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<title>The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells</title>
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Ajore et al. BMC Molecular Biology 2010, 11:38
http://www.biomedcentral.com/1471-2199/11/38
Open Access
RESEARCH ARTICLE
The leukemia associated ETO nuclear repressor
gene is regulated by the GATA-1 transcription
factor in erythroid/megakaryocytic cells
Research article
Ram Ajore1, Rakesh Singh Dhanda*2, Urban Gullberg1 and Inge Olsson1
Abstract
Background: The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also
containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein
1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of
morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been
examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding
sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.
Results: A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong
ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/
megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp
revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636
consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced
activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic
cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the
GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the
conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a
role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells.
Conclusions: We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal
promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/
megakaryocytic differentiation govern ETO expression.
Background
The human ETO co-repressor family comprises the
homologous nuclear proteins ETO (Eight-Twenty-One),
MTG16 (Myeloid Translocation Gene on chromosome
16) and MTGR1 (Myeloid translocation Gene-Related
protein1) evolutionary related to the Drosophila protein
Nervy [1]. The ETO homologues do not interact directly
with DNA but are recruited by transcription factors such
as PLZF, BCL6, TAL1/SCL, Gfi1and Heb [2-7] to become
partners of multi-protein complexes on a gene promoter
* Correspondence: Rakesh.Singh@med.lu.se
2
Protista Biotechnology AB, IDEON, Ole Römers väg 12, SE 223 70 Lund,
Sweden
Full list of author information is available at the end of the article
[8,9]. The ETO homologues of the complexes recruit
nuclear co-repressors such as N-CoR, [9-11] SIN3
[9,10,12] and SMRT [8,13], which in turn interact with
histone deacetylase (HDAC) compelling transcriptional
repression.
Importantly, ETO homologue genes are commonly
involved in reciprocal chromosomal translocation (t)
characteristic of acute leukemia. For example, the ETO
gene becomes fused to the AML1 (Runx1) transcription
factor gene by t(8;21) resulting in the biosynthesis of the
AML1-ETO fusion protein [14,15]. Similarly, the MTG16
gene becomes fused to the AML1 gene by t(16;21) resulting in the production of the AML1-MTG16 fusion protein [16]. The oncogenic fusion proteins interfere with
© 2010 Ajore et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Ajore et al. BMC Molecular Biology 2010, 11:38
http://www.biomedcentral.com/1471-2199/11/38
hematopoietic gene regulation by transcriptional repression mediated by ETO and MTG16, respectively. Corepressors-HDAC recruited by the ETO portion of
AML1-ETO diminishes chromatin accessibility leading to
transcriptional repression at AML1 targets [8-10], contributing to the cellular differentiation block of the leukemic cells.
Gene expression involves both trans-acting factors
such as transcription factors and cis-acting elements such
as promoter, enhancer and silencer regions whose accessibility to the trans-acting factors is governed by the chromatin packing. The mechanisms for transcriptional on/
off switching of ETO homologue genes have not been
examined. ETO and MTG16 show distinct cell-type-specific expression suggesting differences in gene regulation.
ETO is present in many normal tissues with the highest
transcript level detected in brain and heart [17,18].
MTG16 is expressed for example in hematopoietic tissues, placenta and pancreas [18]. Furthermore, the ETO
homologues are differently expressed during hematopoietic differentiation; ETO is transiently expressed during
erythropoiesis, MTG16 is expressed in progenitor cells
and downregulated during myeloid and erythroid differentiation and MTGR1 is ubiquitously expressed, further
suggesting differences in gene regulation among the ETO
homologues [19]. Results from gene targeting reveals
involvement in hematopoietic development of MTG16
[20] but not of ETO [21] or MTGR1 [22]. The leukemogenic fusion protein AML1-ETO promotes self renewal
of primary erythroid cells [23] concomitant with an
AML-ETO-induced block of erythroid lineage commitment. This block correlates to blockade of p300/CBP
coactivation complex-mediated acetylation of the erythroid regulatory transcription factor GATA-1 [24]. As
ETO is expressed in human erythroid cells [19] it may be
affected by AML1-ETO.
The restriction of hematopoietic expression of ETO to
erythroid cells [19] suggests an involvement in lineagespecific gene regulation. In order to study lineage-specificity it is essential to identify structural and functional
promoter elements upstream of the coding sequences of
the ETO gene. As erythroblasts and megakaryocytes
derive from a common bipotent erythroid/megakaryocyte progenitor [25] studies were done in both cell types.
Our results show a critical role for an evolutionary conserved GATA binding site in transcriptional regulation of
the ETO gene in cells of erythroid/megakaryocytic potential.
Results
Homologous non-coding ETO sequence in human, mouse
and rat
In order to identify the location of the proximal ETO promoter, the transcription start site was identified by use of
Page 2 of 15
5'-rapid amplification of cDNA-ends (RACE) with
mRNA extracted from HEL cells. The amplified cDNA
was cloned, sequenced and aligned to genomic DNA (Fig.
1A). A complete sequence match with an upstream
region of the ETO transcript variant-3 (NCBI Ref. Seq:
NM_175635.1) was observed and the transcription start
site was identified at -318 bp (translational start codon at
+1). Orthologous genes may be subject to similar regulatory mechanisms in conserved regions of different species [26]. A search for homologies within the gene
upstream of the transcription start revealed a region at 659 to -432 bp that was highly conserved between
human, mouse and rat (Fig. 1B). This region may carry
important cis-acting regulatory elements. Examination
by bioinformatics analysis revealed potential ETS1 binding sites (5'-TTTCCT-3') at -705 and -661; GATA consensus transcription factor-binding sites at -651 (5'CCATCT-3'), -636 (5'-TGATA-3'), and -619 (5'TGATGC-3') and a CAAT binding site at -633 (5'TATTG-3') (Fig. 1B).
