From the
CCAAT displacement protein (CDP)/cut is implicated in several
systems as a transcriptional repressor of developmentally regulated
genes. In myeloid leukemia cells, CDP/cut binding activity as assayed
on the promoter of the phagocyte-specific cytochrome heavy chain gene
gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves
as a repressor of gp91-phox gene expression. First, we used
transient transfection assays in 3T3 cells to demonstrate that the
CDP/cut binding site from the gp91-phox promoter acts as a
negative regulatory element in artificial promoter constructs. Second,
we isolated a stable transformant of HL-60 myeloid cells constitutively
expressing transfected CDP/cut cDNA. Stable transformants carrying
expression vector alone or expressing CDP/cut mRNA were induced to
differentiate along the macrophage lineage with phorbol ester or along
the neutrophil lineage with dimethyl sulfoxide or retinoic
acid/dimethylformamide. Northern blot analysis was used to assess
induction of mRNAs encoding gp91-phox, and the myeloid oxidase
cytosolic components, p47 and p67. In the stable transformant
expressing transfected CDP/cut cDNA, gp91-phox induction was
selectively reduced, whereas morphologic differentiation and induction
of mRNA for myeloid oxidase components p47 and p67 were unaffected.
These data provide persuasive evidence that CDP/cut acts to repress the
gp91-phox gene.
All organisms require a means of preventing expression of genes
for potentially costly or toxic functions where and when these genes
are not needed. Terminal differentiation in many tissues is accompanied
by acquisition of specialized, tissue-specific functions which are
stringently repressed in immature cells or cells of other lineages.
This is the case for the myeloid oxidase system, which is responsible
for bacterial killing but also generates toxic oxygen metabolites. We
have studied the mechanism of repression of the myeloid cytochrome
heavy chain gene, gp91-phox, whose expression is limited to
terminally differentiated phagocytic cells of the monocyte/macrophage
or granulocyte lineages. Defects in the gp91-phox gene are
responsible for the X-linked form of chronic granulomatous disease.
Skalnik et al.(2) proposed that CCAAT displacement
protein (CDP),
CDP DNA binding to
the gp91-phox promoter is observed in nuclear extracts of
immature HL-60 promyelocytic or PLB-985 myelomonocytic leukemia cell
lines in continuous culture. With terminal differentiation induced by
treatment with retinoic acid/dimethyl formamide (RA/DMF) or phorbol
12-myristate 13-acetate (PMA), CDP DNA binding decreases, and
transcription of the gp91-phox target gene ensues. The
proximal promoter of the gp91-phox gene appears weak, but
exhibits some myeloid specificity
(13) . Deletion of the CDP
binding site from this promoter increases transcription in transient
assays using undifferentiated PLB-985 cells
(2) .
We have
taken two complementary approaches to examine the potential repressor
role of CDP/cut. First, artificial promoters were prepared to study the
ability of CDP/cut binding sites to serve as negative regulatory
elements in reporters transiently introduced into fibroblasts. In this
assay, we have tested CDP/cut binding sites from the human
gp91-phox
To demonstrate that this was
the case, we repeated these transient transfection assays using the CDP
site from the
Since the difference in gp91-phox induction
between RA/DMF and PMA induction might have been due to the differences
in granulocyte versus monocyte differentiation, we also
measured gp91-phox and p67 induction in response to dimethyl
sulfoxide. Me
We have demonstrated that constitutive expression of CDP cDNA
in a myeloid leukemia cell line inhibits expression of the target gene,
gp91-phox, upon terminal myeloid differentiation. Furthermore,
the CDP/cut binding site from the gp91-phox promoter acted as
a negative regulatory domain in artificial promoter constructs
transfected into non-myeloid cells. Constitutive CDP/cut expression did
not prevent normal morphologic differentiation, nor did it limit
induction of other myeloid oxidase components which are not thought to
be regulated by CDP/cut. We infer, therefore, that CDP/cut expression
is likely to act directly on the gp91-phox promoter, and that
lack of induction of gp-91phox in the stably transformed cells
was not due to a general failure of terminal differentiation. These
data provide direct evidence of CDP/cut repressor function and
demonstrate that decreased CDP/cut DNA binding is not a ``master
switch'' for terminal myeloid differentiation.
Homologs of
Drosophila cut have now been described in several eukaryotes.
