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INTRODUCTION |
The 39 human and murine HOX genes encode
homeodomain transcription factors necessary for embryogenesis (1, 2)
and definitive hematopoiesis (3). Genes of the HOX A and
B paralog groups are preferentially expressed in CD34+ bone
marrow progenitor cells and are activated 3' to 5' during hematopoiesis
(3). Expression of 3' HOX A and B genes increases
in early CD34+ cells and decreases in CD34+ committed progenitors. In
contrast, transcription of the 5' genes (i.e.
HOX9-13) is invariant in CD34+ cells, and
decreases in mature phagocytes (3).
In comparison with normal, mature myeloid cells, expression of
HOXA10 is increased in acute myeloid leukemia, chronic
myeloid leukemia, or myelodysplasia (4). Consistent with this,
overexpression of HoxA10 in murine bone marrow induces a
myeloproliferative disorder, which evolves to acute leukemia (5). These
results suggest that HoxA10 is involved in progression of myeloid
differentiation. Although the most abundant HoxA10 transcript in human
myeloid cells encodes a 406-amino acid protein (predicted molecular
mass of 50 kDa) (6), alternatively spliced transcripts have been described at various stages of murine embryogenesis (7) and in
transformed cell lines (4). In myeloid leukemia cell lines, a HoxA10
transcript is present encoding a protein that initiates 20 amino acids
N-terminal to the homeodomain (6). It is hypothesized that the 80-amino
acid (15-kDa) "short A10" may contribute to immortalization of
myeloid cell lines, although the role of short A10 in normal
myelopoiesis is unknown.
Similar to the other Abd-like Hox proteins (Hox9-13), DNA binding
affinity of HoxA10 is increased by partnering with Pbx proteins (8).
Although consensus sequences for Pbx-HoxA10 binding have been derived
(8-10), genuine Pbx-HoxA10 target genes have not been identified. It
has been hypothesized that HoxA10 regulates myeloid differentiation by
activating transcription of genes that are necessary for progression of
myelopoiesis. Conversely, HoxA10 might repress transcription of genes
characteristic of differentiated myeloid cells, or HoxA10 might
activate transcription at one stage of myelopoiesis and repress
transcription at another, as has been described for homologous
Drosophila proteins, during embryogenesis (11).
We have been studying regulation of genes encoding the respiratory
burst oxidase proteins, gp91phox (the CYBB gene)
(12) and p67phox (the NCF2 gene) (13). These genes
are transcribed in cells differentiated beyond the promyelocyte stage
and therefore provide a model for gene regulation during late
myelopoiesis. Both the CYBB and NCF2 genes
contain sequences similar to the Pbx-HoxA10 binding consensus sequence.
One of these CYBB sequences is within a previously described
repressor element (14), which binds the CCAAT displacement protein
(CDP)1 in electrophoretic
mobility shift assays (EMSA) with HeLa or K562 nuclear proteins (14,
15). In NIH 3T3 cells, overexpression of CDP represses an artificial
promoter construct containing the CYBB element (16).
In contrast, our previous investigations demonstrated that CDP is
not a component of the complex binding to the CYBB repressor element in EMSA with nuclear proteins from the myeloid lines PLB985 and
U937 (17). Since HoxA10 mRNA is present in these myeloid cell
lines, but not in HeLa or K562 cells (4), our current studies
investigate the hypothesis that, in committed myeloid progenitors,
HoxA10 interacts with repressor elements and suppresses transcription
of some myeloid-specific genes until later stages of myelopoiesis.
Previously, we demonstrated that IFN-
-induced myeloid
differentiation decreases in vitro protein binding to the
CYBB repressor element, coincident with increased
CYBB transcription (17). Therefore, we also investigate the
effect of IFN--induced differentiation on HoxA10 DNA binding and
functional activity in myeloid cells.
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MATERIALS AND METHODS |
Plasmids and Site-directed Mutagenesis: Reporter Constructs and
Plasmids for Protein Expression--
Artificial promoter/reporter
constructs were generated as described previously (18), in the minimal
promoter/reporter vector, p-TATACAT (19) (obtained from Dr. A. Kraft,
University of Colorado, Denver). Constructs were generated with three
copies (in the forward orientation) of the consensus sequence for
HoxA10-Pbx binding (p-a10TATACAT) (8) or four copies (in the forward
direction) the 
94 to 134 bp sequence from the CYBB
promoter (p-cybba10TATACAT). This CYBB promoter sequence has
previously been demonstrated to function as a repressor element in
myeloid cell lines (16). An artificial promoter construct with five
copies of the Gal4 DNA binding site and a minimal promoter from the
thymidine kinase gene linked to a chloramphenicol acetyltransferase
(CAT) reporter (p-gal4TKCAT) was obtained from T. Gabig (Indiana
University, Indianapolis).
The cDNAs for human HoxA10 and "short A10" were obtained from
C. Largman (University of California, San Francisco) and subcloned in
to the mammalian expression vector pSR
(20). The human Pbx1 cDNA
and a FLAG epitope-tagged HoxA10 cDNA were obtained from M. Cleary
(Stanford University, Stanford, CA) (8) and also subcloned into the
pSR vector. The cDNAs for HoxA10 and "short A10" were also
subcloned into the vector pM2(GAL4), for expression as a fusion protein
with the DNA binding domain of GAL4 (21).
Oligonucleotides--
Oligonucleotides were synthesized by the
Core Facility of the Comprehensive Cancer Center (University of
Alabama, Birmingham) or the Riley Pediatric Research Center (Indiana
University, Indianapolis). Oligonucleotides used were as follows:
derived consensus sequence for Pbx-HoxA10 binding (dsA10),
5'-tgcgatgatttatgaccgc-3'; the similar sequence
from the CYBB promoter (94 to 134 bp) (dscybbA10) (14),
5'-ttcagttgaccaatgattattagccaattttctgataaaa-3'; a mutant sequence from the CYBB promoter (94 to 134 bp)
(dscybbmut), 5'-ttcagttgaccaatgattcggcgccaatttctgataaaa-3'; the similar sequence from the NCF2 gene (600 to 637 bp)
(dsncf2A10), 5'-aaaaggcattagtcaagagataattaattgggaaagag-3'; a
mutant sequence from the NCF2 gene (600 to 637 bp)
(dsncf2A10mut), 5'-aaaaggcattagtcaagagataatgccgtgggaaagag-3'; an
irrelevant sequence from the NCF2 gene (535 to 575 bp)
(dsncf2irf),
5'-cactctaggtcacgggtttcatttgggaccactagcctagt-3'; another
CYBB promoter sequence similar to the Pbx-HoxA10 binding consensus sequence (194 to 242 bp) (dscybb5'A10),
5'-agaaattggtttcattttccactatgtttaattgtgactggatcatta-3'; the CCAAT box from the -globin gene (urccaat) (22),
5'-ccgggctccgcgccagccaatgagcgccgcgg-3'. In these
oligonucleotides, the HoxA10 core (or mutated core) is in boldface
type, the Pbx core is in italics, and ccaat boxes are underlined.
