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Molecular and Cellular Biology, September 2001, p. 5935-5945, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.5935-5945.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The E2A-HLF Oncoprotein Activates
Groucho-Related Genes and Suppresses
Runx1
Jinjun
Dang,1
Takeshi
Inukai,1,
Hidemitsu
Kurosawa,1
Kumiko
Goi,1
Toshiya
Inaba,2
Noel T.
Lenny,3
James R.
Downing,3
Stefano
Stifani,4 and
A.
Thomas
Look1,5,*
Departments of Experimental
Oncology1 and
Pathology,3 St. Jude Children's
Research Hospital, Memphis, Tennessee 38105; Department of
Molecular Biology and Hematology, Jichi Medical School, Tochigi 329-04, Japan2; Montreal Neurological Institute,
McGill University, Montreal, Quebec H3A 2B4,
Canada4; and Pediatric Oncology
Department, Dana-Farber Cancer Institute, and Harvard Medical School,
Boston, Massachusetts 021155
Received 10 April 2001/Accepted 23 May 2001
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ABSTRACT |
The E2A-HLF fusion gene, formed by the
t(17;19)(q22;p13) chromosomal translocation in leukemic pro-B cells,
encodes a chimeric transcription factor consisting of the
transactivation domain of E2A linked to the bZIP DNA-binding and
protein dimerization domain of hepatic leukemia factor (HLF). This
oncoprotein blocks apoptosis induced by growth factor deprivation or
irradiation, but the mechanism for this effect remains unclear. We
therefore performed representational difference analysis (RDA) to
identify downstream genetic targets of E2A-HLF, using a murine FL5.12
pro-B cell line that had been stably transfected with E2A-HLF cDNA
under the control of a zinc-regulated metallothionein promoter. Two RDA
clones, designated RDA1 and RDA3, were differentially upregulated in
E2A-HLF-positive cells after zinc induction. The corresponding cDNAs
encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are
related to the Drosophila protein Groucho, a
transcriptional corepressor that lacks DNA-binding activity on its own
but can act in concert with other proteins to regulate embryologic
development of the fly. Expression of both Grg2 and Grg6 was
upregulated 10- to 50-fold by E2A-HLF. Immunoblot analysis detected
increased amounts of two additional Groucho-related proteins, Grg1 and
Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a
disabled DNA-binding region also mediated pro-B cell survival and
activated Groucho-related genes. Among the transcription factors known
to interact with Groucho-related protein, only RUNX1 was appreciably downregulated by E2A-HLF. Our results identify a highly conserved family of transcriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogenesis.
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INTRODUCTION |
Transcription factor genes that
regulate blood cell development are the most frequent targets of
chromosomal translocations in the acute leukemias (31,
45). The chimeric or dysregulated proteins resulting from these
rearrangements contribute to leukemogenesis, most likely by aberrantly
controlling the expression of downstream genes critical for the growth,
differentiation, or survival of hematopoietic progenitors. The
E2A gene, a member of the basic helix-loop-helix (bHLH)
family, encodes two alternatively spliced bHLH transcription factors,
E12 and E47, which act synergistically to promote B-lymphocyte
maturation (2, 3, 53, 54). E2A is targeted by
two translocations in the human leukemias. The t(1;19) generates the
E2A-PBX1 chimera, consisting of the AD1 and AD2 transactivation domains
of E2A fused to the homeobox DNA-binding domain of PBX1 (26,
38), while the t(17;19) links the same two domains of E2A with
the bZIP DNA-binding and protein dimerization domain of hepatic
leukemia factor (HLF) (17, 21).
To elucidate the mechanism by which E2A-HLF subverts normal
developmental pathways, we programmed t(17;19)-positive leukemia cells
to express a dominant-negative suppressor of the oncoprotein (20). When E2A-HLF function was blocked, the transformed
cells died by apoptosis, suggesting that the oncoprotein affects cell survival rather than cell growth. This interpretation was substantiated by additional experiments in which E2A-HLF reversed both apoptosis by
interleukin-3 (IL-3) deprivation and p53 activation in murine pro-B
lymphocytes (20). To determine the structural motifs that contribute to the antiapoptotic activity of E2A-HLF, we constructed a
panel of E2A-HLF mutants and programmed their expression in IL-3-dependent murine pro-B cells, using a zinc-inducible vector (22). Neither the E12 nor the E47 product of the
E2A gene nor the wild-type HLF protein was able to protect
the cells from apoptosis induced by IL-3 deprivation. Surprisingly,
different combinations of disabling mutations within the HLF bZIP
domain had little effect on the antiapoptotic property of the chimeric
protein, so long as the amino-terminal portion of E2A was left intact.
In the context of an intact HLF bZIP domain, the AD1 but not the AD2
transactivation domain was required for antiapoptotic activity.
However, in constructs with a defective bZIP domain, either
transactivating region (AD1 or AD2) could promote cell survival after
growth factor deprivation. Thus, the block of apoptosis imposed by
E2A-HLF in pro-B lymphocytes depends critically on the transactivating
regions of E2A. Our findings suggest dual mechanisms of E2A-HLF action,
one in which the AD1 and bZIP domains act cooperatively to block
apoptosis and another in which protein-protein interactions with the
amino-terminal region of E2A act to block the expression of genes that
normally control the apoptotic machinery of pro-B cells
(22).
In this study, we used representational difference analysis (RDA) to
identify genes that are differentially regulated in response to
enforced expression of E2A-HLF in murine FL5.12 pro-B cells. Two such
genes, designated Grg2 and Grg6, are related to
the Drosophila gene Groucho, which encodes a
protein that acts as a transcriptional corepressor, interacting with
specific subsets of DNA-binding transcription factors. Immunoblot
analysis identified two other Groucho-related proteins, Grg1 and Grg4,
whose genes were coordinately upregulated with Grg2 and Grg6. Among the
transcription factors known to interact with the Groucho family of
corepressors, only RUNX1 was specifically downregulated by E2A-HLF.
Thus, Groucho-related proteins and the RUNX1 transcription factor are
likely candidates to participate as downstream effectors in
E2A-HLF-mediated oncogenesis.