The nomenclature in these reports and those concerning CDP/cut is not
uniform, and the basis of cloning in each case has been different.
summarizes and compares the nomenclature, experimental
systems, and postulated function of CDP/cut in published reports from
several laboratories. In some, but not all of these cases, CDP/cut has
been shown or postulated to act as a repressor protein. It remains to
be determined how CDP might act to increase or decrease transcriptional
activity. The CDP/cut protein contains four DNA binding sites with
overlapping but distinct sequence
specificities
(25, 26, 27) . It is likely that
CDP/cut recognizes a wide variety of target genes, and the protein
might repress or activate transcription based on any of several
factors: (i) which of its DNA binding sites are in contact with
promoter or enhancer elements; (ii) which other proteins contact
CDP/cut or adjacent elements of target DNA; (iii) regulatory
modifications, which might vary from cell type to cell type. At least
two types of potential regulatory modifications have been reported.
CDP/cut is a phosphoprotein (Ref. 24)
The degree of inhibition of gp91-phox expression in HL-P6
cells compared to control was greater for PMA and Me
In numerous attempts with
several different vectors, we isolated only a single cell line with
stable, constitutive expression of CDP/cut: HL-P6. The difficulty in
selecting additional lines is apparently unique to myeloid cells. Using
several different expression vectors and two selectable markers, we
found that with the exception of HL-P6, screening for stable lines by
drug resistance yielded either cells with rearranged or deleted CDP/cut
transgenes or cells which failed to express transgene CDP from an
apparently intact construct. This occurred not only in HL-60 cells, but
in the human myelomonocytic leukemia cell line PLB-985. On the other
hand, stable transformants of human erythroid-like K562 cells
expressing high levels of CDP mRNA and protein were readily selected
(data not shown). We postulate that high level expression of CDP/cut is
either toxic to myeloid cells or leads to slow growth. The results with
line HL-P6 are unlikely to be due to a nonspecific toxic effect of the
transformation or stable selection process, because myeloid
differentiation is otherwise normal in this line.
The present
results illustrate one way in which a ubiquitous, general repressor can
participate in tissue-specific gene expression, acting on only a subset
of myeloid-specific genes, not on the overall differentiation program.
We propose that in myeloid and non-myeloid cells, de-repression of a
subset of genes controlled by CDP could serve as a final step toward
fully differentiated function in cells already committed to a
particular lineage.
One may also speculate on the relationship
between control of cell growth and CDP/cut function. In the systems for
which CDP de-repression has been implicated (), terminal
differentiation is accompanied by the arrest of cell growth. Cessation
of cell growth alone, either by serum starvation or pharmacologic cell
cycle arrest, is not sufficient for CDP de-repression of gp91-phox in myeloid leukemia cells in culture.
We thank Barbara Aufiero for performing the binding
assay comparison of
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
a DNA binding activity first
described in sea urchin
(1) , serves as a repressor of the
promoter of the gp91-phox gene. To investigate this
possibility, we previously purified CDP from HeLa cells and isolated
human CDP cDNA
(3) . The CDP gene encodes a ubiquitous
homeodomain-containing protein of 180-190 kDa, closely homologous
to the Drosophila protein Cut. We will refer to the human gene
and its protein as CDP/cut. Investigators studying diverse
developmental systems have suggested that CDP/cut serves as a repressor
of developmentally regulated
genes
(1, 2, 4, 5, 6, 7, 8) .
This hypothesis is in general accord with the proposal of Barberis
et al.(1) that CDP binding to promoters of
tissue-specific genes is limited to tissues or developmental stages in
which the target genes are not expressed. With terminal
differentiation, CDP/cut promoter binding is lost, and transcription of
tissue-specific genes is activated. Genetic experiments in
Drosophila show that Cut is involved in determination of cell
fate in several tissue types (9-12), but direct transcriptional
regulatory effects of Cut have not been reported.
-globin promoters. Second, as a more
appropriate in vivo assay, we have asked whether constitutive
expression of transfected CDP/cut cDNA in cultured myeloid leukemia
cells might inhibit expression of the putative target gene,
gp91-phox. Results in both expression systems support the
hypothesis of CDP/cut repressor function.