Cell Culture--
All cell lines were of human origin. The
promyelocytic leukemia cell line PLB985 was obtained from Thomas Rado
(University of Alabama, Birmingham). The myelomonocytic cell line U937
(23) was obtained from Andrew Kraft (University of Colorado, Denver). Cell lines were maintained and differentiated as described (17, 18).
U937 cells were treated with 200 units/ml human recombinant IFN-
(Roche Molecular Biochemicals).
EMSAs--
Nuclear extract proteins were prepared by the method
of Dignam et al. (24), with protease and phosphatase
inhibitors, as described (18). Oligonucleotides probes were prepared,
and EMSA and antibody supershift assays were performed as described
(18). Antiserum to HoxA10 (not cross-reactive with other Hox proteins) was obtained from Covance Research Products (Richmond, CA). Pbx antibodies (C-20 and P-20), blocking peptides, and an antibody to CDP
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Rabbit polyclonal antiserum raised to whole CDP protein (purified from
HeLa cells) was a generous gift of Ellis Neufeld (Boston Children's
Hospital, Boston, MA).
In Vitro Translated Proteins and Tyrosine
Dephosphorylation--
In vitro transcribed HoxA10, short
A10, and Pbx1 mRNA were generated from linearized template DNA,
using the Riboprobe System, according to the manufacturer's
instructions (Promega, Madison, WI). In vitro translated
proteins were generated in rabbit reticulocyte lysate, according to the
manufacturer's instructions (Promega). Control (unprogrammed) lysates
were generated in similar reactions in the absence of input RNA.
In vitro translated proteins and nuclear proteins were
tyrosine-dephosphorylated with Yop protein-tyrosine phosphatase (New England Biolabs, Beverly, MA). Proteins (either 10 µl of in vitro translated protein or 2 µg of nuclear
proteins) were incubated 30 min at 30 °C, in a 20-µl reaction
volume with 50 units of Yop and 1× reaction buffer,
according to the manufacturer's instructions. Control proteins were
incubated, similarly, in 1× reaction buffer without
Yop.
EMSA with the in vitro translated proteins was performed as
described (18). Binding assays with in vitro translated
proteins and the dscybbA10 oligonucleotide were performed in the
presence of a 200-fold molar excess of the urccaat oligonucleotide. The urccaat oligonucleotide competes for binding of CP1, found in reticulocyte lysate, to the dscybbA10 probe.
Transient Transfection and Reporter Gene Assays--
Cells were
transfected by electroporation as described (18). U937 cells (32 × 106/sample) were transfected with 70 µg of p-TATACAT,
p-a10TATACAT, or p-cybba10TATACAT; 30 µg of pSR, HoxA10/pSR,
shortA10/pSR, or Pbx1/pSR or 15 µg each of HoxA10/pSR plus
Pbx1/pSR; and 15 µg of p-CMV
-gal (to normalize for transfection
efficiency). In other experiments, U937 cells were transfected with 20 or 2 µg of p-gal4TKCAT; 20 µg of HoxA10/pM2(GAL4),
shortA10/pM2GAL4, or control pM2GAL4; and 15 µg of
p-CMV-gal. Transfectants were incubated for 24 h at 37 °C,
5% CO2, followed by 24 h with or without IFN- (200 units/ml). Preparation of cell extracts, -galactosidase, and CAT
assays were performed as described (25, 26).
In other experiments, U937 cells were transfected with 30 µg of
pSR, HoxA10/pSR, or HoxA10(FLAG)/pSR. The cells were incubated for 48 h at 37 °C, 5% CO2 and harvested for
extraction of nuclear proteins or total cellular RNA.
Immunoprecipitation and Western Blotting--
Western blots were
performed with 30 µg of nuclear proteins extracted from U937 cells,
with or without 48 h IFN- incubation. Proteins were separated
on 12% SDS-PAGE, transferred to nitrocellulose, and probed with HoxA10
antiserum (Covance Research Products) or control rabbit pre-immune
serum, and proteins were detected by chemiluminescence, according to
the manufacturer's instructions (Amersham Pharmacia Biotech). In other
experiments, Western blots of nuclear proteins from U937 cells
transiently transfected with pSR or FLAG epitope-tagged
HoxA10/pSR were performed with anti-FLAG antibody.
To determine HoxA10 phosphorylation state, U937 cells, with or without
48 h of IFN- differentiation, were incubated for 4 h at
37 °C with 32P-orthophosphate, as described (27). Cells
were lysed, under denaturing conditions, and proteins were
immunoprecipitated for 4 h at 4 °C, with 2 µl of HoxA10
antiserum or 2 µl of control rabbit preimmune serum, followed by a
1-h incubation with 30 µl of 50% staph protein A-Sepharose bead
slurry, as described (27). Immunoprecipitated proteins were washed with
radioimmune precipitation buffer, eluted in SDS sample buffer, and
identified by autoradiography of 12% SDS-PAGE.
Immunoprecipitation experiments were also performed with 100 µg of
nuclear proteins extracted from U937 cells, with or without 48-h
IFN- incubation. Nuclear proteins were diluted into radioimmune precipitation buffer, with protease and phosphatase inhibitors, as
described (28), and incubated with either 1 µl of
anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake
Placid, NY) or irrelevant antibody (mouse anti-rabbit IgG), for 4 h at 4 °C, followed by a 1-h incubation with 15 µl of 50% staph
protein A-Sepharose bead slurry. Beads were washed with radioimmune
precipitation buffer, and proteins were eluted in SDS sample buffer,
separated on 12% SDS-PAGE, and transferred to nitrocellulose. Blots
were probed with HoxA10 antibody (Covance Research Products) or control rabbit preimmune serum.
Similar experiments were performed to determine tyrosine
phosphorylation of in vitro translated HoxA10. In
vitro translated proteins (10 µl), with or without
Yop treatment, and control lysate were diluted into
radioimmune precipitation buffer and immunoprecipitated as described
above. The proteins were detected by autoradiography of SDS-PAGE.
RNA Extraction and Northern Blotting--
Total cellular RNA was
extracted from U937 cells, as described (17), 48 h after
transfection with HoxA10/pSR, FLAG epitope-tagged HoxA10/pSR, or
control pSR. Northern blots were performed with 20 µg of total
cellular RNA, as described (17). Probes for Northern blots were
generated by random primer labeling of cDNAs encoding human
gp91phox, p67phox and chicken -actin, as described
(17).
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RESULTS |
HoxA10 DNA Binding Decreases during IFN--induced Myeloid
Differentiation--
The consensus sequence for HoxA10 binding to DNA
as a heterodimer with Pbx1 (8) consists of a Pbx1 site adjacent to a
HoxA10 site: 5'-ATGATTTATGA-3' (HoxA10 site in
boldface type, the Pbx site in italics) (9). Inspection of the promoter
regions of the genes encoding gp91phox (the CYBB
gene) and p67phox (the NCF2 gene) identified
sequences similar to the Pbx-HoxA10 consensus (Fig.