Functional promoter upstream of the ETO coding region
with erythroid/megakaryocytic specificity
The factors regulating the ETO gene expression have to
our knowledge not been determined. Therefore, we
aimed at identifying major regulatory cis- acting regions
and trans- acting factors regulating human ETO expression in hematopoietic cells. To examine whether the
sequence upstream of the transcription start of the ETO
gene is transcriptionally active, we cloned an -1820 to 259 bp region (translational start codon at +1), which was
inserted upstream of the luciferase reporter gene in promoterless pGL3/Basic vector, creating the plasmid pGL3/
-1820-259. We transfected plasmids into hematopoietic
cell lines and determined luciferase activity. Transcriptional activity was normalized to pGL3/SV40-promoter;
promoterless pGL3/Basic served as negative control.
Renilla vector was used as internal control for transfection efficiency. pGL3/-1820 to -259 showed an approximately 3-fold increased reporter signal compared to
pGL3/SV40-promoter both in erythroid (HEL) and
megakaryocytic (MEG-01) cell lines (Fig. 2, top) suggesting the presence of strong cis-regulatory elements in this
particular region.
In order to identify the functionally important regulatory DNA sequences, sequential deletions were made
from the 5'end of the -1820 to -259 bp region. The deletions were inserted upstream of the luciferase reporter
gene in promoterless pGL3/Basic thus generating pGL3 1326-259, pGL3 -839-259, pGL3 -729-259, pGL3 -579259, and pGL3 -429-259 reporter constructs, which were
transfected into erythroid HEL and megakaryocytic
MEG-01 cell lines. The region between -729 and -259 bp
was found to retain the transcriptional activity (Fig. 2).
Ajore et al. BMC Molecular Biology 2010, 11:38
http://www.biomedcentral.com/1471-2199/11/38
Page 3 of 15
A
Transcription
start site
GATAAGACCAGGAGAAGTGAAGATGTAACATGTTATCTGTCGCTCCTCTTAGCTGGCGGAGAGAATTTACA
-318 GATAAGACCAGGAGAAGTGAAGATGTAACATGTTATCTGTCGCTCCTCTTAGCTGGCGGAGAGAATTTACA
TTTAAAGATTAGCAGAGTGAGAAAGAGAAATCTGCCTTTTGTTGTGTGGGGTGAGGAGGAGGCATCTACCC
-247 TTTAAAGATTAGCAGAGTGAGAAAGAGAAATCTGCCTTTTGTTGTGTGGGGTGAGGAGGAGGCATCTACCC
CTGGCCTTGACGCTATCTCCCATCACCTCTGCTATCCAGACAGGACTCACCGAGGTGAGAAT---------176 CTGGCCTTGACGCTATCTCCCATCACCTCTGCTATCCAGACAGGACTCACCGAGGTGAGAATACCGGAGGG
-----------------------------------------------------------------------105 CCTTATCTTTAATTGGGTTTAGTTTTGCCAGTCTGAATAGGTTTAAAGAGACTCGATAAAGGGGGAACAAT
------------------------------------------ cDNA
- 34 AGATTATTTATTGACTGGACGCTGAAGCCTTTAGATGAAGAA Genome
+1
B
ETS1 - 705
ETS1 - 661
-729 TGTCTTCTCTCTCTCTCTCCCACTTTTCCTCCCTCTCCCGCTCCGTCTCACACGCACCCTCTGTTTATTTT
GATA - 651
CAAT - 633
GATA - 636
GATA - 619
-658 CCTGCCTCCATCTGGGCCCTGCTGATATTGTAATCACCCTGATGCACGTTGGCTTCTCTCCTCTCCCTCCT
-587 GCGCTCACACACTCACTCACACACAATGTGCCATCCTGACAAGGCTTTTACTTCTGATAAGCTCCAATGTG
-516 TGTTTAATGAATACAAAGCCGCGGTCTGGGTGCCGCCTCGGCCGCGGCCGCTCTCCCGCGCTCCTTTGCCA
-445 GAAGGTAATCTCCGTGAACAGGGGAGGGAGGCGAGCAGGGAGGAAGGAGGGGTGGCCAGGAGGAAGGGGGG
-374 CGTGGGGAGGCGGCTTTTCTCTCTCCCTCTCTCCCTTTCCAAATGATTCAGAAGTCGATAAGACCAGGAGA
-303 AGTGAAGATGTAACATGTTATCTGTCGCTCCTCTTAGCTGGCGGAGAGAATTTACATTTAAAGATTAGCAG
-232 AGTGAGAAAGAGAAATCTGCCTTTTGTTGTGTGGGGTGAGGAGGAGGCATCTACCCCTGGCCTTGACGCTA
-161 TCTCCCATCACCTCTGCTATCCAGACAGGACTCACCGAGGTGAGAATACCGGAGGGCCTTATCTTTAATTG
TATA - box
- 90 GGTTTAGTTTTGCCAGTCTGAATAGGTTTAAAGAGACTCGATAAAGGGGGAACAATAGATTATTTATTGAC
- 19 TGGACGCTGAAGCCTTTAGATGAAGA
+1
Figure 1 The sequence of the 5'flanking region of ETO. (A) Alignment with genomic DNA of ETO cDNA from HEL cells amplified by RLM-RACE.
Nucleotide +1 indicates the translational start site (ATG). The transcription start site is shown to be at -318 bp. (B) The sequence of the 5'flanking region
of the ETO promoter region was amplified by PCR with genomic DNA as template. Putative consensus binding sites for transcription factors identified
with MatInspector http://www.genomatix.de/matinspector.html are marked. Alignment of the human sequence with mouse and rat genomic sequences showed a region at -659 to -432 bp (underlined) to be highly conserved suggesting the presence of a proximal promoter.
