause inhibition of hematopoietic differentiation and/or abnormal proliferation of primitive bone marrow
cells by deregulating expression of downstream target genes.
Another type of homeobox gene involvement in human leukemias is the
synthesis of a fusion protein owing to specific chromosomal translocation. Gene fusion between E2A and PBX1 was
identified in pre-B ALL with t(1;19)(q23;p13.3).17,18
Expression of E2A-PBX1 induces both myeloid and T-cell leukemia
in a transgenic mouse model,19 and expression of
E2A-PBX1 in cultured bone marrow cells blocks differentiation
of myeloid progenitors.20,21 We and another group have
recently found that the nucleoporin gene NUP98 is fused to
HOXA9 in human acute myelogenous leukemia (AML) with the
chromosomal translocation t(7;11)(p15;p15).22,23 As a
result of the gene fusion, the major part of the HOXA9
including the DNA binding homeodomain was fused in frame to a
NUP98 domain encoding the N-terminal GLFG repeat. Both mouse
and human data indicated that abnormal expression of the HOXA9
homeodomain might play an important role in leukemogenesis.
NUP98 has been found fused to the putative RNA helicase gene
DDX10 in a few cases of human AML with
inv(11)(p15;q22),24 suggesting that the N-terminal domain
of NUP98 might also have a leukemogenic potential. In this study, we
have shown that the NUP98 is involved in the t(1;11)(q23;p15)
translocation in a case of the secondary AML and that the gene is fused
to another homeobox gene PMX1 located at chromosome 1q23.
NUP98-PMX1 fusion transcripts were detected, and our data
suggested that the chimeric protein consisting of the NUP98 N-terminal
docking site and the PMX1 homeodomain might function as an oncogenic
transcriptional activator.
| |
MATERIALS AND METHODS |
Patient material.
Leukemia cells were obtained from a 55-year-old man with AML of an M2
subtype associated with a t(1;11)(q23;p15) translocation. Details of
clinical data and karyotypic analysis will be reported elsewhere
(Hatano et al, manuscript submitted). The patient had a
history of treatment for non-Hodgkin's lymphoma as the primary malignancy and had received chemotherapy, including the use of topo II
inhibitors. He developed AML 3 years after the initial chemotherapy
against the lymphoma.
DNA extraction and Southern blot analysis.
High molecular weight genomic DNA was extracted from frozen leukemia
cell suspensions. Samples of 5 µg of genomic DNA were subjected to
restriction endonuclease digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization according to the methods previously described.25 A 350-bp HaeIII
fragment22 was used as a probe to detect NUP98 rearrangements.
cDNA cloning of the NUP98 fusion partner.
Poly(A)+ RNA was isolated from frozen leukemia cell
suspensions using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA).
To isolate the unknown gene at chromosome 1q23,
3'-rapid amplification of cDNA ends
(3'-RACE)26 was performed using the
Marathon cDNA Amplification kit (Clontech, Palo Alto, CA) with
NUP98 gene-specific primers 3R-1
(5'-TCTTGGTACAGGAGCCTTTGGG-3') and 3R-2
(5'-CTCTTGGTGCTGGACAGGCATC-3') and adaptor primers AP-1
(5'-CCATCCTAATACGACTCACTATAGGGC-3') and AP-2
(5'-ACTCACTATAGGGCTCGAGCGGC-3'). The 3'-RACE
products were subcloned into the pCR2.1 vector (Invitrogen), and
plasmid clones were sequenced using a Thermo Sequenase dye terminator
cycle sequencing kit (Amersham, Cleveland, OH).
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was extracted from leukemic cell suspensions by using the
RNAzol method (TelTest, Friendswood, TX). First-strand cDNA was
synthesized from 1 µg total RNA using random hexamers and Superscript
II reverse transcriptase (Life Technologies, Rockville, MD) in a total
volume of 20 µL. The mixture was diluted to 50 µL, and 2 µL was
used for PCR. PCR was performed using 2.5 U of Taq DNA polymerase
(Boehringer Mannheim, Mannheim, Germany) and a Perkin Elmer Thermal
Cycler (Perkin Elmer, Norwalk, CT) using the following temperature
cycling protocol: 94°C for 30 seconds, 60°C for 1 minute, and
72°C for 2 minutes for 35 cycles. PCR products were separated
by electrophoresis through a 2.5% GTG gel (FMC Bioproducts,
Rockland, ME), subcloned into the pCR2.1 plasmid, and sequenced. The
PCR primers for NUP98 and PMX1 used were as follows:
n1, 5'-CTCTTGGTGCTGGACAGGCATC-3'; n2,
5'-CTAGGGATGGTTCATCGTC-3'; p1,
5'-AGCTACGGGCACGTTCTGG-3'; P2,
5'-AGGGGTCTGCGATGGTGGTGTGG-3'; and P3,
5'-GCACAAAAGCATCAGGATAGTG-3'.