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MATERIALS AND METHODS |
Cell culture and cell survival assay.
IL-3-dependent FL5.12
murine pro-B cells were cultured in RPMI 1640 medium supplemented with
10% fetal calf serum and 10% WEHI-3B-conditioned medium (as a source
of IL-3). The cell number was maintained below 106/ml to
avoid IL-3-independent growth. Cell lines expressing E2A-HLF or E2A-HLF
mutants from the pMT-CB6+ eukaryotic expression vector (a gift from F. Rauscher III, Wistar Institute, Philadelphia, Pa.) have been described
previously (22).
For cell survival assays, protein expression was induced by treating
cells with 100 µM ZnSO4 for 16 h prior to growth
factor deprivation. IL-3 was removed by repeated centrifugation in
fresh medium lacking the cytokine, and the cells were adjusted to
5 × 105 per ml on day 0 and cultured without IL-3.
Viable cell counts were determined in triplicate by trypan blue dye exclusion.
RDA.
RDA was performed according to the method of Hubank and
Schatz (16) with additional modifications.
Poly(A)+ RNAs were isolated from FL5.12 cells harboring the
E2A-HLF construct and grown in the presence or absence of 100 µM
ZnSO4 by use of the FastTrack 2.0 mRNA isolation system
(Invitrogen). Double-stranded cDNAs were synthesized with the
Superscript cDNA synthesis kit (GIBCO-BRL) according to the
manufacturer's protocol. Double-stranded cDNA (2 µg) isolated from
both cells grown in the presence of zinc and cells deprived of zinc was
digested with DpnII, ligated with adapter, and amplified by
PCR (16, 28). RDA fragments were isolated after three
cycles of subtractive hybridization and selective PCR amplification
with different adapters. The third difference products were digested
with DpnII and cloned into the BamHI site of the
pBluescript phagemid (Stratagene). Double-stranded plasmid DNA was
prepared and sequenced, and findings were compared with the GenBank
database by use of the BLAST program.
Screening of cDNA library.
The full-length cDNAs of RDA1 and
RDA3 (designated Grg2 and Grg6, respectively) were isolated by
screening a cDNA library that was constructed with mRNA isolated from
E2A-HLF-expressing Baf-3 mouse pro-B cells. The pBK-CMV phagemid was
purified from positive clones by in vivo excision from the lambda ZAP
II vector, using ExAssist helper phage (Stratagene). The GenBank
accession numbers for Grg2 and Grg6 are AF145958
and AF145957, respectively.
Northern blot analysis.
Two micrograms of mRNA or 10 µg of
total RNA was subjected to denaturing agarose electrophoresis in 1 M
formaldehyde and transferred to a nylon membrane (Stratagene). The
blots were then hybridized with cDNA probes labeled with
[32P]dCTP. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as a control in quantifying the levels of mRNA
expression. After hybridization, the blots were analyzed with a
PhosphorImager (Molecular Dynamics).
Protein expression.
Expression plasmids for human
TLE1 and TLE2 and mouse Grg6 were
generated by subcloning the full-length coding sequences of these cDNA
inserts into the pMT-CB6+ eukaryotic expression vector, which provides
inducible gene expression under control of the sheep metallothionein
promoter. This vector also contains the neomycin resistance gene
(neo) driven by the simian virus 40 (SV40) promoter. A Kozak
sequence was placed in front of ATG in these cDNA inserts.
Antibodies and Western blot analysis.
Rabbit polyclonal
antibodies were raised against the N terminus (3 to 68 amino acids;
Bio-80) and middle portion (186 to 272 amino acids; Bio-81) of Grg6. A
rabbit polyclonal antibody against HLF was made as previously described
(23). The pan-transducin-like enhancer-of-split (TLE)
monoclonal antibody C597.4A and rabbit polyclonal antibodies against
amino acids unique to each human TLE protein (TLE1, TLE2, TLE3, and
TLE4) were prepared as indicated previously (18, 50). A
rabbit polyclonal antibody against Runx1 (AML1/RHD; Ab-2) was obtained
from Calbiochem, and a goat polyclonal antibody against actin (C-11)
was obtained from Santa Cruz Biotechnology.
Cells were lysed in NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM
Tris [pH 8.0]), and total cellular proteins were separated
by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
After
electrotransfer onto nitrocellulose membranes, the blots
were stained
either with specific TLE antibodies (1:2,000 dilution)
and anti-Grg6
(1:400 dilution) followed by horseradish peroxidase-conjugated
anti-rabbit immunoglobulin G (IgG) or with a pan-TLE antibody
(1:400
dilution) followed by an anti-rat IgG. The bands were visualized
by
enhanced chemiluminescence according to the manufacturer's
instructions
(Amersham).
RNase protection analysis.
An RNase protection assay (RPA)
was performed with the In Vitro Transcription/Ribonuclease Protection
Assay Kit (PharMingen) according to the manufacturer's instructions.
In brief, total RNA (20 µg) isolated from FL5.12 cells was hybridized
with [32P]UTP-labeled antisense RNA probes
(~107 cpm per reaction mixture) at 56°C for 16 h,
then heated at 90°C for 3 min, and digested with RNase. The templates
from Grg6 and Runx1 for RPA were prepared by
inserting a 199-bp PCR fragment of Grg6 cDNA (bp 152 to 350)
and a 310-bp Runx1 fragment (bp 822 to 1131) into the pcDNA3
vector in antisense orientations relative to the T7 polymerase
transcription origin. The 95-bp mouse Gapdh cDNA fragment
(PharMingen) was used as an internal control.
RT-PCR.
Total RNA was extracted from cells using the Trizol
reagent (LTI, Inc.), by following the manufacturer's directions.
Reverse transcription was performed on 2 µg of total RNA. RNA was
resuspended in a final volume of 11 µl with 50 ng of random hexamer
(Pharmacia Biotech), incubated at 80°C for 5 min, and cooled on ice.