CDP Expression Constructs
For stable
transformation experiments, CDP cDNA was cloned into the expression
vector RC-CMV as follows: the cDNA was excised from pBluescript KS II
with Asp-718 (KpnI site in polylinker 5` to cDNA) and
Spe1 (position 5293 in the 3`-untranslated region of CDP cDNA;
Ref 3). The overhanging ends were filled in with Klenow DNA
polymerase
(14) . EcoRI/NotI adapters
(Pharmacia Biotech Inc.) were then added with T4 DNA ligase. This cDNA
fragment was then digested with NotI and inserted into RC-CMV
(Invitrogen) digested with NotI and treated with alkaline
phosphatase (Promega Biotech). The CMV promoter was expected to direct
synthesis of a 6.3-kb mRNA transcript from this construct. For
transient co-transfection assays, full-length CDP or fragments thereof
were cloned into the expression vector pMT2, driven by the adenovirus
major late promoter (15). These effector constructs, shown in
Fig. 1
, included full-length CDP in sense or antisense
(``CDP anti'') orientation, the 5` 1.0-kb EcoRI
fragment encoding 334 nucleotides at the N terminus (Nterm); a 2.9-kb
3` EcoRI fragment including cut repeats 2 and 3, and the
homeodomain region (``CR2-Cterm'').
Figure 1:
Effector constructs used for stable and
transient transfections. Constructs were cloned into expression vectors
RC-CMV (for stable transformants) and pMT2-ATG (for transient
transfections) in the direction shown by the arrows, as
described under ``Experimental Procedures.'' Domain positions
(see Ref. 3) are shown as boxes. Nterm, 1.0-kb
EcoRI fragment; CR2-Cterm, 3.0 kb to 3` end;
CDP, 5.1 kb encompassing entire coding
region.
Probes and Plasmids
-Actin cDNA
(16) was a gift from Dr. M. C. Simon. p47
(17) and p67
cDNA
(18) were kindly provided by Dr. S. Chanock.
Myeloid Differentiation
HL-60 cells were obtained
from the American Type Culture Collection. Cells were maintained in
RPMI 1640 medium supplemented with 10% fetal bovine serum, 2
mM glutamine, 50 units/ml penicillin, and 50 µg/ml
streptomycin. To induce monocyte-macrophage differentiation, growing
cells (5 10
/ml) were suspended in 30 ml of medium
containing 2 µM PMA (Sigma) in each of five P150 dishes.
Cells adhered to the dishes within 24 h and were harvested by scraping
into phosphate-buffered saline at 48 h of induction. For granulocytic
differentiation, HL60 cells at a density of 5
10
/ml
were treated with 1.3% (v/v) dimethyl sulfoxide (Me
SO) for
4 days or with 1 µM retinoic acid for 4 days plus 0.5%
(v/v) dimethylformamide added to the medium 48 h before harvesting.
Fresh medium containing inducing agent was added to the cells 48 h
prior to harvesting as needed to keep the cell density
5
10
/ml.
RNA Analysis
Total RNA from 0.5-1
10
induced or uninduced HL-60 cells was extracted with
RNAzol (Tel-Test B; Friendswood Tx) as described
(19) . RNA in
0.5% SDS was adjusted to 20 mM Tris-HCl, pH 7.4, 0.5
M NaCl, 1 mM EDTA, and subjected to two rounds of
selection on columns containing 25 mg (0.2 ml bed volume) of
oligo(dT)-cellulose (Pharmacia) as described
(14) . RNA was
quantified by UV absorbance, and equal amounts of each sample were
loaded on 1% agarose formaldehyde gels. RNA was blotted onto Magnagraph
nylon membranes as suggested by the manufacturer (Micron Technologies,
Westborough, MA). Northern blots were performed in standard fashion
(14). For sequential probing, each blot was stripped by washing two to
three times in 0.1% SDS at 100 °C.
Quantitation
Northern blots were quantified by
PhosphorImager analysis, using ImageQuant software (Molecular
Dynamics). For each band analyzed, background PhosphorImager counts
were subtracted from an adjacent area of the scan. -Actin signal
was used to normalize for differences in gel loading.
Transient Transfection Reporter
Constructs
Artificial promoter fragments were cloned into the
human growth hormone reporter plasmid TATA-GH. This minimal promoter
construct includes the rabbit -globin TATA box in
-GH
(20) . Complementary oligonucleotides for
double-stranded fragments were synthesized on an ABI model 400 DNA
synthesizer with overhanging ends to facilitate cloning. These
sequences (upper strand shown for clarity) were as follows.