Northern blot analysis.
The full-length human PMX1 cDNA was used to probe Human
Multiple Tissue Northern blots (Clontech). Membranes were hybridized and washed according to the method of Church and Gilbert.27
Construction of expression plasmids.
cDNAs containing full-length NUP98-PMX1 and wild-type
PMX1 coding sequences were obtained by RT-PCR on RNA samples
extracted from leukemia cells and by PCR of the human heart cDNA
(Clontech), respectively. The 5' and 3' oligonucleotides
used for PCR contained unique EcoRI and BamHI
restriction sites to facilitate cloning into the pM eukaryotic
expression plasmid in frame with sequences encoding the GAL4 DNA
binding domain.28 These full-length cDNAs were also
subcloned into pCDNA3.1 expression vector (Invitrogen). Truncated
NUP98 and PMX1 sequences were obtained by PCR
amplification of full-length clones. To generate the pMCK1-1 reporter
plasmid that contains the 246-bp murine muscle creatine kinase (MCK)
basal enhancer,29 the region from bp  1250 to
1048 relative to the MCK transcription start site was amplified
by PCR and subcloned into the pGL3 promoter vector (Promega, Madison,
WI). All constructs were verified by sequencing.
Transient transfection assays.
HeLa cells were seeded 18 to 24 hours before transfection at 5 × 105 cells per 60-mm dish in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. One microgram of each
pM fusion construct, 1 µg of 5 × GAL4-pGL2
plasmid,30 and 0.1 µg of pRL-SV40 plasmid (Promega) were
cotransfected by using Lipofectamin and Opti-MEM (Life Technologies).
The pCDNA vectors containing full-length cDNAs of PMX1 or
NUP98-PMX1, and pMCK1-1 were cotransfected into Jurkat cells (2 × 106 cells) using DIMRIE-C reagent (Life
Technologies) and Opti-MEM. Cells were harvested 24 hours
posttransfection in 1× passive lysis buffer (Promega). Luciferase
activity was measured in a luminometer using a Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturer's protocol.
The firefly luciferase activity derived from the reporter constructs
was normalized to levels of renilla luciferase activity as an internal
control for transfection efficiency. Experiments were repeated three
times. The expression of GAL4 fusion proteins was analyzed by Western
blotting as described below. The 5 × GAL4-pGL2 expression plasmid
was a generous gift of Cory Abate-Shen (UMDNJ, Piscataway, NJ).
Western blot analysis.
Each transfectant (5 × 105 cells) was lysed in 100 µL of sodium dodecyl sulfate (SDS) sample buffer containing 50 mmol/L
Tris (pH 6.8), 2% SDS, 10% glycerol, 100 mmol/L dithiothreitol, and 0.1% bromophenol blue, and 10-µL aliquots were subjected to
SDS-polyacrylamide gel electrophoresis. The size-fractionated proteins
were transferred by electrical field (Trans-Blot cell; Bio-Rad,
Hercules, CA) onto a nitrocellulose membranes. Membranes were
blocked in Trisbuffered saline (pH 8.0) containing 5% nonfat
dry milk. The monoclonal anti-GAL4 DNA binding domain antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) was used as a primary antibody. The
signals were detected using ECL Western blotting detection reagents (Amersham).
| |
RESULTS |
Cloning the breakpoint gene at 1q23.