1A). The CYBB
sequence includes the core sequence preferred in HoxA10 binding site
selection experiments (TTAT) and the Pbx consensus (7). However, unlike
sequences identified by binding site selection, there was overlap of
the Pbx and Hox binding cores. The NCF2 sequence includes an
alternative HoxA10 core, identified by binding site selection (TAAT)
(7), and a Pbx consensus, altered at position 3.
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Fig. 1.
Sequences in the CYBB and
NCF2 genes, similar to the derived Pbx-HoxA10
consensus, have cross-competitive binding specificities.
A, sequence analysis identifies sequences from the
CYBB and NCF2 promoters similar to the derived
consensus for Pbx1-HoxA10 binding. The HoxA10 binding core is indicated
in boldface type, and the Pbx core is shown in
italics. Note that, in the CYBB promoter
sequence, there is a 1-bp overlap between the HoxA10 and Pbx core
sequences. B, a specific protein complex, binding to the
derived Pbx-HoxA10 binding site consensus, decreases during
IFN--induced U937 differentiation and is competed for by the
sequences from the CYBB and NCF2 genes. EMSA was
performed with the dsA10 probe and nuclear proteins from U937 cells (2 µg) without (lanes 1-5) and with
(lane 6) 48-h IFN- differentiation in the
presence of unlabeled, synthetic oligonucleotide competitor (200-fold
molar excess). Lane 1, no competitor;
lane 2, homologous dsA10 oligonucleotide;
lane 3, dscybbA10 oligonucleotide;
lane 4, dsncf2A10 oligonucleotide;
lane 5, urccaat unrelated oligonucleotide;
lane 6, no competitor. The arrowhead
indicates the A1 complex. C, binding of a
specific protein complex (complex A) to the dscybbA10 probe is
decreased during IFN--induced U937 differentiation. EMSA was
performed with the dscybbA10 probe and nuclear proteins from U937 cells
(2 µg) without (lane 1) and with
(lane 2) 48-h IFN- differentiation. The
arrowhead represents binding of specific complex A (17), and
the asterisk shows binding of protein complex, previously
demonstrated to represent the classical CCAAT binding complex, CP1
(14). D, binding of complex A to the dscybbA10 probe is
competed for by the Pbx-HoxA10 binding consensus and other similar
sequences. EMSA was performed with the dscybbA10 probe and nuclear
proteins from U937 cells (2 µg) in the presence of unlabeled,
synthetic oligonucleotide competitor (200-fold molar excess).
Lane 1, no competitor; lane
2, homologous dscybbA10 oligonucleotide; lane
3, mutant dscybbA10mut oligonucleotide; lane
4, unrelated dsncf2irf oligonucleotide;
lane 5, similar dsncf2A10 oligonucleotide;
lane 6, mutant dsncf2A10mut
oligonucleotide; lane 7, consensus dsA10
oligonucleotide; lane 8, unrelated urccaat
oligonucleotide. Complex A is indicated by an
arrowhead.
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The Pbx-HoxA10-like sequence in the CYBB promoter is within
a 30-bp region previously demonstrated to function as a repressor element in undifferentiated cells (16). In EMSA with nuclear proteins
from the promyelocytic leukemia cell line PLB985, an unidentified,
specific protein complex interacts with this CYBB repressor
element (referred to as complex A) (14, 15). We previously demonstrated
that IFN--induced differentiation of PLB985 cells abolishes in
vitro binding of complex A to the repressor element, coincident
with an increase in CYBB transcription (17).
We hypothesized that HoxA10 is a component of complex A binding the
CYBB repressor element and that myeloid differentiation decreases HoxA10 DNA binding. To pursue this hypothesis, we
investigated whether in vitro protein binding to the derived
Pbx-HoxA10 consensus sequence decreases during IFN--induced myeloid
differentiation. In these experiments, we used U937 cells, a
monocyte-committed cell line that expresses HoxA10 mRNA (4).
IFN- treatment of U937 cells results in monocyte differentiation and
increased CYBB and NCF2 transcription (17). EMSAs
were performed, using nuclear proteins from U937 cells and a
radiolabeled probe with the derived consensus sequence for Pbx-HoxA10
binding (referred to as dsA10 oligonucleotide) (Fig. 1B). A
complex binds to dsA10 that is of similar mobility to complex A
generated by U937 nuclear proteins and the homologous CYBB
sequence (the dscybbA10 oligonucleotide). Binding of this complex to
the dsA10 probe (referred to as complex A1) is competed for
by excess, unlabeled, homologous oligonucleotide and by
oligonucleotides with the similar sequences from the CYBB and NCF2 genes (the dsncf2A10 oligonucleotide) but
not by several dissimilar oligonucleotides (Fig. 1B).
Binding of both complex A1 to the dsA10 probe and of A to
the dscybbA10 probe is decreased in EMSA with nuclear proteins from IFN--treated U937 cells, in comparison with nuclear proteins from
undifferentiated cells (Fig. 1, B and C). These
results are consistent with our previous data with nuclear proteins
from PLB985 cells and the dscybbA10 probe (17). In EMSA with U937
nuclear proteins, binding of the A complex to the dscybbA10 probe was competed for by homologous oligonucleotide and the dsncf2A10 and dsA10 oligonucleotides, but not by unrelated oligonucleotides or by
dscybbA10 or dsncf2A10 oligonucleotides with mutations in the
TTAT or TAAT sequences (Fig. 1D).
However, there are differences in the complexes shifted by the dsA10
and dscybbA10 probes. Binding of complex A1 to the dsA10
probe is of lower affinity than complex A binding to the dscybbA10
probe (in terms of pmol of shifted probe/µg of nuclear proteins; Fig.
1, compare B and C). Also, the dsA10 probe binds
several higher mobility protein complexes, in addition to A1, which are competed for by homologous oligonucleotide or
dscybbA10 but not dsncf2A10 (Fig. 1B). These higher
mobility complexes may represent proteins encoded by alternatively
spliced HoxA10 transcripts (6, 7). Alternatively, these complexes may
represent proteolytic fragments of HoxA10, although U937 nuclear
proteins used in these experiments were tested for integrity in other
EMSAs with characterized binding sites.
To determine if HoxA10 is a component of either complex A1
binding to the dsA10 probe or complex A binding to the dscybbA10 probe,
we used an antibody raised to the HoxA10 C terminus that does not
recognize other Hox proteins. In preliminary experiments, we determined
that this antibody does not recognize recombinant CDP (data not shown).
In EMSA with the dsA10 probe and nuclear proteins from U937 cells,
binding of complex A1 is disrupted by HoxA10 antibody but
not preimmune serum (Fig. 2A).
Antibody to HoxA10 also disrupts binding of complex A to the dscybbA10
probe (Fig. 2B). Identical results were obtained with
nuclear proteins from PLB985 cells (not shown). In addition, HoxA10
antibody disrupts two high mobility complexes binding the dsA10 probe,
suggesting that they contain the HoxA10 C terminus, consistent with
previously described, alternatively spliced messages (6, 7).
Interestingly, the dscybbA10 probe does not bind high mobility species,
cross-immunoreactive with HoxA10.
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Fig. 2.