``
'', human
-globin promoter fragment:
TGCCTTGACCAATAGCCTTGACAAGGCAAACTTGACCAATAGTCTTAGAGTATCCAGTG; 5`
restriction site EcoRI, 3` site BamHI.
``gp91,'' gp91-phox promoter fragment:
GCTTTTCAGTTGACCAATGATTATTAGCCAATTTCTGATAAAAGAAAAGGAAACCGATTGC;
BamHI sites were at both ends. ``SP1,''
GAGGCGTAACATAGGGGCGTAGCATAGAGGCGGAA. 5` restriction site SalI,
3` site HindIII.
Transient Transfection Assays
NIH 3T3 or HeLa
cells (from ATCC) were plated in Costar six-well dishes at 5
10
cells/well. 24 h later, calcium phosphate-mediated
transfection was performed as described
(22) . In each case, 5
µg of reporter construct was added to each well. For effector
plasmids, either 10 µg of pMT2 CDP or, as control, pMT2 alone or
pBluescript II (Stratagene) were included to keep the total DNA amount
to 15 µg. We used the plasmid pSV-
gal, 2 µg/transfection,
to control for transfection efficiency. 48 h after transfection, medium
was collected, centrifuged briefly, and assayed for human growth
hormone by radioimmunoassay (Nichols Institute, San Juan Capistrano,
CA). Cells were collected by trypsinization, the trypsin was
neutralized with serum, cells were lysed by freeze-thawing, and
-galactosidase was assayed as described (14).
Selection of Stable Transformants
HL-60 cells were
transfected with RC-CMV-CDP or with RC-CMV vector
alone by electroporation at 960 µF in a Bio-Rad gene pulser as
described
(23) . After 24 h of recovery in 20% fetal calf serum,
G418 (Life Technologies, Inc.), 1.8 µg/ml, was added to cultures
for selection, and cells were plated at limiting dilution. Twenty
clones from the CDP
transformation were obtained. Of
these, 10 had intact transgenes, and one, HL-P6, expressed the expected
6.3-kb transgene mRNA. This line, plus vector-only line HL-V4 were used
for subsequent experiments.
CDP Binding Sites from gp91-phox and
Skalnik et al.(2) demonstrated that deletion of the CDP binding site of the
gp91-phox promoter led to increased transcriptional activity
in transient assays in undifferentiated PLB-985 myeloid leukemia cells.
To test the ability of CDP/cut binding sites to act as negative
regulatory domains in non-myeloid cells, we created artificial
``active'' promoters into which we placed CDP/cut binding
sites from the gp91-phox gene
(2, 3) or from
the human -Globin
Promoters Are Negative Regulatory Domains When Assayed in Artificial
Promoter Constructs in Fibroblasts
-globin gene (Ref. 6; see Fig. 2A). For a
basal promoter, we chose the TATA-growth hormone plasmid described by
Martin et al.(20) . For an activation domain, we used a
triplet of consensus sites for the ubiquitous transcriptional activator
SP1. Constructs were transfected into 3T3 murine fibroblasts, which
contain abundant CDP/cut (not shown).
Figure 2:
CDP
binding site is a negative regulatory domain in artificial promoter
constructs in non-myeloid cells. A, reporter constructs were
prepared from double-stranded oligonucleotides in TATA-GH as described
under ``Experimental Procedures.'' hGH, human growth
hormone gene; TATA, TATA region from rabbit -globin gene;
SP1, artificial triplet of consensus SP1 binding sites (21);
gp91, CDP binding site from duplicated CCAAT box region of
gp91-phox proximal promoter (``FP'' in Ref. 3);
, CCAAT-box region of human
-globin gene (Ref. 6 and
``Experimental Procedures''). B, results of
transient transfections in 3T3 cells. Reporter plasmids as shown in
A were transfected in combination with CDP in PMT-2 or control
plasmid (pBluescript II), and human growth hormone assays were
performed on duplicate or triplicate wells. To correct for transfection
efficiency which varied among experiments, results are normalized to
the SP1-TATA positive control (mean = 100%). Bars represent mean ± S.D. of three or more determinations. Note
that constructs including the gp91 site are repressed with or without
co-transfection of exogenous CDP. C,
-globin CDP binding
site depends on expression of exogenous CDP cDNA. The experiment was
performed as in B. Effectors included CDP, antisense CDP cDNA
(anti), a construct including the 3` half of the cDNA, encompassing CR2
through the homeodomain (CR2-Cterm) in sense and antisense orientation
as shown in Fig. 1.