Chromosomal analysis of the patient bone marrow cells at the onset of
AML showed a t(1;11)(q23;p15) (Hatano et al, manuscript submitted). This finding suggested the involvement of
NUP98 in chromosome 11 break. Southern blot analysis using a
NUP98 intron probe showed the DNA rearrangement in the t(1;11)
patient sample (Fig 1A). The result
indicated that the chromosome 11 break was within the same intron as
was seen in t(7;11) AML, resulting in a NUP98-HOXA9
fusion.22 Because the vast majority of human AML with
reciprocal chromosomal translocations have shown that chimeric transcripts of the fused genes are
synthesized,8,31 we attempted to isolate the
affected gene on chromosome 1q23 by using the 3'-RACE method.25 Two RACE products of different sizes were
identified (Fig 1B), and the t(1;11) DNA rearrangement was
observed using the 3'-end of the shorter product as a probe (Fig
1C), indicating that the product contained a cDNA fragment derived from
chromosome 1q23.
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| Fig 1.
(A) Southern blot analysis of the leukemia cell sample
derived from t(1;11)(q23;p15) AML. Genomic DNA samples from the
t(7;11)(p15;p15) AML patient as well as healthy control were also
shown. Rearranged bands were detected both in t(1;11) patient as well
as t(7;11) by Sac I digestion using a NUP98 probe. (B)
3'-RACE products of NUP98 fusion cDNA. MWM, 123-bp ladder
molecular weight marker; RT(+), reverse transcribed and PCR-amplified
patient poly(A)+ RNA sample; RT(), patient RNA without
reverse transcription. (C) Southern blot analysis of the patient
genomic DNA with the 3'-fragment of shorter RACE product as a
probe. Rearranged band was detected in patient sample digested with
BamHI.
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Sequence analysis confirmed that both the two 3'-RACE products
contained unique sequences. Genetic database searching showed that the
3'-region of the longer product was identical to human PMX1 (PHOX1).32 The shorter product did not
show significant homology to PMX1; however, further analysis
showed that both products were located on the same genomic DNA fragment
derived from chromosome 1, which was produced by Sanger Centre Human
Chromosome 1 Genome Project (EMBL/GenBank Accession No. Z97200). The
chromosome 1 derived parts of the RACE products were situated at 50 kb (Fig 2A). The shorter
fragment was thought to contain the noncoding exon 1b of the
PMX1 gene that is located 5.2 kb downstream of exon 1a (Fig
2A). The longer product contains exons 2 and 3 that encode the DNA
binding homeodomain of PMX1.
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| Fig 2.
(A) Breakpoint at chromosomes 1 and 11. The 5'
exons of NUP98 were previously described.22 The
location of the primers used for RT-PCR is indicated. (B) Detection of
fusion transcripts between NUP98 and PMX1 exon 2 and
between NUP98 and PMX1 exon 1b. A 270-bp product of
NUP98-PMX1 exon 2 fusion and a 168-bp product of
NUP98-PMX1 exon 1b fusion were observed, whereas no
PMX1 exon 1a-NUP98 fusion was seen. MWM, 123-bp ladder
molecular weight marker. + and indicate the presence and absence
of RT reaction, respectively. (C) Nucleotide and deduced
amino acid sequences of NUP98-PMX1 fusions.
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Gene fusion between NUP98 and PMX1.
RT-PCR analysis showed two NUP98-PMX1-derived chimeric
transcripts, suggesting that the NUP98-PMX1 protein may have a
biological function; however, no reciprocal PMX1-NUP98 fusion
transcript was detected (Fig 2B). Sequence analysis of 3'-RACE
and RT-PCR products showed that NUP98-PMX1 exon 1b encoded a
protein that lacked PMX1 functional domain (Fig 2C).
NUP98 and PMX1 exon 2 were fused in frame, resulting in
production of a chimeric protein containing the N-terminal half of
NUP9833 and most of the coding region of PMX1, including
the entire homeodomain (Figs 2C and 3). HOX
proteins, which show cooperative DNA binding with PBX, contain short
conserved peptide motifs, including a key tryptophan required for the
interaction with PBX.34,35 The PBX-interaction domain is
conserved in NUP98-HOXA9, and we observed cooperative DNA binding with
PBX (Fujino and Nakamura, unpublished data). However,
neither wild-type PMX1 nor NUP98-PMX1 contained such a motif.
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| Fig 3.
Structure of the wild-type proteins and predicted
chimeric protein. HD, homeodomain; RNA binding, putative RNA binding
domain of NUP98.