Ajore et al. BMC Molecular Biology 2010, 11:38
http://www.biomedcentral.com/1471-2199/11/38
Page 4 of 15
-259
– 1820
-259
– 1326
-259
– 839
-259
– 729
– 579
– 429
pGL3/Promoter
pGL3/Basic
-259
-259
-259
-259
Luc
Luc
Luc
Luc
Luc
Luc
Luc
Luc
Figure 2 Effects of 5'deletions on ETO promoter activity in erythroleukemia HEL and megakaryocytic MEG-01 cells. The following reporter
constructs were examined: pGL3 -1820-259 (-1820), pGL3 -1326-259 (-1326), pGL3 -839-259 (-839), pGL3 -729-259 (-729), pGL3 -579-259 (-579), and
pGL3 -429-259 (-429) after transfection into erythroid (HEL) and megakaryocytic (MEG-01) cell lines. Nucleotide +1 indicates the translational start site
(ATG) and nucleotides 5'and 3' thereof have a "-" and "+" designation. The promoterless pGL3/basic and the pGL3/SV40-promoter are used as negative
and positive control, respectively. Firefly and Renilla luciferase (internal standard) activities were assayed 24 h post-transfection. The luciferase activity
is normalized against pGL3/SV40-promoter activity. The transcriptional activity of the full-length promoter was retained by the -729 to -259 bp region,
which is therefore likely to contain the proximal ETO promoter. The -579 to -259/-429 to -171 bp regions showed lack of transcriptional activity. Firefly
was normalized to Renilla luciferase as internal control for transfection efficiency and the results are given as adjusted Relative Luciferase Units (AdjRLU). Bars represent the mean of results from 3 to 5 separate transfections and the error bars show SEM.
The -579 to -259 and the -429 to -259 bp regions obtained
by further deletions showed no transcriptional activity
and may not play a significant role in ETO gene expression in HEL or in MEG-01 cells. Hence, the -729 to -259
bp region represents the smallest fragment generated
herein that retained full transcriptional activity. Thus, the
results from both phylogenetic footprinting and deletional analyses reveal a region, which is likely to contain
the proximal ETO promoter.
The likely proximal ETO promoter region (-729 to -259
bp) was investigated for cell specificity. The pGL3 -729259 reporter plasmid gave a strong signal in erythroid
(HEL) and megakaryocytic (MEG-01) cell lines and a low
signal in promyelocytic HL-60, myelomonocytic U-937
and monkey kidney COS-7 cells (Fig. 3). As shown by
results from real-time PCR, ETO transcripts were
detected in the erythroid/megakaryocytic cell lines but
not in the myeloid cell lines U-937 and HL-60 or in COS7 cells (Fig. 3). Thus, even if a limited number of cell lines
were investigated a robust correlation is suggested
between transfected promoter activity and endogeneous
ETO gene activity. This result suggests cell-type-specific
activation of the ETO promoter. However, transfected
promoter activity did not correlate directly with the ETO
mRNA levels detected with real-time PCR. For example,
MEG-01 cells showed higher luciferase activities than
HEL cells but lower transcript levels (Fig. 3). The relationship between promoter activity and mRNA activity is
affected by the endogeneous environment. Gene regulation involves more than promoter activity. Differences in
repressor elements could explain the lack of correlation
between promoter activity and mRNA.
Ajore et al. BMC Molecular Biology 2010, 11:38
http://www.biomedcentral.com/1471-2199/11/38
HEL
Page 5 of 15
MEG-01
U-937
HL-60
COS-7
Luc ife ras e a c tiv ity (Adj RLU)
12
10
8
6
4
2
0
Re la tiv e mRNA lev e l
1,4
1,2
1
0,8
0,6
0,4
0,2
0
Figure 3 Relative activity of the proximal ETO promoter (-729 to -259 bp) reporter expressed in various cell lines. The pGL3/basic and pGL3/
SV40-promoter are used as negative and positive control, respectively. The luciferase activity is normalized against pGL3/promoter activity. The ETO 729 to -259 bp region reporter shows strong activity in the erythroleukemia HEL and in the megakaryocytic MEG-01 cell lines. The myelomonocytic
U-937, the promyelocytic HL-60 and the monkey kidney COS-7 cell lines show only low luciferase activity upon expression of ETO -729 to -259 bp region. ETO transcripts detected by real-time PCR were found in cell lines showing increased luciferase expression upon transfection of the ETO -729 to
-259 bp region. Lack of ETO expression in COS-7 cells was shown before [54]. Firefly was normalized to Renilla luciferase as internal control for transfection efficiency and the results are given as adjusted Relative Luciferase Units (AdjRLU). Luciferase results are shown for 3 to 5 separate transfections;
bars represent the mean and the error bars show SEM. Real-time PCR results are from two experiments in triplicate samples. Relative luciferase unit
(RLU) represents experimental value for luciferase activity.
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Page 6 of 15
Mutagenesis of the GATA -636 consensus binding site
represses and mutagenesis of the ETS1 -705 binding site
increases transactivation of the ETO promoter in HEL/MEG01 cells
As mentioned above several potential transcription factor
binding sites were detected on the conserved region of
the promoter (Fig 1). ETS and GATA factors play a role in
erythroid differentiation and CAAT-binding sites are
often involved in promoter regulation. Therefore, we
choose to determine whether the identified potential
transcription factor binding sites of the conserved region
contribute to transactivation of the promoter each element was disrupted by site-directed mutagenesis (Fig. 4).
Disruption of the GATA -636 binding site led to a 4-fold
reduction in reporter gene activity in HEL/MEG-01 cells
relative to intact ETO promoter. Conversely, mutation of
the ETS1 -705 binding site (outside the evolutionary conserved region) increased the luciferase signal twice. Disruption of the GATA -651, GATA -619, ETS1 -661, and
the CAAT -633 binding sites did not significantly affect
the reporter signal. CAAT -633 was mutated because
CAAT plays a role in promoter regulation. The results
suggest that the GATA -636 site may have a role in activation and that the ETS1 -705 sites may have a role in
repression of the ETO gene activity in cells with erythroid/megakaryocytic potential.
GATA-1 binds to consensus sites in the ETO promoter in
vitro and in vivo of HEL/MEG-01 cells but not in in vitro in
G1E cells
Electrophoretic Mobility Shift Assays (EMSA) and antibody supershift assays were used to examine interactions
of the putative GATA binding sequences (GATA -651,
GATA -636 and GATA -619) using nuclear extracts from
HEL or MEG-01 cells. Biotin-labeled probes, which
include the various GATA binding sequences were used
for EMSA. Binding of proteins from nuclear extracts to
biotinylated probe that includes the GATA -636 sequence
was indicated by gel shift (Fig. 5). Specificity of the shift
was shown by lack of binding to a probe with mutations
within the core consensus sequence and by inhibition of
binding of biotinylated probe by excess unlabeled probe.