HoxA10 from U937 nuclear proteins interacts
in vitro with DNA probes containing the derived
Pbx-HoxA10 DNA-binding consensus or the similar CYBB
promoter sequence. A, a U937 nuclear protein,
cross-immunoreactive with HoxA10, binds in vitro to the
dsA10 probe. EMSA was performed with the dsA10 probe and nuclear
proteins from U937 cells (2 µg), preincubated with the following.
Lane 1, rabbit preimmune serum (2 µl);
lane 2, HoxA10 specific rabbit serum (2 µl).
The arrowhead indicates complex A1, and the
double arrows indicate higher mobility complexes,
also cross-immunoreactive with HoxA10. B, a U937 nuclear
protein, cross-immunoreactive with HoxA10, binds in vitro to
the dscybbA10 probe. EMSA was performed with the dscybbA10 probe and
nuclear proteins from U937 cells (2 µg), preincubated with the
following. Lane 1, rabbit preimmune serum (2 µl); lane 2, HoxA10-specific rabbit serum (2 µl). The arrowhead indicates complex A; the
asterisk indicates CP1 binding to a CCAAT box in the probe.
C, a U937 nuclear protein, cross-immunoreactive with Pbx1,
binds in vitro to the dsA10 probe. EMSA was performed with
the dsA10 probe and nuclear proteins from U937 cells (2 µg),
preincubated with the following. Lane 1, Pbx
antibody (2 µg) and blocking peptide (1 µl); lane
2, Pbx antibody alone (2 µg). The arrowhead
indicates complex A1. D, a U937 nuclear protein,
cross-immunoreactive with Pbx1, binds in vitro to the
dscybbA10 probe. EMSA was performed with the dscybbA10 probe and
nuclear proteins from U937 cells (2 µg), preincubated with the
following. Lane 1, Pbx antibody (2 µg) and
blocking peptide (1 µl); lane 2, Pbx antibody
alone (2 µg). The arrowhead indicates complex A; the
asterisk indicates CP1 binding to a CCAAT box in the
probe.
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EMSAs were also performed to determine if Pbx1 is a component of either
complex A1 (binding to dsA10) or complex A (binding to
dscybbA10). EMSAs were performed with U937 nuclear proteins and Pbx
antibodies, with or without blocking peptides. In EMSA with an antibody
to a peptide in the N terminus of Pbx1, inconsistent disruption of both
complexes A and A1 is demonstrated (not shown). However, in
EMSA with an antibody to a peptide in the Pbx C terminus, binding of
complex A1 to the dsA10 probe and of complex A to the
dscybbA10 probe is disrupted (Fig. 1, C and D).
However, these results do not exclude the possibility that additional,
unidentified proteins bind to dsA10 or dscybbA10 and participate in
these complexes.
Consistent with our previously reported EMSA with PLB985 nuclear
proteins and the dscybbA10 probe (17), neither complex A binding to the
dscybbA10 probe nor complex A1 binding to the dsA10 probe
is disrupted by either of two antibodies to CDP (not shown).
HoxA10 Represses Transcription of Artificial Promoter Constructs
with Pbx-HoxA10 Binding Sites--
To determine if HoxA10 represses
transcription in myeloid cells, U937 cells were transfected with
artificial promoter constructs containing multiple copies of the
derived Pbx-HoxA10 binding site (p-a10TATACAT) or the repressor element
from the CYBB gene (p-cybba10TATACAT). Transfectants with
these constructs demonstrated increased promoter activity that is
statistically significant in comparison with control, empty vector
(p-TATACAT) transfectants (p < 0.05 for both) (Fig.
3A). Reporter gene expression
from U937 transfectants with the p-a10TATACAT construct is
significantly repressed by overexpression of HoxA10 (p = 0.012, n = 6). Overexpression of HoxA10 also
represses the p-cybba10TATACAT construct in U937 transfectants (p = 0.001, n = 5). However, HoxA10 has
no significant effect on p-TATACAT control vector expression
(p > 0.5, n = 6) (Fig. 3A).
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Fig. 3.
In undifferentiated U937 cells, HoxA10
represses transcription from artificial promoter constructs containing
a Pbx-HoxA10 binding site. A, overexpression of HoxA10
in U937 cells represses reporter gene expression from artificial
promoter constructs with either the derived Pbx-HoxA10 binding
consensus or the similar CYBB promoter sequence. U937 cells
were transfected with an artificial promoter construct containing
either three copies of the derived consensus for Pbx-HoxA10 binding, a
minimal promoter, and a reporter (p-a10TATACAT), four copies of the
similar CYBB promoter sequence (p-cybba10TATACAT), or
control vector (p-TATACAT) (70 µg); a vector to overexpress either
HoxA10 (HoxA10/pSR), short A10 (SA10/pSR), Pbx (Pbx/pSR),
control vector (pSR) (30 µg), or HoxA10 and Pbx1 (15 µg each);
and a vector to control for transfection efficiency (CMV/gal) (15 µg). Results are reported as absolute CAT activity (in cpm), and each
experiment was repeated at least four times. B,
overexpression of HoxA10 in IFN--treated U937 cells does not repress
transcription from artificial promoter constructs with either the
derived Pbx-HoxA10 binding consensus or the similar CYBB
promoter sequence. U937 transfections were performed as in
A, except that the transfectants were treated with IFN-
(200 units/ml) for 48 h. Note the difference of the x
axis scale in comparison with Fig. 3A. C, HoxA10
either has endogenous repression domains or recruits repressor
proteins. U937 cells were transfected with an artificial promoter
construct with five copies of the DNA-binding site for the GAL4
transcription factor (p-gal4TKCAT) (30 µg); a vector to over express
the DNA-binding domain of GAL4 as a fusion protein with HoxA10
(A10Gal4) or short A10 (sA10Gal4) or vector control (20 µg); and a
vector to control for transfection efficiency, p-CMV/gal (15 µg).
Results were reported as absolute CAT activity (in cpm), and each
transfection was performed at least four times. D, increased
amounts of HoxA10 fusion protein, relative to reporter construct,
resulted in increased reporter repression in U937 transfectants. U937
cells were transfected with an artificial promoter construct with five
copies of the DNA-binding site for the GAL4 transcription factor
(p-gal4TKCAT) (3 µg); a vector to overexpress the DNA-binding domain
of GAL4 as a fusion protein with HoxA10 (A10Gal4) or short A10
(sA10Gal4) or vector control (20 µg); and a vector to control for
transfection efficiency p-CMV/gal (15 µg). Results were reported
as absolute CAT activity (in cpm), and each transfection was performed
three times. Transfectants were assayed with and without 48-h IFN-
treatment. Note the difference in the x axis scale in
comparison with Fig. 3C.
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Since the "short A10" form of HoxA10, present in myeloid cell
lines, contains the DNA-binding homeodomain, we investigated whether
overexpressed short A10 represses transcription. U937 cells were
co-transfected with either p-a10TATACAT, p-cybbTATACAT, or control
p-TATACAT and a vector to overexpress short A10. Short A10 also
significantly represses reporter gene expression from the p-a10TATACAT
and p-cybba10TATACAT constructs (p < 0.05 for both)
but less than full-length HoxA10 (Fig. 3A).