Reverse transcription was performed using 200 U of SuperScriptII
reverse transcriptase (GIBCO-BRL) in the manufacturer's buffer in the presence of 0.5 mM deoxynucleoside triphosphates (dNTP)-1.0 mM dithiothreitol in a final volume of 20 µl, which was incubated at
42°C for 1 h. After the reaction, each sample was added to 1 µl of RNase H (GIBCO-BRL) and incubated at 37°C for 20 min. For the
negative control, the reaction was also performed on each sample
without reverse transcriptase (RT). cDNA samples were stored at
20°C. PCRs were performed using a DNA Thermal cycler
(Perkin-Elmer). PCR mixtures contained 1 µl of cDNA, 1× reaction
buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 10 pmol of each primer,
and 2.5 U of Taq polymerase (Perkin-Elmer) in a total volume
of 50 µl. The thermocycling parameters and the sequences of the
specific primers for PCR are as follows: for TLE1, 30 cycles at 94°C
for 30 s, 50°C for 30 s, and 72°C for 1 min with sense
primer 5'-AATCGCCAAGAGATTGAATA-3' and antisense primer
5'-GTGCCTCTTAGACTGTCGG-3'; for TLE2, 40 cycles at 94°C for
30 s, 52°C for 30 s, and 72°C for 1 min with sense primer 5'-AGCAGAGAGCAGATGAGAAG-3' and antisense primer
5'-GAGAGGTCTCCGTTGAGAGT-3'; for TLE3, 30 cycles at 94°C
for 30 s, 52°C for 30 s, and 72°C for 1 min with sense
primer 5'-TACGACAGTGATGGAGACAA-3' and antisense primer
5'-CATTATACCTATCGGGTCCA-3'; for TLE4, 30 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min with sense primer 5'-GTTTGTAAGCACTGGAAAGG-3' and antisense primer
5'-AAAAAGGATGACAGAGCAAA-3'; for AML-1, 40 cycles at 94°C
for 30 s, 54°C for 30 s, and 72°C for 1 min with sense
primer 5'-AGACATCGGCAGAAACTAGA-3' and antisense primer
5'-CCAGGTATTGGTAGGACTGA-3'; for c-ABL, 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min with sense primer 5'-GTATCATCTGACTTTGAGCC-3' and antisense primer
5'-GTACAGGAGTGTTTCTCCA-3'. Amplification of c-ABL mRNA was
performed to assess the quality of RNA extraction. These primer sets
amplify a 342-bp product of TLE1, a 501-bp product of TLE2, a 501-bp
product of TLE3, a 501-bp product of TLE4, a 253-bp product of AML-1,
and a 288-bp product of c-ABL. Ten microliters of the PCR products was
electrophoresed in a 2.0% agarose gel and transferred onto a nylon
membrane (NEN Life Science) with 0.4 N NaOH. Membranes were hybridized
with an internal probe end labeled with 32P using
polynucleotide kinase, and autoradiography was performed. The sequences
of the internal probes were as follows: for TLE1, 5'-ACCCTCCCTGACCTGTCTCG-3'; for TLE2,
5'-CTCATTGTGGCCCTTATGTA-3'; for TLE3,
5'-GAGAAATTGACTGCTCGAGT-3'; for TLE4,
5'-TAACTAAAGGTGAAAAGCTCC-3'; for AML-1,
5'-AACCCTCAGCCTCAGCGTCA-3'; and for c-ABL,
5'-TAACTAAAGGTGAAAAGCTCC-3'.
Nucleotide sequence accession numbers.
The GenBank accession
numbers for Grg2 and Grg6 are AF145958 and
AF145957, respectively.
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RESULTS |
Identification of genes that are upregulated by E2A-HLF.
Overexpression of E2A-HLF protects pro-B cells from death induced by
growth factor deprivation or irradiation (20). We have previously shown that IL-3-dependent murine pro-B cells (FL5.12 or
BAF-3 cell lines) can survive in the absence of the cytokine when
E2A-HLF expression is controlled by a metallothionein-regulated vector
(20, 22). To identify downstream genes targeted by the
E2A-HLF oncoprotein, we performed RDA on the mRNAs of FL5.12 pro-B
cells shortly after removal of IL-3 from the culture medium. The
so-called "tester" E2A-HLF+ mRNAs were extracted from
cells that had been incubated in the presence of both IL-3 and 100 µM
ZnSO4 for 18 h to induce E2A-HLF expression and
maintained in the same concentration of zinc but deprived of IL-3 for
an additional 12 h. The control or "driver" mRNAs were
extracted from the same cell clone after identical treatment, except
that E2A-HLF expression was not induced by the addition of zinc to the medium.
After three rounds of sequential hybridization and PCR amplification
according to the RDA protocol of Hubank and Schatz (
16),
we isolated three clones (RDA1, RDA3, and RDA9) that were
differentially
expressed in E2A-HLF-positive cells compared with
controls. RDA1
contains a 239-bp cDNA fragment that hybridized with an
mRNA of
approximately 3 kb (Fig.
1). This
transcript was expressed at
high levels in FL5.12 cells induced to
express E2A-HLF, whether
or not IL-3 was present in the culture medium
(Fig.
1A, lanes
6 and 8). The same cells did not express detectable
levels of
the RDA1 mRNA in the absence of zinc (Fig.
1A, lanes 5 and
7).
The 300-bp RDA3 cDNA fragment hybridized with an mRNA of
approximately
2 kb in E2A-HLF-transfected FL5.12 cells (Fig.
1). RDA3
mRNA was
also undetectable in control FL5.12 cells but was strikingly
upregulated
after addition of zinc in the presence or absence of IL-3
(Fig.
1A, lanes 6 and 8). A 454-bp RDA9 cDNA fragment also hybridized
with a 2-kb transcript (data not shown). Subsequent analysis of
the
complete sequence of the cDNA clones identified by these probes
showed
that both RDA3 and RDA9 are represented in the 3' and middle
portions
of the same mRNA.
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FIG. 1.