Proteins bound to the GATA -636 probe were "supershifted" by antibody to GATA-1 but not by antibody to
GATA-2 indicating binding of GATA-1 to the consensus
site (Fig. 5). GATA -619 and -651 probes also showed a
shift that was competed for by excess unlabeled probe
indicating specific binding of nuclear extract protein (Fig.
ETS1 ETS1 GATA GATA CAATA GATA
-619
-705 -661 -651 -636 -633
pGL3/Basic
Luc
HEL
MEG-01
Luc
*
*
Luc
Luc
*
*
*
Luc
Luc
Luc
Luc
(-729)
(-259)
0
0,5
1
1,5
2
2,5
Luciferase activity (Adj RLU)
Figure 4 Mutagenesis of consensus transcription factor binding sequences in the 5'ETO. Erythroid HEL and megakaryocytic MEG-01 cells were
transfected with the -729 to -259 bp ETO promoter reporter construct with or without mutations of transcription factor binding sites as indicated (X).
The mutations are described in "Materials and Methods". The pGL3/basic and pGL3/SV40-promoter are used as negative and positive control, respectively. Firefly and Renilla luciferase (internal standard) activities were assayed 24 h post-transfection. The luciferase activity of mutated ETO promoter
is normalized against luciferase activity of wildtype ETO promoter. Mutation of the GATA -636 site strongly represses transactivation of the ETO promoter reporter. Mutation of the ETS1 -705 binding site increased the luciferase signal twice. Firefly was normalized to Renilla luciferase as internal control for transfection efficiency and the results are given as adjusted Relative Luciferase Units (AdjRLU). Bars represent the mean of results from 3 to 5
separate transfections and the error bars show SEM. **, p < 0.01; ***, p < 0.0001
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Page 7 of 15
Figure 5 Detection of DNA-protein interactions using electrophoretic mobility shift/supershift assays in vitro of consensus GATA binding
sequences in the 5'promoter of ETO and nuclear extracts from HEL/MEG-01/G1E cells. Sequences for oligonucleotide probes of core consensus
and mutated GATA -651, GATA -636 and GATA-619 sites are shown. Arrows marked shift demonstrate primary DNA-nuclear protein interactions; arrows
marked supershift demonstrate DNA-nuclear protein-antibody interactions. Results for HEL cells (A-C) are to the left, results for MEG-01 cells (D-F) to
the right and results for the G1E (G) is at bottom. For the GATA -636 probe a shift is shown in HEL cells (A2) that is competed for by excess unlabelled
probe (competitor) (A3-A5) indicating binding of nuclear extract protein to the biotinylated probe that contains the GATA -636 sequence. In support
of this no binding was observed to a probe that contains a mutated GATA -636 sequence (A9). Proteins bound to the GATA -636 probe were "supershifted" by antibody to GATA-1 (A7) but not with antibody to GATA-2 (A6) indicating specificity of the DNA-protein interaction. Similar results are
shown for the GATA -636 probe in MEG-01 cells (D). For GATA -619 and -651 probes a shift is shown (B, C, E, F; lane 2) that is competed for by excess
unlabeled probe (competitor) indicating binding of nuclear extract protein to the biotinylated probe that contains GATA -619 or -651 sequences. A
supershift is shown with antibody to GATA-1 (B, C, E, F; lane 4) but not with anti-GATA-2 (B, C, E, F; lane 5). To try to distinguish between the binding
of GATA-1 and GATA-2 to the ETO promoter EMSA was performed with nuclear extract of G1E cells, which lack GATA-1. MEG-01 nuclear extract was
used as positive control (G2). No binding of GATA-2 protein to the consensus ETO promoter was observed (G3) suggesting lack of GATA-2 interaction.
These experiments were repeated twice.
Ajore et al. BMC Molecular Biology 2010, 11:38
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5). Furthermore, proteins bound to the GATA -651 and
GATA -619 elements were "super-shifted" by antibody to
GATA-1 but not by antibody to GATA-2 (Fig. 5). This
indicates that GATA-1 can bind to all three GATA consensus sites within the conserved region of the ETO promoter.
The lack of GATA-2 binding to the -636 probe could
result from competition from GATA-1. To determine
whether GATA-2 binding can be competitively inhibited
by GATA-1, EMSA was performed with nuclear extract
of the G1E cell line, which is GATA-1- (null) but GATA-2+
[27]. No primary interaction of GATA-2 with probe that
included the GATA -636 sequence was seen (Fig 5G, Lane
3). In lack of primary GATA-2 protein-DNA interaction
no supershift was observed with anti-GATA-2 (not
shown). The results argue against strong binding of
GATA-2 to the -636 site, and therefore do not support
competition from GATA-1 for GATA-2 binding,
although this can not be entirely ruled out for the experiments with MEG-01/HEL cells.
An additional band besides the one labeled "shift" is
present in the EMSA experiments of Fig. 5. It is still present with the GATA mutant oligo (Fig 5A, lane 9) indicating that it is non-specific, but the band is lost by
supershift (Fig 5A, lane 7), suggesting that it contains
GATA-1 protein. It may represent a non-specific GATA-1
interaction.
Chromatin immunoprecipitation (ChIP) assays were
used to examine in vivo binding of GATA-1 and GATA-2
to the putative ETO gene promoter. ChIP assays were
performed using chromatin isolated from HEL or MEG01 cells and antibodies towards GATA-1 or GATA-2. The
presence of GATA-1 and GATA-2 in HEL and MEG-01
cells was confirmed by Western blotting (Fig. 6). The precipitated DNA was examined by PCR amplification of the
ETO promoter fragment using gene specific oligonucleotides. By using primers specific for the evolutionary
conserved region of the ETO promoter, PCR products of
90 bp were generated from the anti-GATA-2- and antiGATA-1-immunoprecipitated chromatin of MEG-01
cells (Fig 6, top, lanes 6 & 11). ETO promoter amplification with anti-GATA-2 was also obtained in HEL cells
(Fig 6, bottom, lane 6). ChIP with anti-GATA-2 and
amplification of control region with primers B was negative in both MEG-01 (top, lane 7) and HEL cells (bottom,
lane 7) and amplification with control primers B was also
negative with anti-GATA-1 in both HEL and MEG-01
cells (not shown). ChIP controls with anti-actin or without antibody were negative (both panels, lane 2-5), while
positive PCR controls with genomic DNA were positive
(both panels, lanes 8-9. Thus, specific amplification was
achieved by precipitation with anti-GATA-1 or antiGATA-2 only. No amplification was seen in the absence
of antibody or in the presence of anti-actin antibody.