In contrast, overexpression of Pbx1, in U937 cells co-transfected with
either p-a10TATACAT, p-cybba10TATACAT, or control p-TATACAT, did not
significantly alter reporter gene expression (Fig. 3A). However, in U937 cells co-transfected with either p-a10TATACAT or
p-cybba10TATACAT and vectors to overexpress both HoxA10 and Pbx1,
repression of reporter gene expression is significantly greater than
with HoxA10 alone (Fig. 3A, difference in reporter gene
activity with HoxA10 versus HoxA10 plus Pbx,
p = 0.015 for p-a10TATACAT and p = 0.026 for p-cybba10TATACAT). Short A10 does not include the HoxA10 Pbx1
interaction domain (6). Consistent with this, repression of the two
HoxA10-Pbx-containing constructs in U937 transfectants overexpressing
short A10 and Pbx1 is not significantly different from the repression
with short A10 alone (data not shown).
Since IFN- treatment of U937 cells decreases Pbx-HoxA10 binding to
both the derived Pbx-HoxA10 consensus sequence and the CYBB
repressor element, we investigated whether overexpressed HoxA10
represses transcription in IFN--treated U937 cells. U937 cells were
co-transfected with p-a10TATACAT, p-cybba10TATACAT, or empty vector
control and a vector to overexpress HoxA10, short A10, Pbx1, or empty
vector control. U937 transfectants incubated for 48 h with IFN-
(200 units/ml) were compared with transfectants incubated for the same
time without IFN-. Treatment with IFN- significantly increases
expression from the p-a10TATACAT and p-cybba10TATACAT constructs
(p < 0.05) but not p-TATACAT control transfectants (Fig. 3B).
In contrast to undifferentiated U937 transfectants, overexpression of
HoxA10 did not significantly repress reporter gene expression from
these constructs in IFN--treated cells, with or without Pbx1
(p > 0.40 for all combinations in comparison with
empty expression vector control). However, overexpressed short A10
represses reporter gene expression from p-a10TATACAT and
p-cybba10TATACAT constructs in IFN--treated U937 transfectants (Fig.
3B). Reporter gene activity in short A10 overexpressing U937
cells co-transfected with p-a10TATACAT is decreased 78.3% without
versus 64.1% with IFN- treatment. In U937 transfectants
with p-cybbTATACAT, overexpression of short A10 decreases reporter
activity 49.3% without versus 88.9% with IFN-
treatment. Overexpression of short A10 does not repress p-TATACAT
reporter expression in U937 cells with IFN- treatment.
HoxA10 Contains Transcriptional Repression Domains That Are Not
Functionally Altered by IFN---
To determine if HoxA10 protein
possesses endogenous repression domains, the full-length protein and
short A10 were expressed as fusion proteins with the DNA binding domain
of GAL4 (A10gal4DB and SA10gal4DB, respectively). U937 cells were
co-transfected with these fusion protein constructs (or empty gal4DB
vector control) and an artificial promoter construct with multiple
copies of a GAL4 DNA-binding site, linked to a minimal promoter and a
CAT reporter (p-gal4TKCAT) (20 µg). Reporter gene expression is
significantly repressed by overexpression of A10gal4DB
(p = 0.00029, n = 11, Fig.
3C). This repression is not significantly altered by 48 h of IFN- treatment (p = 0.000017, n = 8; p value for reporter activity with or without IFN-
was 0.949). However, overexpression of SA10gal4DB does not
significantly repress reporter gene activity in U937 transfectants,
with or without 48 h of IFN- (without IFN-,
p = 0.50, n = 7; with IFN-,
p = 0.77, n = 11; p value for reporter activity with or without IFN- was 0.65, Fig.
3C). U937 transfections were also performed with one-tenth
the amount of reporter plasmid and the same amounts of A10gal4DB,
SA10gal4DB, and control gal4DB plasmids. In these experiments,
A10gal4DB repressed reporter expression to a greater extent than in the
previous transfections with 10-fold more input reporter plasmid DNA
(Fig. 3D).
Overexpressed HoxA10 Repressed Endogenous gp91phox and
p67phox Expression in U937 Cells--
RNA isolated from
U937 transfectants was analyzed by Northern blot to determine if
overexpression of HoxA10 was associated with decreased abundance of
gp91phox or p67phox mRNA. U937 cells were
transfected with either control pSR expression vector,
HoxA10/pSR, or FLAG epitope-tagged HoxA10/pSR. Total cellular RNA
from the U937 transfectants was analyzed for abundance of
gp91phox, p67phox, or -actin control mRNA by
serially probing Northern blots. Message abundance of both
gp91phox and p67phox was decreased in U937
transfectants with either HoxA10/pSR (Fig. 4A) or FLAG epitope-tagged
HoxA10/pSR (not shown). Both tagged and full-length, untagged
proteins were overexpressed because of the possibility that epitope
tagging alters the function of HoxA10.
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Fig. 4.
Overexpressed HoxA10 represses
gp91phox and p67phox expression in U937 cells.
A, overexpression of HoxA10 in U937 cells decreases
abundance of gp91phox and p67phox mRNA. U937 cells
were transfected with HoxA10/pSR or control pSR (30 µg) and
harvested 48 h later for extraction of total cellular RNA. RNA (20 µg) was analyzed by Northern blot, probed for gp91phox,
p67phox, and -actin mRNA as indicated. U937 cells
transfected with HoxA10/pSR demonstrated decreased gp91phox
and p67phox mRNA abundance in comparison with control
transfectants. Blots were probed for -actin mRNA to control for
loading. B, overexpressed HoxA10 is detected by Western blot
in U937 transfectants. U937 cells were transfected with FLAG
epitope-tagged HoxA10/pSR or control pSR (30 µg) and harvested
48 h later for extraction of nuclear proteins. Nuclear proteins
(30 µg) were separated by 12% SDS-PAGE and Western blots performed
with anti-FLAG antibody.
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To demonstrate that HoxA10 protein was expressed in the U937
transfectants, nuclear proteins from epitope-tagged HoxA10/pSR and
control pSR transfectants were analyzed by Western blot. Nuclear
proteins from transfectants with HoxA10/pSR, but not control pSR,
demonstrated a 50-kDa epitope-tagged protein by Western blot (Fig.
4B). No immunoreactive protein species were detected when
the blot was probed with irrelevant antibody (mouse anti-rabbit IgG)
(not shown).
HoxA10 Is Tyrosine-phosphorylated during IFN--induced U937
Differentiation--
Several mechanisms might decrease HoxA10 DNA
binding, and therefore transcriptional repression, during myeloid
differentiation. Decreased HoxA10 abundance might result in successful
competition for an adjacent or overlapping element by transcriptional
activators. Conversely, increased activator abundance, during
differentiation, might result in successful competition for the DNA
binding site, or post-translational modification of HoxA10, such as
phosphorylation, might decrease HoxA10 affinity for the DNA-binding site.