Upregulation of expression of the Grg2 (RDA1) and Grg6
(RDA3) genes induced by E2A-HLF. (A) Northern blot analysis of
poly(A)+ RNA (1 µg per lane) prepared from FL5.12 cells
stably transfected with the pMT empty vector (lanes 1 to 4) or with
metallothionein promoter-regulated constructs containing cDNAs for
E2A-HLF (lanes 5 to 8), the  AD1 E2A-HLF mutant lacking the AD1
transactivation domain of E2A (lanes 9 and 10), the BX E2A-HLF mutant
with a disabled HLF DNA-binding domain (lanes 11 and 12), E2A (E12
[lanes 13 and 14] and E47 [lanes 15 and 16]), HLF (lanes 17 and
18), or TEF (lanes 19 and 20). Cells were cultured in IL-3-containing
medium with (+) or without () 100 µM ZnSO4 for 18 h to induce expression of the transfected cDNA and then continued in
the same concentration of zinc and either maintained in IL-3 (+) or
deprived of the cytokine () for an additional 12 h before RNA
extraction. The blot was hybridized with the Grg2 (RDA1), Grg6 (RDA3),
or GAPDH cDNA probe as indicated. The mobilities of the 18S and 28S
rRNAs are shown. (B) Immunoblot analysis of FL5.12 cells transfected
with E2A-HLF, E12, E47, HLF, or TEF protein. To verify that expression
of the indicated genes was upregulated by zinc addition, lysates were
prepared from FL5.12 cells and analyzed by immunoblotting with E2A
rabbit antisera ( E2A) (top) and HLF(C) rabbit antisera (HLF)
(bottom). Cells were stably transfected with the pMT empty vector
(pMT-CB6+) or with metallothionein promoter-regulated constructs
containing cDNAs for E2A-HLF, E2A (E12 and E47), HLF, or TEF, with or
without the addition of zinc as described for panel A.
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There was no accumulation of RDA1 or RDA3 mRNAs in cells transfected
with cDNAs encoding the intact E2A protein E12 or E47,
wild-type HLF,
or the HLF-related TEF, a bZIP transcription factor
(Fig.
1A, lanes 13 to 20), indicating that the genes detected
by RDA were specifically
upregulated by E2A-HLF. To exclude the
possibility that expression of
these RDA fragments was induced
nonspecifically by zinc, we analyzed
mRNAs from cells transfected
with the empty pMT-CB6+ expression vector.
As shown in Fig.
1A,
neither RDA1 nor RDA3 mRNAs were detectable in
these cells, whether
or not zinc was added to the medium (lanes 1 to
4).
In a recent study, we found that at least one of the transactivation
domains of E2A is required for the antiapoptotic activity
of E2A-HLF,
regardless of the functional status of the HLF bZIP
domain
(
22). To relate this observation to the ability of E2A-HLF
to upregulate RDA1 and RDA3, we analyzed the mRNA levels of both
genes,
using FL5.12 cell lines that had been stably transfected
with cDNAs
encoding two mutant E2A-HLF proteins. As shown in Fig.
1A, the
AD1
mutant, which lacks the entire AD1 transactivation
domain but possesses
an intact HLF DNA-binding domain, failed
to induce expression of either
RDA1 or RDA3 (lanes 9 and 10).
By contrast, a mutant defective in
DNA-binding activity [E2A-HLF
(BX)], as a result of substitutions for
six critical amino acids
in the HLF basic region (
22),
stimulated RDA1 and RDA3 expression
as efficiently as the wild-type
protein (Fig.
1A, lanes 11 and
12). Thus, activation of these candidate
genes requires an intact
AD1 domain but not sequence-specific DNA
binding through the bZIP
domain of HLF, mirroring the ability of these
mutants to prolong
cell survival in the absence of
cytokine.
The timing of RDA3 mRNA accumulation relative to the induction of
E2A-HLF expression is shown in Fig.
2.
Low baseline levels
of E2A-HLF maintained by the metallothionein vector
increased
markedly within 2 h after zinc addition, while
expression of RDA3
mRNA was first detected 8 h after incubation of
the cells with
zinc, reaching maximal levels within 12 h. A
similar kinetic profile
was observed for RDA1 mRNA (data not shown).
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FIG. 2.
Time course of RDA3 (Grg6) mRNA expression in
response to E2A-HLF. Northern blot analysis was performed of total RNA
(10 µg per lane) isolated from FL5.12 cells stably transfected with
the metallothionein promoter-regulated E2A-HLF construct. Cells were
cultured in IL-3-containing medium for the indicated times in hours
after addition of 100 µM ZnSO4. The blot was hybridized
with the RDA3 (Grg6), E2A-HLF, or
Gapdh cDNA probe as indicated.
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Both RDA1 and RDA3 are homologous to the Drosophila
gene Groucho.
DNA sequence analysis revealed that RDA1
is closely related to the human gene TLE2, one of several
mammalian genes related to the Drosophila gene
Groucho (27, 29, 36, 50). Although murine
homologues have been identified for most of the TLE genes, none has been found for TLE2 (29). To clone the
full-length coding sequence of RDA1, we isolated multiple cDNA clones
from a library prepared from a Baf-3 murine pro-B cell line programmed to express E2A-HLF. The longest cDNA consisted of 2,749 bp, including one long open reading frame that encoded a 767-amino-acid protein (GenBank accession number AF145958) which did not correspond to any of
the previously identified murine Grg proteins (29). Because of the high degree of amino acid sequence identity (87%) between the murine protein and human TLE2, we concluded that RDA1 represents the murine homologue, Grg2 (Fig.
3). The predicted Grg2 protein contains a
23-amino-acid insertion near its amino terminus that is not shared with
TLE2, but otherwise these proteins are very highly conserved (90%
amino acid identity).
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FIG. 3.
Alignment of the Grg protein WD repeat domains. The
C-terminal amino acid sequences of the newly identified Grg2 and Grg6
proteins are shown in comparison with the previously reported mouse (m)
Grg1, -3, and -4 proteins and the human TLE2 sequences. Identical
residues are boxed. Dashes indicate gaps in the alignment. The WD
repeat domain is underlined.