Page 8 of 15
In conclusion, results from EMSA/supershift assays
demonstrate GATA-1 binding in vitro to the GATA -636
binding site supported by ChIP assays demonstrating
binding in vivo of GATA-1 to the putative ETO promoter.
These results are consistent with a function of GATA-1 in
activation of the ETO promoter suggested by the results
of the mutagenesis studies depicted in Fig. 4.
Overexpression of GATA-1 stimulates the ETO promoter
GATA-1 was transiently overexpressed in HEL/MEG-01
cells to determine the effect on co-transfected ETO -729
to -259 bp promoter. The ETO promoter was stimulated
in a dose-dependent manner by GATA-1 (Fig. 7A). This
result is consistent with a role of GATA-1 in transactivation of the promoter.
Expression of AML1-ETO represses the ETO gene reporter in
HEL/MEG-01 cells
AML1-ETO was transiently expressed in HEL/MEG-01
cells to determine the effect on the co-transfected ETO 729 to -259 bp proximal promoter reporter. The ETO
promoter reporter was strongly repressed in a dosedependent manner by expression of AML-ETO (Fig. 7B)
Discussion
The goal of this work was to feature the promoter of the
ETO co-repressor gene. To this end, we identified essential cis-acting elements and trans-acting factors that govern ETO expression within hematopoietic cells. We
identified the likely proximal promoter of the ETO gene
whose expression within hematopoiesis seemed to be
restricted to erythroid/megakaryocytic cells. Examination for regulatory cis-elements of a 1.5-kb region
upstream of the transcription start site of the 5' flanking
region of the ETO gene revealed an initial 400 bp stretch
to be required for maximal ETO promoter reporter signal
when examined in erythroid/megakaryocytic cell lines,
which have endogeneous ETO expression. Conversely,
the ETO promoter gave no reporter signal when examined in hematopoietic cell lines with lack of endogeneous
ETO. Phylogenetic footprinting revealed a 196 bp region
at -659 to -462 bp (+1 indicates translational start codon)
containing cis-acting elements with GATA binding sites
required for regulation of ETO transcription.
Disruption of the GATA -636 site within the conserved
region repressed transactivation whereas disruption of
the ETS1 -705 binding site activated the ETO promoter.
Examination in vitro with EMSA revealed binding of
GATA-1 but not of GATA-2 to a probe that included
GATA -636 site sequences, the disruption of which abolished transactivation of the ETO gene. Our demonstration that EMSA from the G1E cell line, which expresses
GATA-2 but lacks GATA-1, also did not show GATA-2
binding to the -636 site, suggests that GATA-2 does not
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Page 9 of 15
ETS1 ETS1 GATA GATA CAATA GATA
-619
-705 -661 -651 -636 -633
(1929)
(-684)
A
(-595)
B
(2032)
90 bp
HEL MEG-01
MEG-01
HEL
Figure 6 Chromatin immunoprecipitation (ChIP) assay for examining interactions in vivo of consensus GATA binding sequences in the
5'promoter of ETO. The forward and reverse primers used to amplify the proximal promoter region from -684 to -595 (primers A, solid arrows) and
forward and reverse primers for a downstream region from 1929 to 2032 as control (primers B, dashed arrows) are shown. ChIP assays were carried
out as described in Methods using chromatin isolated from HEL and MEG-01 cells. PCR products were separated on a 2% gel and representative results
are shown. Top and bottom gel figures represent MEG-01 and HEL cells, respectively, except that lane 10 in top gel represents HEL cells. Lane 1, 100bp ladder; lane 2, actin antibody and primers A; lane 3, actin antibody and primers B; lane 4, no antibody and primers A; lane 5, no antibody and primers B; lane 6, GATA-2 antibody and primers A; lane 7, GATA-2 antibody and primers B; lane 8, genomic DNA and primers A; lane 9, genomic DNA and
primers B. GATA-1 precipitated chromatin amplified with primers A in HEL and MEG-01 cells is shown in lane 10 and lane 11, respectively (top gel). By
using primers specific for the evolutionary conserved region of the ETO promoter, a PCR product is generated both from the anti-GATA-1 and the antiGATA-2 immunoprecipitated chromatin. No amplification is seen in the absence of antibody or in the presence of anti-actin. The experiment was repeated twice.