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Because murine Pmx1 is expressed mainly in embryonic mesoderm,
adult heart, and adult muscle tissue,36 we analyzed the
expression pattern of PMX1 in human normal tissue. Northern
blot analysis showed that PMX1 was expressed in heart, muscle,
thymus, prostate, testis, and ovary tissues, whereas no expression was
detected in peripheral blood leukocytes
(Fig 4). This suggested that PMX1 may be downregulated in mature myeloid cells. Previous reports have
shown ubiquitous NUP98 expression,23,24 and the
expression is observed throughout the myeloid differentiation of murine
32D cells (Nakamura et al, unpublished data). Thus,
expression of the C-terminal region of PMX1 as part of the fusion gene,
which includes the entire homeodomain, may be driven by the
NUP98 promoter, leading to constitutive upregulation.
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| Fig 4.
Expression of PMX1 in human tissues. The gene is
highly expressed in heart, skeletal muscle, thymus, prostate, testis,
and ovary, although RNAs of heart and muscle were overloaded. No
expression was detected in peripheral blood leukocytes.
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The N-terminal domain of NUP98 modulates the transcriptional
activity of PMX1.
PMX1 encodes a homeodomain protein that acts as a
sequence-specific transcription factor.32 To assess the
transcriptional activities of both wild-type and fusion PMX1 proteins,
transient-transfection assays were performed. Wild-type PMX1,
chimeric NUP98-PMX1, and deletion constructs of both
NUP98 and PMX1 were subcloned into the pM eukaryotic
expression vector in frame with sequences encoding the GAL4 DNA binding
domain.28 Constructs and the 5 × GAL4 pGL2 reporter
plasmid, which contains GAL4 binding sequences, were cotransfected to
HeLa cells. After production of GAL4 fusion proteins was confirmed by
Western blotting (Fig 5A), luciferase
activity was determined using a luminometer. As shown in Fig 5B, the
wild-type PMX1 construct showed weak repression of the luciferase
activity. The N-terminal part of PMX1, removed by the translocation,
did not significantly modulate the luciferase activity. The N-terminal NUP98 encodes a strong transcriptional activation domain that resulted
in significant enhancement of transcriptional activity by the fusion
protein construct. Similar results were seen when the human
T-lymphocyte cell line Jurkat was transfected with the same series of
constructs (data not shown).
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| Fig 5.
The N-terminal NUP98 contains a transactivation domain.
(A) Western blot analysis of GAL4 DNA binding domain fusion proteins
that were expressed in transiently transfected HeLa cells. Fusion
proteins were detected by using a monoclonal antibody directed against
the GAL4 DNA binding domain. (B) Transfection assay of HeLa cells using
the GAL4 luciferase reporter plasmid and expression plasmids encoding
GAL4 fusion proteins (left panel). (C) Jurkat cells were cotransfected
with pMCK1-1 and pCDNA-PMX1 or pCDNA-NUP98-PMX1 expression plasmids as
described in the text. Data in (B) and (C) are expressed as the fold
difference in luciferase activity obtained with test constructs
compared with that obtained with the GAL4 DNA binding domain alone (B)
or the empty pCDNA3.1 vector (C) (right panels). The assays were
repeated three times and error bars are indicated.
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Transcriptional properties of PMX1 and NUP98-PMX1 were also assessed
using the murine MCK regulatory element, a known target of the PMX1
protein.29,37 PMX1 showed only slight activation (1.9-fold)
of luciferase activity, whereas NUP98-PMX1 showed significantly enhanced transactivation (7- to 8-fold; Fig 5C). Thus, NUP98-PMX1 fusion resulted in transactivation against the endogenous enhancer for PMX1.
| |
DISCUSSION |
We show here a novel fusion gene encoding a homeodomain protein that is
synthesized due to a t(1;11) reciprocal chromosomal translocation. This
is the fourth example of chimeric NUP98 gene in leukemia. To
date, this is the only case of a NUP98-PMX1 gene fusion;
however, there are similarities between the predicted NUP98-PMX1
protein structure and the NUP98-HOXA9 and NUP98-HOXD1338 fusion. Our data showed that the addition of the NUP98 N-terminal domain significantly altered the transcriptional activity of PMX1. In
addition, upregulation of PMX1 homeodomain, under the control of the
ubiquitously expressed NUP98 promoter, may be relevant. The
functional alterations of PMX1 activity might result in deregulation of
target genes in bone marrow cells. Recently, Kasper et al39 also reported potent transactivation of the NUP98-HOXA9 fusion protein.