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Sequence analysis of two overlapping cDNA clones hybridizing to RDA3
and RDA9 revealed a span of 2,015 bp, including a single
open reading
frame that encodes a 581-amino-acid protein with
a predicted molecular
mass of 65 kDa (GenBank accession number
AF145957). The main region of
homology with previously identified
Grg proteins was within the
C-terminal domain containing the so-called
WD40 repeats (
29,
37). Short regions showing identity with
the highly conserved
amino acids of other Grg family members were
also noted. Alignment of
this protein, which we have designated
Grg6, with the murine Grg1, -2, -3, and -4 proteins is shown in
Fig.
3.
The tissue distribution of Grg2 and Grg6 mRNAs was determined in adult
mice as well as in embryos. The 3.5-kb Grg2 transcript
was detected
predominantly in the heart, brain, and liver, with
much lower levels
found in the lung, muscle, kidney, testis, and
spleen (Fig.
4). A larger Grg2 mRNA of approximately 6 kb was
identified in the heart. The 2.2-kb Grg6 transcript was
expressed
predominantly in the heart, lung, liver, muscle, and testis
and
at very low levels in the brain, spleen, and kidney (Fig.
4).
Like
other
Groucho-related genes (
33), neither Grg2
nor Grg6
was expressed in embryos until 11 days postconception, with
both
mRNAs detected by day 15 (Fig.
4).
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FIG. 4.
Expression of the RDA1 (Grg2) and RDA3
(Grg6) genes in adult mouse tissues and embryos. Northern
blot analysis was performed of poly(A)+ RNA (2 µg per
lane) from adult mouse tissues, including the heart, brain, spleen,
lung, liver, skeletal muscle, kidney, and testis (left panel) and from
mouse embryos from days 7, 11, 15, and 17 postconception (right panel).
The blot was hybridized with the RDA1 (Grg2), RDA3
(Grg6), or human  -actin cDNA probe as indicated.
Mobilities of size standards (in kilobases) are shown.
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Upregulation of a family of Groucho-related proteins in response to
E2A-HLF expression.
Identification by RDA of two Grg
genes that are upregulated by E2A-HLF suggested that increased amounts
of other Grg family members might be detectable after enforced
expression of the oncoprotein. As shown in Fig.
5, E2A-HLF expression was apparent within
6 h after addition of zinc to FL5.12 cells bearing the fusion
construct and reached maximal levels by 24 h. To analyze the
pattern of Grg protein expression, we first used a monoclonal antibody
(C597.4A) (50) that recognizes all the human TLE proteins
and then used antibodies specific for each of these proteins. The Grg
proteins recognized by the pan-TLE antibody were expressed at low
levels in murine FL5.12 cells before induction of E2A-HLF expression, with increased expression apparent 12 h after zinc addition (Fig. 5). Maximal expression of TLE proteins occurred after 24 h and was
maintained through 48 h. The Grg1, Grg2, and Grg4 proteins showed
similar expression patterns (Fig. 5), while Grg3 was not detected at
any time (data not shown).
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FIG. 5.
Immunoblot analysis of Grg proteins induced by E2A-HLF.
Extracts were prepared from cells stably transfected with either the
pMT-CB6+ vector (left) or the metallothionein promoter-regulated
E2A-HLF construct (right) after the cells were cultured in
IL-3-containing medium for the indicated times in hours after the
addition of 100 µM ZnSO4. The blots were immunostained
with the HLF(C) rabbit antiserum, the pan-TLE rat monoclonal antibody
C597.4A, TLE1-, TLE2-, or TLE4-specific rabbit antisera, or the Grg6
(RDA3)-specific rabbit antiserum. Relative mobilities of molecular size
markers (in kilodaltons) are shown.
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Grg6 encodes a predicted 65-kDa protein that was not
recognized in FL5.12 cells by antibodies to the human TLE proteins.
However,
it was detectable by Western blot analysis with a polyclonal
antibody
to a recombinant polypeptide from the N-terminal region of the
protein (see Materials and Methods). Grg6 expression was clearly
upregulated by enforced expression of E2A-HLF, reaching maximal
levels
18 h after zinc addition (Fig.
5). In contrast to results
for the
Grg1, Grg2, and Grg3 proteins, Grg6 expression was markedly
reduced
48 h after zinc addition, despite continued expression
of E2A-HLF.
Lack of antiapoptotic activity by Grg proteins.
Since E2A-HLF
protects IL-3-dependent murine pro-B cells from apoptosis due to IL-3
deprivation (20, 22), we asked whether any of the the
Groucho-related proteins TLE1, TLE2, and Grg6 could replace this
function in the absence of the oncoprotein. We therefore transfected
FL5.12 cells with a zinc-regulated vector encoding either the murine
Grg6 protein or human TLE1 or TLE2. Independent clones isolated by G418
selection expressed each of the proteins in the presence of 100 µM
ZnSO4 at levels approximately 10-fold higher than the
background levels in medium lacking the metal (Fig.
6A). As shown in Fig. 6B, FL5.12 cells
expressing E2A-HLF survived with little loss of viability, while those
expressing TLE1, TLE2, or Grg6 rapidly underwent apoptosis after
removal of IL-3 from the growth medium. These observations indicate
that enforced expression of the individual Groucho-related proteins cannot replace the antiapoptotic function of E2A-HLF.
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FIG. 6.
Effects of E2A-HLF, TLE1, TLE2, and Grg6 on the survival
of FL5.12 cells deprived of IL-3. (A) Immunoblot analysis of three
independently derived clones of G418-resistant FL5.12 cells stably
transfected with the indicated cDNAs under the control of the
zinc-regulated metallothionein promoter, as well as controls
transfected with the empty vector pMT-CB6+ or the vector containing
E2A-HLF. Blots were stained with antisera recognizing E2A-HLF, TLE1,
TLE2, and Grg6 (see the legend to Fig. 5). (B) Survival of FL5.12 cells
expressing E2A-HLF, TLE1, TLE2, or Grg6 in the absence of IL-3. Cells
growing exponentially in IL-3-supplemented medium for 16 h in the
presence or absence of zinc (100 µM) were adjusted to 5 × 105 cells per ml on day 0, and viable cell counts were
determined at the indicated times after the removal of IL-3.
|
|
Analysis of RUNX1 (PEBP2b) and other transcription factors known
to interact with Grg proteins.