***
Luciferase activity (Adj RLU)
A
3
2,5
2
1,5
1
0,5
0
pGL3/–729
0
2
4
6
10
GATA-1 Plasmid, Pg
B
0
100
500
1000
Luciferase activity (Adj RLU)
AML1-ETO , ng
1,2
1
0,8
0,6
0,4
0,2
0
pGL3/Basic
pGL3/SV40 pGL3/ –729
0
10
50
300
AML1-ETO , ng
1000
Figure 7 Effects of overexpression of GATA-1/GATA-2 or AML1-ETO on the ETO promoter reporter. (A) HEL/MEG-01 cells were co-transfected
with 15 μg ETO -729 to -259 bp promoter plasmid and 0 to 10 μg of GATA-1 plasmid. The luciferase activity is normalized against the ETO -729 to -259
promoter. The ETO promoter is activated by overexpression of GATA-1 in a dose-dependent manner. Similar results were obtained by co-transfection
of HEL cells (data not shown). (B) MEG-01 cells were co-transfected with 15 μg ETO -729 to -259 bp promoter plasmid and 0 to 1 μg of AML1-ETO
plasmid. The pGL3/basic and pGL3/SV40-promoter are used as negative and positive control, respectively. The luciferase activity is normalized against
ETO -729 to -259 bp promoter. The ETO promoter is strongly repressed in a dose-dependent manner by AML-ETO. Western blotting shows exogeneous AML1-ETO (detected with anti-MTG) and endogeneous ETO expression (detected with anti-ETO). These experiments were repeated three times
with similar results. Firefly was normalized to Renilla luciferase as internal control for transfection efficiency and the results are given as adjusted Relative Luciferase Units (AdjRLU). ***, p < 0.0001
Ajore et al. BMC Molecular Biology 2010, 11:38
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bind to this site. However, the lack of binding of GATA-2
to this probe is of uncertain significance and it is not possible to definitely distinguish between GATA-1 and
GATA-2 interactions at the ETO promoter. Examination
with ChIP assay revealed binding in vivo of GATA-1 to
elements within the conserved region of the ETO promoter. Furthermore, the promoter was stimulated by
overexpression of GATA-1. Collectively, our results demonstrate that the GATA-1 transcription factor binds to
the ETO proximal promoter and is involved in ETO gene
expression. The GATA-1 transcription factor is a master
regulator in erythroid/megakaryocytic development
[28,29].
Role of GATA-1/ETO in hematopoiesis
GATA-1 belongs to a family of GATA transcription factors, which bind to DNA sequences within the internal
GATA-motif A/T(GATA)A/G [30]. GATA-1/GATA-2
recognize similar DNA-binding motifs; their expression
profiles overlap for example in the erythroid lineage.
GATA-1-mediated ETO activation is in agreement with
GATA-1 being a critical direct repressor of several target
genes including GATA-2 [31,32], the repression of which
facilitates erythroid differentiation [31,33]. In addition to
the cis-acting GATA elements, a putative ETS1 binding
element was also identified within the conserved region
of the ETO proximal promoter and shown to mediate
suppressor activity. Many members of the ETS family for
example PU.1, Fli1 and ETS1 are known to play an important role in megakaryocytic and erythroid differentiation
[34].
The GATA family of transcription factors contains
important regulators of gene expression in hematopoietic
cells [35,36]. GATA-1 is essential for the development of
early and definitive erythropoiesis/thrombopoiesis
[28,29]. GATA-1 deletion results in blocked terminal
erythroid and megakaryocytic maturation [37-39].
GATA-1 and GATA-2 are expressed reciprocally during
erythropoiesis, GATA-1 levels rise when GATA-2 levels
decrease [40]. What role does ETO have in erythroid/
megakaryocytic development and differentiation? The
regulation of the ETO promoter by GATA-1 suggests a
role of ETO-mediated gene suppression at a phase of
erythropoiesis/thrombopoiesis when GATA-1 is up
[41,42]. GATA-1 is expressed at high levels during terminal maturation of erythroid/megakaryocytic cells [43].
Thus, ETO-mediated gene suppressor action may have a
role during terminal erythroid/megakaryocytic maturation as a result of GATA-1-mediated ETO transactivation.
GATA-1 has a role in erythroid/megakaryocytic cell
proliferation and differentiation [44] by activating erythroid-specific genes [33] or megakaryocyte-specific genes
[38,39,45] and repressing genes associated with prolifera-
Page 11 of 15
tion [31,33,46,47]. The expression level of one member of
the ETO homologues, murine MTG16 (ETO2) has
already been shown to regulate expansion of erythroid
progenitors [3]. Likewise, ETO2 expression in megakaryocytic cells is restricted to immature megakaryocytes
and restrains their differentiation [48]. Therefore, ETO2
is suggested to repress inappropriate early expression of
terminal megakaryocyte genes by binding to GATA-1
[48]. We have observed that MTG16 decreases during
early in vitro-induced human erythropoiesis whereas
ETO is increased transiently during the peak of erythropoiesis [19]. Therefore, it is possible that ETO, in contrast
to MTG16 (ETO2), has a role in repressing genes associated with self renewal and proliferation and that GATA1-activation of the ETO gene might be viewed in this context.
ETO homologue functions
The ETO homologues are expressed in hematopoietic
cells in a more or less cell-type-specific manner [19]. This
is supported by the present work, which indicates differences in promoter regulation among the ETO homologues as a possible explanation for lineage-specific
expression. We find that the ETO promoter is regulated
by cis-acting elements contained within an evolutionary
conserved region, which is lacking in the 5' flanking
region of both MTG16 and MTGR1 (in silico, data not
shown). The 5' flanking region of MTGR1 contains an
evolutionary conserved region lacking in the two other
ETO homologues (in silico, data nor shown). The celltype-specific hematopoietic expression of ETO is much
tighter than that of MTG16 and MTGR1 suggesting specific ETO functions. However, even though their genes
are differently regulated, the ETO homologues could have
redundant functions if they are expressed in the same
cell-type-specific context.
Suppression of ETO promoter by AML1-ETO
The AML1-ETO fusion protein, which is a gene product
of the (8;21) chromosomal translocation of acute leukemia [14,15], binds the promoter region of many genes
mostly causing transcriptional suppression [49]. However, some genes regulated by AML1-ETO do not show
binding of the fusion protein to the promoter, the transactivation of which is instead affected indirectly [49,50].
The AML1-ETO-mediated suppression of the ETO promoter observed is unlikely to be due to a direct competition for AML1-binding sites, which are not detectable on
the promoter. Nevertheless, our observation may be relevant to the reported AML-ETO-induced block of erythroid development [23,24]. If ETO is normally involved in
repressing genes associated with self renewal and proliferation, suppression of the ETO gene by AML1-ETO
Ajore et al. BMC Molecular Biology 2010, 11:38
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could facilitate the AML1-ETO-induced block of erythroid lineage commitment.
Conclusion
In conclusion, we report that the GATA-1 transcription
factor binds to the ETO proximal promoter and transactivates the gene in cells of erythroid/megakaryocytic
potential in a cell-type-specific manner. The same transacting factors that are essential in ETO expression are
essential in erythroid/megakaryocytic differentiation.