Full- or partial-length
CDP/cut expression constructs used for co-transfection were prepared as
described under ``Experimental Procedures,'' and are shown in
Fig. 1
. Reporter constructs tested are shown in
Fig. 2A. Growth hormone expression results of constructs
containing the gp91-phox CDP site are shown in
Fig. 2B. As expected, the TATA box alone (TATA) or
gp91-TATA constructs resulted in minimal human growth hormone
expression, and the positive control construct SP1-TATA exhibited
abundant activity (set to 100%). Exogenous CDP effector plasmid had no
effect on this reporter plasmid in the co-transfection assay. However,
reporters containing the gp91-phox CDP binding site had
substantially lower transcriptional activity, less than 25% of the
relevant control without a CDP binding site. We infer that this
decrease was due to binding of endogenous murine CDP/cut to the CDP
binding site (Fig. 2B).
-globin promoter, which binds approximately 40-fold
less tightly to CDP than does the gp91-phox site. In this
context, repression of reporter constructs requires exogenous CDP
expression. As shown in Fig. 2C, constructs SP1-TATA and
SP1-
-TATA had similar expression in the absence of exogenous
CDP/cut effectors. Co-transfection with CDP/cut cDNA or the truncated
CR2-Cterm expression construct (Fig. 1) resulted in repression of
transcription from SP1-
-TATA, while inactive antisense effectors
(``CDP anti'' and ``CR2-Cterm anti'') had no effect
on basal transcription. Similarly, the Nterm construct, which lacks DNA
binding activity, had no effect on transcription from SP1-
-TATA.
gp91-phox mRNA Induction Is Reduced in an HL-60 Stable
Transformant Constitutively Expressing CDP mRNA, While Induction of
Other Terminally Differentiated Myeloid Genes Is Unaffected
Cell
lines were selected as described under ``Experimental
Procedures.'' RNA from a CDP-expressing stable transformant line
of HL-60 cells, designated HL-P6, and a vector-only stably transformed
control line (HL-V4) were subjected to Northern blot analysis to
demonstrate expression of transgene CDP mRNA in the constitutively
expressing line (Fig. 3A). The expected 6.3-kb
transcript was found in HL-P6 (lanes 1-3, see
arrow), but not in HL-V4 (lanes 4-6). We
examined the induction of gp91-phox and two cytosolic
components of the myeloid oxidase system, p47 and p67, in HL-P6 and the
control line HL-V4. Three different inducing agents were tested as
described under ``Experimental Procedures'': RA/DMF or
MeSO (granulocyte differentiation) and PMA
(monocyte/macrophage differentiation).
Figure 3:
Constitutive CDP expression reduces
gp91-phox mRNA induction in myeloid leukemia cells treated
with phorbol ester or retinoic acid/dimethylformamide. HL-P6 stable
transformants (lanes 1-3) or HL-V4 control (lanes
4-6) were grown in medium alone (lanes 1 and
4) or induced toward monocyte/macrophage differentiation with
2 µM PMA for 48 h (lanes 2 and 5) or
with RA/DMF (1 µM retinoic acid for 4 days, plus 0.5%
dimethylformamide for 48 h) (lanes 3 and 6).
Oligo(dT)-selected mRNA, 2 µg/lane, was subjected to Northern blot
analysis and probed sequentially with each of the following cDNA
species. A, CDP 5` 1-kb EcoRI fragment. Endogenous
CDP transcripts, constitutively expressed with induction, co-migrate
with 28 S RNA and at 12 kb. The 3.4-kb band represents an alternatively
spliced product of the CDP gene. The 6.3-kb transgene (arrow)
is limited to HL-P6 stable transformants (lanes 1-3).
B, gp91-phox mRNA. Arrow denotes the
expected transcript. Induction is evident in HLV4 controls treated with
PMA (lane 5) or RA/DMF (lane 6). Induction is
substantially less in HL-P6 (lanes 2 and 3).
C, p67 component of myeloid oxidase. Induction in HL-P6 and
HL-V4 are similar. D, p47 component of myeloid oxidase.
Arrow denotes the expected transcript. E,
-actin, which serves as a control for RNA recovery and gel
loading.