The Groucho protein and its
mammalian homologues lack DNA-binding domains and act as
transcriptional corepressors, that is, they interact with DNA-binding
transcription factors to downregulate gene expression (11,
41). Thus, we reasoned that E2A-HLF might block programmed cell
death by regulating both a subset of the Grg proteins and one or more
of the transcription factors with which these proteins interact. This
would account for the failure of individual Grg proteins to block
apoptosis in cells deprived of IL-3. In Drosophila
melanogaster, Groucho interacts with discrete families of
transcription factors, including Hairy, Runt, Engrailed, Dorsal, and
Tcf, and acts as a corepressor with widespread roles in development
(1, 5, 9, 25, 51).
In mammalian cells, Groucho-related proteins repress gene expression by
interacting with several transcription factors, including
RUNX1, LEF-1,
Tcf and a family of Hes (Hairy-enhancer-of-split)
proteins (
15,
19,
30). Thus, we examined E2A-HLF-positive
FL5.12 cells for
evidence of such transcription factors. The only
detectable candidate
was RUNX1, which was markedly downregulated
by E2A-HLF. As shown in
Fig.
7,
RUNX1 mRNA levels in
FL5.12 pro-B
cells transfected with the pMT-CB6+ vector were not
altered in
response to IL-3 deprivation, whether or not zinc was added
to
the medium (Fig.
7, lanes 1 to 4). However,
RUNX1 mRNA
levels
were strikingly decreased in E2A-HLF-transfected cells grown for
30 h in the presence of zinc (Fig.
7, lanes 6 and 8), compared
with the same cells grown in the absence of the metal. They were
unaffected by the presence or absence of IL-3 (Fig.
7, lanes 5
and 7).
We also tested the regulation of RUNX1 expression in FL5.12
cells
transfected with two E2A-HLF mutants (
AD1 and BX). The
AD1 domain of
the E2A portion was required for this inhibitory
effect, as deletion of
this region completely abolished the downregulation
of RUNX1 by
E2A-HLF. By contrast, the E2A-HLF(BX) mutant was as
potent as wild-type
E2A-HLF in mediating RUNX1 downregulation,
indicating that
sequence-specific DNA binding by E2A-HLF is not
required for this
effect of the protein, as was previously observed
for its antiapoptotic
activity and its ability to upregulate the
expression of
Groucho-related genes. Enforced expression of E12
or
wild-type HLF had little discernible effect on
RUNX1 mRNA
levels,
whereas TEF, a transcription factor that recognizes the same
DNA-binding
motif as HLF, markedly downregulated
RUNX1 mRNA
levels.
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FIG. 7.
Downregulation of expression of the RUNX1
gene induced by E2A-HLF. Northern blot analysis was performed on
poly(A)+ RNA (1 µg per lane) prepared from FL5.12 cells
expressing the indicated cDNAs under the control of a zinc-regulated
metallothionein promoter. Cells were cultured in the presence or
absence of zinc (100 µM) and IL-3 as described in the legend to Fig.
1. The blot was hybridized with the RUNX1 or
Gapdh cDNA probe as indicated. Mobilities of the 18S and 28S
rRNAs are shown.
|
|
To determine the kinetics of the downregulation of
Runx1 in
relation to the increase in the expression of
Groucho-related
genes induced by E2A-HLF, we used the RPA to
examine their mRNA
levels in E2A-HLF-expressing cells at serial times
after zinc
induction. Expression of Grg6 was induced 6 h after the
addition
of zinc (Fig.
8), with kinetics
similar to those of Grg2 (data
not shown) and the E2A-HLF protein (Fig.
5). By contrast, the
decrease in the level of
RUNX1 mRNAs
did not occur for an additional
6 h (Fig.
8), indicating that its
change was temporally delayed
compared with the increase in Groucho
expression.
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FIG. 8.
Time course of RUNX1 and Grg6 mRNA
expression in E2A-HLF-transfected FL5.12 cells. (A) Total RNA was
isolated at different time points after addition of zinc (100 µM) and
hybridized with RUNX1, Grg6, and Gapdh
antisense mRNA probes. GAPDH was used as an internal control. The
protected RNA fragments (solid arrowheads) were separated by an 8%
sequencing gel. Open arrowheads, RNA probes. (B) Immunoblot analysis of
Runx1 in E2A-HLF-transfected FL5.12 cells. Extracts were prepared from
cells stably transfected with either the pMT-CB6+ vector or the
metallothionein promoter-regulated E2A-HLF construct after the cells
were cultured in IL-3-containing medium for the indicated times in
hours after the addition of 100 µM ZnSO4. The blot was
immunoblotted with Runx1 rabbit antisera, HLF(C) rabbit antisera, and
actin goat antisera (C11), respectively.
|
|
No change in expression of other genes that encode proteins interacting
with Groucho-related proteins was detected in E2A-HLF-expressing
cells.
Most of them, such as those encoding Hes-1, Lef-1, and
Sox4, were not
detectable in FL5.12 cell RNA by RPA, while their
expression was
detected in mouse bone marrow cells, as shown in
Fig.
9. The expression of other genes, such as
Tcf-1 and
En-1,
was detected in FL5.12 cells but
was unchanged by E2A-HLF expression
(data not shown).
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FIG. 9.
RPA of Hes-1, Lef-1, and
Sox4 mRNAs. Total RNAs were isolated from FL5.12 cells
stably transfected with plasmids expressing E2A-HLF or the pMT vector,
with or without a 24-h incubation with zinc (100 µM), and from mouse
bone marrow (BM) cells and were hybridized with Hes-1,
Sox4, Lef-1, and Gapdh antisense RNA
probes. GAPDH was used as an internal control. The protected RNA
fragments (solid arrowheads) were separated by an 8% sequencing gel.
Open arrowheads, RNA probes.
|
|
Runx1 expression levels are unaffected by enforced expression of
Groucho-related proteins.