Methods
Cell culture
The human myelomonocytic U-937, human erythrolukemic HEL, megakaryocytic MEG-01, and promyelocytic
HL-60 cell lines were maintained in RPMI-1640 medium
supplemented with 10% Fetal Bovine Serum (FBS) (Gibco
BRL, Life Technologies, Rockville, MD, USA). Monkey
kidney COS-7 cells were maintained in DMEM medium
with 10% FBS supplemented with high glucose (4.5 g/l)
and L-glutamine. The G1E erythroid cell line, derived
from murine embryonic stem cells [27], was maintained
in IMDM, Pen/Step 20 ml/L, monothioglycerol 12 μl/L,
FCS 20%, human erythropoietin 2U/ml, Stem Cell Factor
50 ng/ml. GIE cells express GATA-2 mRNA at a high
level relative to the expression in wild-type proerythroblasts or erythroleukemia cells but are GATA-1-(null)
[27].
5'-rapid amplification of cDNA-ends (RACE)
The mRNA was prepared from HEL cells using Oligotex
Direct mRNA mini Kit (Qiagen, Hilden, Germany). The
5'-end (transcription start) of the mRNA was identified
by the first choice RNA Ligase mediated (RLM)-RACE
kit (Ambion Inc., TX, USA). Nested PCR of the RACE
reaction was performed with adapter primer 5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3'
and nested gene specific primer 5'-ACTGGTTCTTGGAGCTCCTTGAGTAGT-3'. RACE- products were
cloned into pGEM-T Easy Vector system (Promega Corporation, WI, USA) and sequenced.
Amplification of ETO promoter region
A 5' flanking region of 2049 bp from -2307 to -259 bp was
amplified from human genomic DNA by PCR. The forward and reverse primers used were 5'-TGGAGAAATATTACCTTGTTCTCGTCTCAG-3' and 5'-ACACAA
CAAAAGGCAGATTTCTCTTTCTCAC-3',
respectively. Regions corresponding to -1820 to -259, -1326 to 259 and -839 to -259 bp were amplified from the 2049 bp
fragment by nested PCR with forward primers 5'TAGCTCGGTACCACTTTCTGGCCCCATCC-3' (KpnI
restriction site highlighted in bold),5'- AGCAGGTACCTAAGAGTCACCTGTGGCTAC-3' (KpnI restriction
Page 12 of 15
site highlighted in bold), 5'-ACAGCAGCTAGCGCTACAGTGCACACCATG-3' (Nhe I restriction site highlighted in bold) and a common reverse primer 5'-TC
CGCCAGATCTGAGGAGCGACAGATAAC-3' (BglII
restriction site highlighted in bold). Sequential 5' deletions of the -1820 to -259 bp promoter region were generated by PCR from the cloned genomic DNA as template
to generate -729 to -259, the -579 to -259 and the -429 to
-259 bp regions. Forward primers were 5'-AACAGGTACCGGAGGCGAGCAGGGAGG-3', 5'-ACACGGTA
CCACACACAATGTGCCATCCTG-3' and 5'-TGT CTG
GTACCTCTCTCTCCCACTTTTCC-3'
with
KpnI
restricton sites highlighted in bold. The common reverse
primer is the same as used for amplification of the -2049
to -259 bp region. All sequences were verified.
Site-directed mutagenesis of transcription factor-binding
sites
Oligonucleotide primers including desired mutations
were synthesized and used in two-step spliced overhang
extension PCR. The following potential transcription factor binding sites were mutated: ETS1 sites at positions 705 and -661, 5'- TCC-3' changed to 5'-GAA-3'; GATA
site at position -651, 5'-ATC-3'changed to 5'-GCT-3';
GATA site at position -636 , 5'-GAT-3' changed to 5'ACC-3'; CAAT site at position -633, 5'-ATT-3' changed
to 5'-GCC-3'; GATA site at position -619, 5'-GAT-3'
changed to 5'-AGC-3'. After subcloning into promoterless pGL3/Basic reporter plasmid, the mutations were
verified by sequencing.
Luciferase reporter assays
PCR products were cloned into promoterless pGL3/Basic
reporter plasmid employing firefly luciferase as specific
reporter to generate pGL3 -1820-259, pGL3 -1326-259,
pGL3 -839-259, pGL3 -729-259, pGL3 -579-259, and
pGL3 -429-259 reporter constructs. The mutants were
cloned into the same reporter plasmid. Transient transfections of hematopoietic cell lines were performed by
electroporation as previously described by Lennartsson
et al [51]. The pGL3/SV40-promoter vector served as
positive control and promoterless pGL3/Basic vector as
negative control. Renilla luciferase was used as internal
control for transfection efficiency. Thirtyfive μg pGL3
DNA were used for HL-60 target cells and 15 μg for U937, HEL or MEG-01 target cells. At 24 h after transfection cells were disintegrated in 200 μl lysis buffer. Twenty
μl triplicate lysate samples were used for luciferase assays
with the Dual luciferase reporter assay kit (Promega Corporation, WI, USA). Onehundred μl each of firefly and
Renilla substrates were added. Light emission was quantified using standard procedures (Run Promega Protocol,
DLR-0-INJ) on the GLOMAX 20/20 Luminometer. Firefly was normalized to Renilla luciferase as internal con-
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trol for transfection efficiency and the results are given as
adjusted Relative Luciferase Units (AdjRLU). Three to
five independent transfections were performed in each
case.
Electrophoretic Mobility Shift Assay (EMSA)
Three potential GATA sites for positions -651, -636 and 619 were examined by electrophoretic mobility shift assay
(EMSA). The probe sequences were biotin-5-TTCCTGCCTCCATCTGGGCCCTG-3', biotin-5'-GGGCCCTGCTGATATTGTAATCA-3' and biotin-5'-TAATCA
CCCTGATGCACGTTGGC-3', respectively. Nuclear
extracts from HEL and MEG-01 cells were prepared as
described by Andrews and Faller [52]. Three to four μg of
nuclear extract were incubated with biotin-labeled probe
for 20 min at room temperature with LightShift EMSA
optimization kit reagents (Pierce, IL, USA, cat.no.