PMX1 was first identified as a cofactor of a serum response
factor.32 PMX1 has sequence similarity to the
drosophila paired protein; however, it contains a homeodomain
as a unique DNA binding domain and lacks a paired domain.32
The gene is mainly expressed in the embryonic mesoderm and in adult
heart and skeletal muscle36 and binds to the muscle
creatine kinase gene enhancer element.37 Mice homozygous
for a mutant PMX1 allele show defects in the formation and
growth of chondrogenic and osteogenic precursors and defective skeletogenesis.40 However, the function of PMX1 in the
hematopoietic system and leukemogenesis remains unknown. Further study
is required to clarify the physiological role of PMX1 in regulating
myeloid differentiation and proliferation.
The present t(1;11) case of AML belongs to the M2 subtype that was also
found in t(7;11) AML cases.22,23 Although the homeodomain sequences are not highly similar between PMX1 and HOXA9, the
similarities of the disease phenotype and structures of the fusion
proteins suggest that PMX1 functions in a manner equivalent to that of HOXA9 in human AML. However, half of the inv(11) cases
were myelodysplastic syndrome.24 This phenotypic difference
might be ascribed to functional distinctions between DDX10 RNA helicase
and homeodomain proteins. NUP98-PMX1 fusion was not detected in 27 human acute leukemia samples, including AML subclasses M1 to M5, and
B-cell and T-cell leukemias by RT-PCR (data not shown). The result is not very surprising, because genetic abnormalities of acute leukemias are very heterogeneous, with even common mutations seen in less than
1% of all the leukemia cases.8 The t(1;11) case, 2 of 4 inv(11) patients,24 and the t(2;11) case38 were
secondary leukemias and received chemotherapy, including topo II
inhibitors against the primary neoplasms. There is growing evidence
that cancer chemotherapies that include topo II inhibitors are one of
the major causes of human AML.41 Although the nucleotide sequences of genomic DNA at the breakpoints remain unknown, it might be
speculated that topo II-sensitive sequences exist around the breakpoint
of NUP98. The identical NUP98 N-terminal domain seen in the
NUP98-HOXA9 fusion22,23 was contained in the chimeric NUP98-PMX1 protein, suggesting that this domain may play a critical role in leukemogenesis.
The importance of the NUP98 N-terminal domain to leukemogenesis is also
suggested by other examples of nucleoporin involvement in human AML.
NUP214/CAN is fused to DEK in t(6;9)(p23;q34) AML42 and to
SET in a case of acute undifferentiated leukemia with an apparently
normal karyotype.43 In both NUP214/CAN-DEK and
NUP214/CAN-SET fusion, the C-terminal domain of NUP214, which contains
multiple FG repeat-rich docking sites, is retained in the fusion
products. Both NUP98 and NUP214 are components of the nuclear pore
complex.35,44 When proteins with nuclear localization
signals (NLS) are imported to the nucleus, they bind karyopherin  and  . After the binding of the karyopherin heterodimers to the FXFG
motif of nucleoporin docking sites and release caused by GTP bound form
of Ran protein, the NLS-proteins are transferred into the
nucleus.45 This suggests that the docking site may
facilitate the interaction of homeodomain proteins with their partners.
The human homologue of yeast CRM1 protein, which interacts with NUP214,
DEK-NUP214, and SET-NUP214, has been recently identified.46
Although it is currently unknown whether hCRM1 can interact with
homeodomain proteins such as PMX1 and HOXA9, specific cofactors of
these homeodomain proteins may be recruited by the NUP98 docking domain.
| |
ACKNOWLEDGMENT |
The authors thank Neal G. Copeland and Yuriko Saiki for critical
comments, Cory Abate-Shen for 5 × GAL4-pGL2 plasmid, Eric N. Olson for the murine MCK enhancer fragment, and Ryoko Iwata for
technical assistance.
| |
FOOTNOTES |
Submitted November 6, 1998; accepted March 2, 1999.
Supported in part by a Grant-in-Aid for Scientific Research from the
Ministry of Education of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Takuro Nakamura, MD, PhD, PRESTO, JST, The
Cancer Institute, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan; e-mail: takuro-ind{at}umin.u-tokyo.ac.jp.
| |
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ABSTRACT
INTRODUCTION