The Runx1 protein contains a WRPY motif
at its carboxyl terminus, and Groucho-related proteins are known to
interact through this motif to convert Runx1 from a transcriptional
activator into a repressor (1, 19, 30). Furthermore, the
Runx1 promoter contains its own core DNA-binding motif, and
published evidence suggests that Runx1 may in part regulate
its own expression (13). Thus, we tested whether
overexpression of Groucho-related proteins alone in FL5.12 cells was
sufficient to downregulate RUNX1 gene expression through an
autoregulatory mechanism. The expression levels of Runx1 were tested by
RPA in FL5.12 cells expressing zinc-inducible Grg proteins (see Fig. 6)
and were unchanged in TLE1-, TLE2- and Grg6-transfected FL5.12 cells
after addition of the metal (Fig. 10,
lanes 6 to 11). Similarly, equal levels of the 310-bp protected band of
Runx1 were detected in control pMT vector-transfected cells
in the presence or absence of zinc (Fig. 10, lanes 2 and 3). By
contrast, the Runx1 mRNA level in E2A-HLF-transfected cells
was significantly decreased by the oncoprotein after addition of zinc
(Fig. 10, lanes 4 and 5). As expected, the 199-bp protected
Grg6 band was detected only in E2A-HLF-positive and
Grg6-positive cells, not in TLE1- and TLE2-transfected cells. These
results suggested that expression of TLE1, TLE2, and Grg6 alone could
not substitute for E2A-HLF in mediating the repression of Runx1.
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FIG. 10.
Expression of RUNX1 and Grg6 in FL5.12 cells stably
transfected with plasmids expressing E2A-HLF, TLE1, TLE2, and RDA3.
Total RNA was isolated from cells with or without a 24-h incubation
with zinc (100 µM) and was hybridized with RUNX1,
Grg6, and Gapdh antisense RNA probes. Solid
arrowheads, protected RNA fragments; open arrowheads, RNA probes.
|
|
Expression of Groucho-related genes in early B lineage
leukemias.
To assess the expression of the human homologues of
Groucho in early B-lineage leukemia cells, we performed
RT-PCR analysis of leukemic cell lines with and without the t(17;19)
translocation. As shown in Fig. 11, all
t(17;19)-positive cell lines, including UOC-B1, YCUB-2, and HAL-01,
expressed mRNAs encoding TLE1, TLE2, and TLE4 (lanes 1 to 3). Moreover,
TLE3 was expressed in HAL-01 cells but not in the other two
t(17;19)-positive cell lines. Examples of RNAs from five other leukemia
cell lines lacking t(17;19) are also shown in Fig. 11, including the
Nalm6, REH, and 697 early B-lineage, Jurkat T-cell, and U937 monocytic
cell lines. Like t(17;19)-positive cell lines, all these
t(17;19)-negative cell lines except U937 expressed TLE1, TLE2, and
TLE4, but only Jurkat and REH cells expressed TLE3. By contrast,
expression level of RUNX1 was very low in all of the cell lines and
could not be detected under the same PCR conditions used for the
analysis of the TLE genes (data not shown). Thus, our analysis suggests
that Groucho-related genes are expressed in human leukemias
including those with the t(17;19) translocation and that the expression
level of Runx1 is very low.
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FIG. 11.
RT-PCR analysis. Each RNA was transcribed with (+) (odd
lanes) or without () (even lanes) RT. PCR products were transferred
to a nylon membrane and analyzed by hybridization with end-labeled
internal oligonucleotide probes specific for each gene. RNAs were
evaluated for the UOC-B1 (lanes 1 and 2), YCUB-2 (lanes 3 and 4), and
HAL-01 (lanes 5 and 6) t(17;19)-positive human leukemic cell lines and
for the t(17;19)-negative Nalm6 (lanes 7 and 8), REH (lanes 9 and 10),
and 697 (lanes 11 and 12) B-lineage leukemic cell lines, the
t(17;19)-negative Jurkat T-lineage leukemic cell line (lanes 13 and
14), and the t(17;19)-negative U937 myeloid cell line (lanes 15 and
16). In addition, a control PCR lacking RNA template was performed
(lanes 17 and 18).
|
|
| |
DISCUSSION |
Many chimeric and dysregulated transcription factors have been
identified in association with chromosomal translocations in the human
acute leukemias (31). In recent years, major efforts have
been made to link these genes and their mutated counterparts in
multistep pathways that regulate programs of gene expression during
development. Recent studies revealed that E2A-HLF protects IL-3-dependent pro-B cells from apoptosis induced by growth factor deprivation or irradiation, suggesting that this chimeric transcription factor may cause leukemia by interfering with apoptotic responses in
normal cells. In this study, we used RDA to identify E2A-HLF-regulated genes, including two murine homologues of the gene encoding the Groucho
developmental regulator in Drosophila, designated
Grg2 and Grg6. Analysis of other members of the
Grg family revealed that Grg1 and Grg4 were also activated by E2A-HLF.
Among transcription factors that have been shown to interact with
Groucho-related proteins, we found that RUNX1, a frequent target of
chromosomal abnormalities and inactivating point mutations in human
leukemia (8, 40, 49), was suppressed by E2A-HLF.
The Drosophila Groucho protein is a widely expressed nuclear
protein that has critical roles in many development processes (11, 41, 42). Groucho lacks a DNA-binding domain but
contains a conserved C-terminal WD repeat domain that is present in a
large number of proteins performing various cellular functions,
including transcriptional regulation and signal transduction, and is
believed to mediate protein-protein interactions (37, 48).
In Drosophila, Groucho acts as a transcriptional corepressor
to modulate gene expression through its associations with numerous
transcription factors including Hairy, Runt, engrailed, Tcf, Dorsal,
and Hkb (1, 5, 9, 14, 25, 51). These sequence-specific transcription factors recruit Groucho to the target gene promoters through different interaction domains. Hairy and Runt proteins interact
with Groucho through their C-terminal WRPW or WRPY motifs (1,
12), whereas Dorsal binds Groucho through its Rel homology domain (52) and Tcf interacts with Groucho through an HMG
domain that also mediates its interaction with CBP (46).