To investigate the effect of myeloid differentiation on HoxA10
abundance, U937 cells were treated with for 48 h with IFN-. By
Western blot, nuclear proteins from treated and untreated U937 cells
demonstrate three HoxA10 cross-immunoreactive species: a 50-kDa
species, the predicted size of the protein encoded by the major
transcript in myeloid cells (4); a 42-kDa species, the predicted size
of a protein encoded by an alternatively spliced HoxA10 mRNA
described in murine tissues (7); and a 15-kDa species, the predicted
size of short A10 (6). IFN- treatment of U937 cells does not alter
the abundance of any of these HoxA10 species (Fig.
5A), although binding to
Pbx-HoxA10 consensus sequences is decreased by IFN- treatment in
EMSA with the same nuclear proteins (Fig. 1, B and
C). No protein species were detected when the blot was
probed with rabbit pre-immune serum (not shown).
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Fig. 5.
HoxA10 is tyrosine-phosphorylated during
IFN--induced U937 differentiation.
A, HoxA10 protein abundance is not altered by
IFN--induced U937 differentiation. Nuclear proteins from U937 cells
(30 µg), either untreated (lane 1) or after
48 h of IFN- (lane 2), were analyzed by
Western blot with a specific antibody to HoxA10. B, HoxA10
is phosphorylated during IFN--induced U937 differentiation. U937
cells, untreated or after 48 h of IFN- treatment (as
indicated), were labeled by incubation with
[32P]orthophosphate. Cell lysate proteins were
immunoprecipitated with HoxA10 antibody or control rabbit preimmune
serum, and phosphorylated proteins were detected by autoradiography of
SDS-PAGE. C, HoxA10 is tyrosine-phosphorylated during
IFN--induced U937 differentiation. Nuclear proteins isolated from
U937 cells (100 µg) that were either untreated or treated for 48 h with IFN- (as indicated), were immunoprecipitated with an
anti-phosphotyrosine antibody or irrelevant control antibody (mouse
anti-rabbit Ig). Western blot of the immunoprecipitated proteins was
probed with a HoxA10-specific antibody.
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To determine if IFN--induced differentiation results in HoxA10
phosphorylation, both IFN--treated and undifferentiated U937 cells
were 32P-labeled, and lysate proteins were analyzed.
IFN- treatment of U937 cells increases the phosphorylation of
anti-HoxA10 immunoprecipitable 50- and 42-kDa HoxA10 species, but not
of short A10 (Fig. 5B). Since HoxA10 includes tyrosine,
threonine, and serine residues (4), we next investigated whether HoxA10
was tyrosine-phosphorylated during myeloid differentiation. U937 cells
were lysed, after 48 h of incubation with or without IFN-, and
lysate proteins were immunoprecipitated with an anti-phosphotyrosine
antibody or irrelevant control antibody. In Western blots of
immunoprecipitated proteins, IFN- treatment of U937 cells increased
immunoprecipitable, tyrosine-phosphorylated 50- and 42-kDa HoxA10 (Fig.
5C). In contrast, no immunoreactive species were detected
when the blot was probed with rabbit preimmune serum (not shown).
HoxA10 Tyrosine Phosphorylation Decreases DNA Binding
Affinity--
To determine if HoxA10 tyrosine phosphorylation
influences DNA binding affinity, in vitro translated
proteins were dephosphorylated and used in EMSA with Pbx-HoxA10 binding
probes. In preliminary experiments, in vitro translated
HoxA10 was immunoprecipitable with anti-phosphotyrosine antibody (Fig.
6A). To generate
tyrosine-dephosphorylated HoxA10, in vitro translated
protein was treated with the specific tyrosine phosphatase, Yop.
Yop-treated in vitro translated HoxA10, was not
immunoprecipitated by anti-phosphotyrosine antibody, although Yop treated protein was intact (Fig. 6A).
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Fig. 6.
Tyrosine phosphorylation decreases DNA
binding affinity of HoxA10 for Pbx-HoxA10 binding sites.
A, in vitro translated HoxA10 is
tyrosine-phosphorylated. In vitro translated HoxA10 (10 µl), either with or without Yop tyrosine phosphatase
treatment, was immunoprecipitated with anti-phosphotyrosine antibody or
irrelevant control antibody. Unprogrammed reticulocyte lysate was
included as a control. Immunoprecipitated proteins, separated by 12%
SDS-PAGE, were detected by autoradiography as indicated. B,
tyrosine phosphatase treatment of in vitro translated HoxA10
increases DNA binding to the derived Pbx-HoxA10 consensus sequence,
with and without Pbx1. EMSA was performed with the Pbx-HoxA10 consensus
sequence probe (dsA10) and in vitro translated HoxA10 or
control rabbit reticulocyte lysate, with and without in
vitro translated Pbx1. Lane 1, control
reticulocyte lysate (1.5 µl); lane 2,
Yop buffer-incubated control reticulocyte lysate (1.5 µl);
lane 3, Yop-treated control
reticulocyte lysate (1.5 µl); lane 4, HoxA10
(1.0 µl) plus control reticulocyte lysate (0.5 µl); lane
5, Yop buffer-incubated HoxA10 (1.0 µl) plus
control reticulocyte lysate (0.5 µl); lane 6,
Yop-treated HoxA10 (1.0 µl) plus control reticulocyte
lysate (0.5 µl); lane 7, Pbx1 (0.5 µl) and
control reticulocyte lysate (1.0 µl); lane 8,
Pbx1 (0.5 µl) and Yop buffer-incubated control
reticulocyte lysate (1.0 µl); lane 9, Pbx1 (0.5 µl) and Yop-treated control reticulocyte lysate (1.0 µl); lane 10, Pbx1 (0.5 µl) and HoxA10 (1.0 µl); lane 11, Pbx1 (0.5 µl) and
Yop buffer-incubated HoxA10 (1.0 µl); lane
12, Pbx1 (0.5 µl) and Yop-treated HoxA10 (1.0 µl). The upper arrowhead indicates a complex
formed by HoxA10 with Pbx1, and the lower
arrowhead represents binding of HoxA10. Control reticulocyte
lysate generates a complex with the dsA10 probe, consistent with
observations of other investigators (8). C, tyrosine
phosphatase treatment of in vitro translated HoxA10
increases DNA binding to the similar CYBB promoter sequence,
with and without Pbx1. EMSA was performed with the CYBB
promoter sequence probe (dscybbA10) and in vitro translated
HoxA10, or control rabbit reticulocyte lysate, with and without
in vitro translated Pbx1. Comparison of DNA binding affinity
with and without Yop treatment was made. Lane
1, Yop buffer-incubated control reticulocyte
lysate (2.5 µl); lane 2, Yop buffer
incubated HoxA10 (2.0 µl) and control reticulocyte lysate (0.5 µl);
lane 3, Yop-treated HoxA10 (2.0 µl)
and control reticulocyte lysate (0.5 µl); lane
4, Pbx1 (0.5 µl) and Yop-treated control
reticulocyte lysate (2.0 µl); lane 5, Pbx1 (0.5 µl) and Yop buffer-incubated HoxA10 (2.0 µl);
lane 6, Pbx1 (0.5 µl) and
Yop-treated HoxA10 (2.0 µl). The upper
arrowhead indicates a complex formed by HoxA10 with Pbx1,
and the lower arrowhead represents binding of
HoxA10. Control reticulocyte lysate generates a complex with the
dscybbA10 probe, similar to the dsA10 probe. D, in
vitro translated HoxA10 binds specifically to the CYBB
promoter sequence, similar to the Pbx-HoxA10 binding consensus. EMSA
was performed with the CYBB promoter sequence probe
(dscybbA10) and in vitro translated, Yop-treated
HoxA10 in the presence of competitor oligonucleotides (200-fold molar
excess). Lane 1, dsA10 oligonucleotide;
lane 2, homologous dscybbA10 oligonucleotide;
lane 3, dsncf2A10 oligonucleotide;
lane 4, dscybb5'A10 oligonucleotide (another
CYBB promoter sequence similar to the Pbx-HoxA10 consensus);
lane 5, dscybbA10mut oligonucleotide (mutant
homologous sequence); lane 6, no competitor. The
specific HoxA10 protein complex is indicated by the
arrowhead. E, in vitro translated
HoxA10, binding to the dscybbA10 probe, is recognized by HoxA10
antibody. EMSA was performed with the CYBB promoter sequence
probe (dscybbA10), in vitro translated,
Yop-treated HoxA10 and in vitro translated Pbx.