In HL-P6 cells induced toward
monocyte/macrophage differentiation with PMA (Fig. 3B,
lane 2) or toward granulocyte differentiation with RA/DMF
(Fig. 3B, lane 3), gp91-phox induction
was reduced compared to the HL-V4 control (Fig. 3B,
lane 5, PMA and lane 6, RA/DMF). However, induction
of mRNA for the cytosolic oxidase components p47 and p67, which are not
thought to be controlled by CDP, was equivalent in the two cell lines
(Fig. 3, C and D). The differences in
gp91-phox mRNA levels were not due to differences in RNA
loading on the Northern blot, as shown by actin recovery
(Fig. 3E). Results from the Northern blot in
Fig. 3
and from duplicate or triplicate experiments were
quantified by PhosphorImager analysis as described under
``Experimental Procedures.'' For RA/DMF, gp91-phox induction in HL-P6 was 44% ± 11% of that observed in HL-V4
cells (mean ± S.D. of three experiments). Compared to the
results with RA/DMF, PMA induction of gp91-phox was more
curtailed in HL-P6 (16 ± 3% of HL-V4 levels; mean of duplicate
experiments). The difference between PMA and RA/DMF induction in HL-P6
cells was significant by two-tailed t test (p <
0.05). p47 and p67 induction as quantified by PhosphorImager revealed
slightly greater induction in both cytosolic oxidase components'
mRNA in HL-P6 compared to control HL-V4 (p67: 108% of control to PMA
(n = 1), 148% to RA/DMF (n = 3); p47:
122% of control to PMA (n = 2), 137% of control
induction to RA/DMF (n = 2). These differences in p47
and p67 induction between HL-V4 and HL-P6 were not statistically
significant.
SO promotes granulocytic induction, as does
RA/DMF. However, the degree of inhibition of gp91-phox induction with
Me
SO was equivalent to that seen with PMA and more
pronounced than that seen with RA/DMF (gp91-phox induction 14%
± 2% of HL-V4 level, mean of duplicate experiments;
autoradiograph not shown).
Morphologic Differentiation Is Normal in HL-P6 Stable
Transformants
We used standard morphologic criteria to assess
differentiation of HL-P6 and HL-V4 cells in response to inducing
agents. For PMA induction toward monocyte-macrophage lineage,
differentiation refers to adhesion to the culture dish and extension of
processes. For RA/DMF induction, differentiation was assessed as
progression from promyelocytic phenotype to myelocytes and
metamyelocytes on Wright-stained Cytospin preparations from the induced
cultures. The degree of differentiation was similar for HL-P6 and
HL-V4. For RA/DMF treatment, approximately 70% of cells in both cell
lines had progressed along granulocytic development from promyelocytic
appearance to the myelocyte or metamyelocyte stages. For PMA induction
along the monocyte/macrophage pathway, floating cells were removed
prior to analysis, so that essentially all of the cells analyzed had
begun to differentiate. We conclude that constitutive expression of
CDP/cut in the HL-P6 cell line had no observable effect on morphologic
differentiation but had a specific negative regulatory effect on the
expression of its target gene, gp91-phox.
(
)
and
changes in phosphorylation may alter DNA binding or function. The
mechanism by which CDP DNA binding decreases with myeloid
differentiation is unknown. Endogenous CDP mRNA levels do not change
substantially with differentiation (Fig. 3A), so
regulation of DNA binding is not at the level of CDP transcription or
message stability. CDP mRNA also exhibits complex alternative splicing,
with at least three major species, and perhaps several minor splice
variants, whose expression with differentiation has not been studied.
Variations among the cut homology family members in rodents
(4, 24) and dog
(8) may represent splice variants as well.
SO
induction (gp91-phox induction approximately 15% of control)
than for RA/DMF induction (about 40% of control). Although the
mechanism for this difference is unknown, it suggests that different
inducing agents may utilize different cellular signaling pathways to
accomplish gp91-phox induction.
However, in
normal differentiation, growth control and CDP/cut de-repression may be
controlled by similar signals. Understanding what these signals may be
has particular relevance to the study of myeloid leukemias, in which
terminal differentiation fails and unregulated growth results.
Table:
Reported Studies of CDP/cut: numerous
designations and postulated transcriptional regulatory roles
SO, dimethyl sulfoxide;
CMV, cytomegalovirus; kb, kilobase(s).
-globin and gp91-phox CDP binding
site affinities and for helpful discussions. We gratefully acknowledge
the support and advice of Stuart Orkin throughout the course of these
studies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.