20148X) as per manufacturer's instruction. Two to four μl
polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA,
USA), monoclonal anti-GATA-2 (Santa Cruz Biotechnology Inc., CA, USA, sc-9008) or polyclonal anti-CD63
(Santa Cruz Biotechnology Inc., CA, USA, sc-7080) antibodies were added to the reaction mixtures and incubated for 15 min at room temperature. A 20 μl binding
reaction mixture contained 1x binding buffer, 2.5% glycerol, 5 mM MgCl2, 50 ng/ul poly(dI.dC), 0.05% NP-40,
and 20 fmol biotin leveled probe. The samples were separated on a 6% DNA retardation gel (Invitrogen, UK) in
0.5% TBE buffer at 90V followed by semi-dry blotting to
0.45 mm Biodyne B pre-cut modified nylon membranes
(Pierce, IL, USA) for 30 min at 20V. Immediately after
blotting, DNA was cross-linked to the membrane in the
GS gene linker UV chamber (Bio-Rad, CA, USA) for 55
sec (120 mJ/cm2). The membrane was processed as per
manufacturer's instruction and the chemiluminescence
was determined on Hyperfilm ECL (Amersham Pharmacia, UK).
Chromatin Immunoprecipitation (ChIP) assay
ChIP was performed by use of an IP assay kit (Millipore,
MA, USA). Chromatin was prepared from 106 HEL/
MEG-01 cells and cross-linked with 1% formaldehyde at
37°C for 10 min. Cells were washed in ice-cold PBS lacking Ca2+ & Mg2+ and supplemented with protease inhibitor (Roche Applied Science, IN, USA). The cell pellet was
resuspended in 200 μl SDS lysis buffer supplemented
with protease inhibitor and incubated on ice for 10 min.
Sonication was performed on ice for 3-4 sets of 10 sec
pulses at 40% amplitude using UP 50 Ultraschallprozessor (LabVision, GmbH) at an interval of 2 min. To
reduce non-specific background, sonicated samples were
pre-cleared with salmon sperm DNA/protein A agarose
slurry. For IP, 4 μl of polyclonal anti-GATA-1 (Active
Motif, Carlsbad, CA, USA) or 0.8 mg monoclonal anti-
Page 13 of 15
GATA-2 antibodies (Santa Cruz Biotechnology Inc., CA,
USA, sc-9008) were added followed by rotation overnight
at 4°C. Then, 60 μl salmon sperm DNA/protein A agarose
slurry was added and incubated for one h at 4° C with
rotation. Agarose-immunoprecipitate was collected by
centrifugation and washed as per manufacturer's instruction. Histone complex was eluted with 250 μl freshly prepared elution buffer (1% SDS, 0.1M NaHCO3). Histone
DNA crosslinks were reversed at 65° C for 4 h in 5M NaCl
followed by digestion with proteinase K. DNA was
extracted with phenol-chloroform-isoamylalcohol. The
recovered DNA was used in duplicate PCR reactions performed on each immunoprecipitated template. Forward
and reverse primers for GATA sites were 5'-TCTCACACGCACCCTCTGTTTATTTTCCTGC-3' and 5'- AG
AGGAGAGAAGCCAACGTGCATCAGGGTG-3'. Control forward and reverse primers were: 5'- TCTGCTCCAATATGAATATTGAACTACTTC-3' and 5'- TTGTT
TTTAAATAACCCACTCACATTAACA-3'. Three different chromatin preparations were used for each IP.
Quantitative real-time PCR
Real-time PCR was performed as described previously
[19]. Based on the Ct values of the samples, transcript
levels were calculated from a standard curve. Relative
quantification based on the ΔCt method [53] was used.
Normalization: ΔCt = Ct (sample) - Ct (HEL cells of corresponding dilution concentraction). Relative quantification = 2-ΔCt. Relative mRNA level presented is relatively
quantified, subtracted Ct value of HEL cells from the
samples Ct value of the corresponding dilution concentration.
Immunoprecipitation (IP) and Western blotting
IP and Western blotting were performed as described
previously [18]. The following antibodies were used:
polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA,
USA), polyclonal anti-GATA-2 (R&D Systems, MN,
USA), polyclonal anti-ETO specifically reactive with ETO
[54], and polyclonal anti-MTG reactive with all ETO
homologues and AML1-ETO [54].
Bioinformatics
Sequences of cDNA were analyzed using the NCBI Blast
program http://www.ncbi.nlm.nih.gov/BLAST/. Conserved regions were searched by multiple alignment of
genomic
sequences
using
ClustalW
http://
www.ebi.ac.uk/Tools/clustalw2/index.html.
Potential
transcription factor binding sites were identified with
MatInspector
http://www.genomatix.de//matinspector.html and the Jaspar database (Jaspar.genereg.net).
Statistical analysis
The statistical significance between two samples was
determined by student's t-test.
Ajore et al. BMC Molecular Biology 2010, 11:38
http://www.biomedcentral.com/1471-2199/11/38
Authors' contributions
RA carried out most of the experiments, analyzed data and was involved in
drafting the manuscript. RSD initiated the project, carried out some experiments, supervised experimental design/data analysis and was involved in
drafting the manuscript. UG supervised experimental design/data analysis and
was involved in drafting the manuscript. IO supervised experimental design/
data analysis and was involved in drafting the manuscript. All critically revised
and approved the final manuscript.
Acknowledgements
This work was supported by the Swedish Cancer Foundation and the Swedish
Childhood Cancer Foundation. We are grateful to Ann-Maj Persson for kind
help and expert guidance.
Author Details
of Hematology, C14, BMC, S-221 84 Lund, Sweden and 2Protista
Biotechnology AB, IDEON, Ole Römers väg 12, SE 223 70 Lund, Sweden
1Department
Received: 5 March 2010 Accepted: 20 May 2010
Published: 20 May 2010
© 2010 Ajore available 2010, 11:38 Central Ltd. the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an OpenBiology from: http://www.biomedcentral.com/1471-2199/11/38
BMC article is et Access article distributed under
Molecular al; licensee BioMed
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