Binding reactions were incubated in the presence of HoxA10 antibody
(HoxA10), rabbit preimmune serum, or no antibody, as indicated.
HoxA10 antibody disrupted both complexes generated by HoxA10 with or
without Pbx1, binding to the dscybbA10 probe. The arrowheads
indicate HoxA10 cross-immunoreactive complexes. F, tyrosine
phosphatase treatment of "short A10" does not increase DNA binding
affinity. EMSAs were performed with the CYBB promoter
sequence probe (dscybbA10) and in vitro translated short A10, or rabbit reticulocyte control. Comparison of DNA
binding affinity, with and without Yop treatment was made.
Lane 1, Yop buffer-incubated control
reticulocyte lysate (0.5 µl); lane 2,
Yop-treated control reticulocyte lysate (0.5 µl);
lane 3, Yop buffer-incubated short A10
(0.5 µl); lane 4, Yop-treated short
A10 (0.5 µl). The arrowhead indicates binding of short A10
to the probe.
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Tyrosine-dephosphorylated HoxA10 and control proteins were used in EMSA
with the dsA10 and dscybbA10 probes. As controls, in vitro
translated HoxA10 was incubated in Yop reaction buffer without enzyme, and control reticulocyte lysate was incubated with and
without Yop. Tyrosine-dephosphorylated HoxA10
demonstrates increased binding to the Pbx-HoxA10 consensus probe (Fig.
6B). Similar results were obtained with the dscybbA10 probe
(Fig. 6C). Binding of in vitro translated HoxA10
to the dscybbA10 probe was competed for by unlabeled homologous
oligonucleotide, by the dsA10 and dsncf2A10 oligonucleotides,
and by an oligonucleotide representing another CYBB promoter
sequence (dscybb5'A10), similar to the derived Pbx-HoxA10 consensus
(Fig. 6D).
To determine if HoxA10 tyrosine dephosphorylation influences
interaction with Pbx, in vitro translated HoxA10, with and
without Yop treatment, was incubated in binding reactions
with in vitro translated Pbx1. Both tyrosine
dephosphorylated HoxA10 and control HoxA10 interact with Pbx1 to form a
low mobility complex with the Pbx-HoxA10 consensus probe. However,
binding affinity of the Pbx-HoxA10 complex is reproducibly increased
with Pbx1-tyrosine-dephosphorylated HoxA10, in comparison with
Pbx1-control HoxA10 (Fig. 6B). Identical results were
obtained with the dscybbA10 probe (Fig. 6C). In addition, the complexes generated by binding of in vitro translated
HoxA10 and HoxA10 plus Pbx1 to the dscybbA10 probe were disrupted by antibody to HoxA10, but not by pre immune serum (Fig. 6E).
Identical results were also obtained with the dsA10 probe (data not shown).
We also generated in vitro translated short A10 protein, in
reticulocyte lysate, and performed similar experiments. In contrast to
our results with HoxA10, binding of short A10 to the dscybba10 probe is
not increased by Yop treatment (Fig. 6F).
Identical results were obtained with the dsA10 probe (not shown).
Since HoxA10 is phosphorylated during IFN--induced U937
differentiation, we hypothesized that tyrosine dephosphorylation of
endogenous HoxA10, from IFN--treated U937 cells, would restore binding to the dsA10 and dscybbA10 probes. U937 nuclear proteins from
undifferentiated U937 cells and from U937 cells treated for 48 h
with IFN- were incubated with Yop. Control extracts were incubated under the same conditions in the absence of the enzyme. Consistent with our hypothesis, Yop treatment of nuclear
proteins from IFN--treated U937 cells increases binding of the
HoxA10-containing protein complex to the dsA10 and dscybbA10 probes
(Fig. 7, A and B).
This complex was verified to contain immunoreactive HoxA10 in EMSA with
the HoxA10-specific antibody (not shown). Yop treatment of
nuclear proteins from undifferentiated U937 cells also increases abundance of the HoxA10-containing protein complex binding these probes.
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Fig. 7.
Specific tyrosine phosphatase treatment
increases endogenous nuclear protein binding to the derived Pbx-HoxA10
binding consensus and to the similar CYBB promoter
sequence. A, tyrosine phosphatase treatment of nuclear
proteins from U937 cells increases HoxA10 DNA binding to the derived
Pbx-HoxA10 consensus. EMSAs were performed with the dsA10 probe and
nuclear proteins (3 µg) isolated from U937 cells that were either
treated for 48 h with IFN- (lanes 1 and
2), or untreated (lanes 3 and
4). Nuclear proteins were either Yop-treated
(lanes 1 and 3) or sham-incubated in
Yop buffer (lanes 2 and 4).
The arrowhead indicates binding of the A1
complex. B, tyrosine phosphatase treatment of nuclear
proteins from U937 cells increases HoxA10 DNA binding to the
CYBB promoter sequence, similar to the derived Pbx-HoxA10
consensus. EMSAs were performed with the dscybbA10 probe and nuclear
proteins (3 µg) isolated from U937 cells that were either treated for
48 h with IFN- (lanes 1 and
2), or untreated (lanes 3 and
4). Nuclear proteins were either Yop-treated
(lanes 1 and 3) or sham-incubated in
Yop buffer (lanes 2 and 4).
The arrowhead indicates binding of the A complex, and the
asterisk indicates CP1 binding to the CCAAT box in the
probe.