Once targeted to the promoter region in a heterocomplex with one of
these transcription factors, Groucho can actively repress both basal
and activated transcription (11, 25). The mechanism
underlying Groucho-mediated repression appears to be linked to the
direct recruitment of histone deacetylase (HDAC) with its associated
chromatin-modifying activities. More recently, it has been shown that
the HDAC Rpd3 activity is required for Groucho-mediated transcriptional
repression during Drosophila development (6).
In addition, NK-3, a homeodomain transcription factor, has recently
been shown to recruit both the Groucho corepressor and an HDAC complex
with HIPK2 to repress gene expression (7). Many mammalian
Groucho homologues have been identified in humans (TLE genes) and mice
(Grg) (27, 29, 32, 36, 50). These mammalian Groucho family
members are highly conserved and have similar functions in
transcriptional repression (11, 15, 37). In mammalian
cells, Groucho proteins have been shown to be involved in both Notch
and Wnt signaling pathways through interaction with Hes and Tcf
transcription factors, respectively (5, 10, 30, 35, 43,
44).
Activation of several Grg family members by E2A-HLF suggests the
possibility that they may contribute to an E2A-HLF-mediated antiapoptotic pathway of leukemogenesis. In FL5.12 cells transfected with Grg genes, however, we did not see protection from cell
death after growth factor deprivation, indicating that increased
expression levels of Groucho proteins alone were insufficient to block
apoptosis. Since Groucho acts as a corepressor in transcriptional
regulation, we considered the possibility that specific DNA-binding
transcription factors might be required to prevent apoptosis. In
Drosophila, a number of transcription factors are known to
interact with Groucho and control cell fate determination. Recently, it
has been shown that Re1/NF-
B, a mammalian homologue of Dorsal, is
involved in suppressing p53-independent apoptosis induced by Ras
(34) and that inhibition of NF-B activity induces
apoptosis (4). Jehn et al. have reported that Notch-1
protects T-cell receptor (TCR)-induced apoptosis (24),
suggesting that Hes, the downstream genes activated by Notch signaling,
may be involved in cell survival. Among these Groucho-interacting
transcription factors (including RUNX1, NF-B, Hes-1, Tcf-1, Lef-1,
and En-1), however, only the expression levels of RUNX1 were affected
in E2A-HLF-expressing cells.
RUNX1 is affected by several of the most frequent chromosomal
abnormalities associated with human leukemia (39, 40, 47). Since this transcription factor controls many genes that are important in hematopoiesis (40), downregulation of RUNX1 may play an
important role in E2A-HLF-mediated leukemogenesis. TLE1 has been
demonstrated to inhibit RUNX1-induced transactivation by interacting
with this protein (19, 30), effectively converting RUNX1
from an activator to a repressor of gene expression. For example, in
conjunction with TLE1, RUNX1 negatively regulates the activity of
TCR and TCR enhancers (30). Binding of TLE1 to RUNX1
was also found to suppress activities of the macrophage
colony-stimulating factor and neutrophil elastase promoters
(19). This binding is necessary for RUNX1-mediated
repression, because RUNX1-induced transactivation was recovered when
the interaction was abolished by deletion of the C terminus of RUNX1.
Thus, downregulation of RUNX1 by E2A-HLF may block Groucho-mediated
repression through AML pathways and provides another mechanism through
which inhibition of RUNX1 contributes to leukemia in humans
(8).
Upregulation of Grg proteins and downregulation of RUNX1 in response to
E2A-HLF emphasize the fact that this chimeric oncoprotein is able to
regulate gene expression both positively and negatively, either
directly or through intermediate pathways of transcriptional control.
Recently, we have shown that E2A-HLF-mediated prevention of apoptosis
in IL-3-dependent pro-B cells depends critically on protein-protein
interactions mediated by the intact transactivation domains of E2A
(23). Data presented here support this mechanism, showing
that E2A-HLF activation of Groucho-related genes depends on
the AD1 transactivation domain of E2A but not on the DNA-binding activity of HLF. We have detected a significant increase in
Grg6 promoter activity in E2A-HLF-transfected cells (data
not shown). Identification of E2A-HLF-responsive elements in the
promoter regions of Grg genes will be required to implicate
a direct effect of E2A-HLF in the activation of Grg genes.
Chimeric and dysregulated transcription factors, created by chromosomal
translocation in the acute leukemias, act in concert with other
mutations in multistep pathways leading to leukemogenesis. Identification of a family of Groucho-related genes and
RUNX1 as potential downstream targets of the E2A-HLF oncoprotein
provide direct evidence for its ability to profoundly alter the
transcriptional status of early B-lineage cells. Although the relevance
of Grg proteins to leukemogenesis has not been conclusively
established, RUNX1 is the gene most commonly disrupted by
chromosomal abnormalities (8) and inactivated by point
mutation in human leukemia (49). The functional analysis
of downstream genes in E2A-HLF transcriptional programs may therefore
provide useful insights into E2A-HLF-mediated survival advantages as
well as leukemogenesis. In addition, identification of the
transcription factor(s) involved in regulation of Grg and/or RUNX1
expression should be valuable for elucidating the mechanism by which
the Groucho corepressors are activated by E2A-HLF.
| |
ACKNOWLEDGMENTS |
We thank Jing Wu and Bart Jones for technical assistance. We also
thank Karen M. Rakestraw and Clayton W. Naeve of the Center for
Biotechnology for DNA sequence analysis.
This work was supported in part by NIH grants CA59571 and CA21765
(Cancer Center CORE Grant), and The American Lebanese Syrian Associated
Charities (ALSAC), St. Jude Children's Research Hospital.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Pediatric
Oncology Department, Mayer-630, Dana-Farber Cancer Institute, Boston,
MA 02115. Phone: (617) 632-5826. Fax: (617) 632-6989. E-mail:
thomas_look{at}dfci.harvard.edu.
Present address: Department of Pediatrics, Yamanashi Medical
University, Tamaho, Yamanashi 409-38, Japan.
| |
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Molecular and Cellular Biology, September 2001, p. 5935-5945, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.5935-5945.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