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DISCUSSION |
Previous investigations suggest that HoxA10 increases
proliferation and blocks differentiation during early myelopoiesis. HoxA10 function may be difficult to determine if there is variation in
protein-protein interactions, protein-DNA interactions, or activity of
functional domains during myelopoiesis. Our investigations determined
that tyrosine phosphorylation of HoxA10 occurs during IFN- induced
myeloid differentiation and that tyrosine-phosphorylated HoxA10 has
decreased DNA binding affinity. Additionally, we demonstrate, in U937
myeloid cells, that HoxA10 represses expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that
HoxA10 has endogenous repression domains, not affected by IFN--induced myeloid differentiation. Perhaps most interestingly, we
identify the CYBB gene as a potential target for Pbx-HoxA10 repression, in undifferentiated myeloid cells.
We found that IFN--induced myeloid differentiation decreases
in vitro HoxA10 DNA binding to the derived Pbx-HoxA10
consensus, and to a similar CYBB promoter sequence. The
CYBB sequence includes Pbx (5'-atgat-3') and HoxA10
(5'-ttat-3') cores, previously identified by binding site selection (7,
8). However, unlike the derived consensus, there was a 1-bp overlap
between the two sites. Despite this difference, the Pbx-HoxA10
consensus and CYBB sequence have cross-competitive binding
specificities, although the derived consensus binds the complex with
lower affinity. Also, the complex shifted by the CYBB probe
migrates as a broader band than the complex shifted by the derived
Pbx-HoxA10 consensus, suggesting that the CYBB sequence
recruits additional proteins to the binding site. Other investigators
found that HoxA9 interacts simultaneously with Pbx1 and Meis1 at
DNA-binding sites (29, 30). It is similarly possible that a Meis
protein participates in Pbx-HoxA10 binding.
We identified a similar sequence in the NCF2 gene (31), with
cross competitive binding specificity to the derived Pbx-HoxA10 consensus. The NCF2 sequence has a bp change in position 3 in the Pbx core (5'-ataat-3') and an alternative HoxA10 core
(5'-taat-3') (7). The NCF2 gene encodes the respiratory
burst oxidase protein p67phox and is transcriptionally
activated at the same point in myelopoiesis as the CYBB
gene, suggesting that HoxA10 interacts with multiple genes activated
during late myeloid differentiation. Previous investigations identified
two other sequences in the CYBB promoter with
cross-competitive binding with the CYBB sequence
investigated in the current studies (17, 32). Therefore, HoxA10 may
exert an effect on transcription by interacting with multiple promoter sites. We are currently investigating the significance of these other
CYBB promoter sequences, and several similar NCF2
sequences (31).
In our investigations, overexpression of HoxA10 represses reporter gene
expression from constructs with the derived Pbx-HoxA10 consensus or the
similar CYBB promoter sequence in U937 cells. Although Pbx1
overexpression augments HoxA10 repression, Pbx1 alone did not repress
reporter gene expression. Since U937 cells have endogenous HoxA10, this
suggests that Pbx1 is not rate-limiting. Our results differ from U937
transfection experiments by other investigators with HoxA9 and an
artificial promoter construct with the Pbx-Meis-HoxA9 consensus (30).
In those studies, HoxA9 overexpression did not alter reporter
expression. Differences in experimental design may explain the
discrepancy, including shorter post-transfection incubation (6 versus 48 h), less transfected DNA (0.5 µg/106 cells versus 3.0 µg/106
cells), and differences in the minimal promoter. Or there may be
differences in HoxA9 and HoxA10 function, despite similarity in
protein-protein interactions and DNA binding specificity. In these
investigations, we determined that overexpression of HoxA10 decreases
endogenous gp91phox and p67phox mRNA abundance.
Although this result is reassuringly consistent with our reporter gene
assays, we cannot exclude the possibility that HoxA10 influences
abundance of these transcripts indirectly by altering transcription of
other genes or disrupting differentiation.
We determined that IFN--induced U937 differentiation is accompanied
by HoxA10 tyrosine phosphorylation, which decreases DNA binding but
does not alter endogenous HoxA10 repression domains. Therefore, HoxA10
tyrosine phosphorylation, reversible by tyrosine phosphatases, is a
mechanism for reversible gene regulation in response to cytokines. We
found that Yop tyrosine phosphatase treatment of U937
nuclear proteins increases binding of the Pbx-HoxA10-containing complex
to both the derived consensus and similar CYBB sequence. However, Yop-treated proteins from IFN- differentiated
U937 cells do not generate the Pbx-HoxA10 complexes as efficiently as
proteins from Yop-treated, undifferentiated U937 cells. This
is consistent with the hypothesis that IFN- treatment increases
kinase activity in U937 cells and antagonizes endogenous (and also
exogenous) phosphatase activity.
We also investigated the significance of the "short A10" transcript
(6). We identified a HoxA10 cross-immunoreactive protein the predicted
size of short A10 but were unable to demonstrate IFN--induced
phosphorylation in U937 cells. Also, DNA-binding of in vitro
translated short A10 is not increased by tyrosine phosphatase
treatment. Consistent with this, short A10-induced repression of
reporter expression from constructs with Pbx-HoxA10 binding sites is
not decreased by IFN- treatment. Since short A10 has insignificant
endogenous repression activity, these results suggest that repression
is due to successful competition with transcriptional activators for
the same (or adjacent) DNA sequences.
Therefore, the mechanism of HoxA10 transcriptional repression is
2-fold: repression due to endogenous HoxA10 domains and binding site
competition with transcriptional activators. Our studies also imply a
function for short A10 in immortalized cell lines. Since short A10
represses transcription but is not modulated by differentiation-induced
phosphorylation, it might contribute to transformation by repressing
transcription of genes that are necessary for differentiation
progression, or characteristic of the differentiated phenotype.
Previous studies of the CYBB promoter indicated that CDP
represses transcription by binding within a 30-bp sequence that
includes the Pbx-HoxA10-like site (15, 16). CDP binds to this sequence in EMSA with nuclear proteins from the epithelial cell line HeLa and
the erythroleukemia cell line K562. Additional studies determined that
CDP DNA binding is modulated by another homeodomain protein, SATB1
(33). These factors are nuclear matrix-associated, and relative
abundance regulates DNA interactions (33). These studies, in
combination with our current observations, suggest a mechanism for
transcriptional repression by homeodomain proteins in cells of various
lineages. In nonmyeloid cells, transcription of CYBB may be
repressed by CDP. In committed myeloid progenitors, SABT1 increases,
decreasing CDP association with the repressor element, coincident with
nuclear matrix disassociation. Transcriptional repression in myeloid
progenitors would be maintained by Pbx-HoxA10 binding until later in
differentiation. This interaction could be rapidly (and reversibly)
modulated by HoxA10 tyrosine phosphorylation, during differentiation or
the inflammatory response (both increasing CYBB and
NCF2 transcription).
Our investigations suggest that one role of HoxA10 during myeloid
differentiation is repression of transcription of genes characteristic
of mature myeloid cells, such as components of the phagocyte
respiratory burst oxidase. It will be of interest to investigate other
genes also transcriptionally activated during late myeloid
differentiation (or actively transcribed during the immune response) to
determine the significance of this HoxA10 function.
-induced
Differentiation of Myeloid Leukemia Cell Lines*
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. E-mail:
elizabeth.eklund@ccc.uab.edu.
