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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2517-2524
Isolation and Characterization of the cDNA for Mouse Neutrophil
Collagenase: Demonstration of Shared Negative Regulatory Pathways for
Neutrophil Secondary Granule Protein Gene Expression
By
Nathan D. Lawson,
Arati Khanna-Gupta, and
Nancy Berliner
From the Department of Biology and Section of Hematology, Department
of Internal Medicine, Yale University School of Medicine, New Haven,
CT.
 |
ABSTRACT |
A characteristic of normal neutrophil maturation is the induction of
secondary granule protein (SGP) mRNA expression. Several leukemic human
cell lines mimic normal morphologic neutrophil differentiation but fail
to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase
(NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to
differentiate into neutrophils and induce expression of LF and NG.
Therefore, to study the normal regulation and function of these genes,
the corresponding murine homologs must be isolated. Using cDNA
representational difference analysis (RDA) to compare a committed
myeloid progenitor cell line (EPRO) with the multipotent stem cell line
from which it was derived (EML), we isolated a fragment bearing
homology to human neutrophil collagenase (hNC). Here, we describe the
cloning and characterization of a full-length (~2 kb) clone that
exhibits nearly 65% nucleotide and 73% amino acid identity to hNC.
Ribonuclease protection analysis (RPA) of the tissues and cell lines
shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the
mouse homolog of hNC. Furthermore, we have demonstrated a shared
negative regulatory pathway for this and other SGP genes. We have
previously shown that CCAAT displacement protein (CDP/cut) binds to a
specific region of the LF promoter, and overexpression of CDP blocks
G-CSF-induced upregulation of LF gene expression in 32D C13
cells. We show here that in these cells, upregulation of both
NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.
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INTRODUCTION |
MATRIX METALLOPROTEINASES (MMPs) are
responsible for the turnover of extracellular matrix associated with a
variety of biologic processes, including tissue regeneration and wound
healing, and may play a role in the acquisition of metastatic potential
in transformed cells.1 A neutrophil-specific MMP, human
neutrophil collagenase (hNC), has been identified and is distinct from
the closely related fibroblast collagenase with respect to peptide and
collagen specificity.2 hNC has been shown to colocalize with lactoferrin (LF) in specific granules within polymorphonuclear neutrophils,3 and its expression is thought to be
coordinately regulated with other secondary granule protein (SGP)
transcripts such as LF and neutrophil gelatinase (NG).4,5
Cell lines used for the study of neutrophil differentiation include
HL60 and NB4 cells, both of which were derived from patients with acute
promyelocytic leukemia.6,7 The NB4 cell line is characterized by t(15;17) involving the retinoic acid receptor  (RAR) and the PML gene,8 and both HL60 and NB4 cells
exhibit neutrophil differentiation upon induction with
all-trans retinoic acid (ATRA).7,9 Although
morphologic maturation into neutrophils is apparent, neither HL60 nor
NB4 cell lines express SGP transcripts characteristic of normal
neutrophils.2,10-13 In contrast, the murine cell line 32D
C1314 is able to undergo complete neutrophil maturation
with stage-specific expression of SGP transcripts upon induction with
granulocyte colony-stimulating factor (G-CSF).4 Therefore,
to study the normal regulation of these genes during neutrophil
maturation and the potential cause of SGP gene dysregulation in
leukemic cells, it is necessary to isolate their murine homologs.
Two murine cell lines have recently been established using a
dominant-negative form of RAR.15-18 The stem cell factor
(SCF)-dependent EML cell line is arrested at an early point in
hematopoiesis and is characterized by the ability to differentiate
along multiple lineages while exhibiting a specific block in neutrophil
maturation that can be overcome by superphysiologic concentrations of
ATRA in the presence of granulocyte-macrophage CSF
(GM-CSF).17 The MPRO cell line is GM-CSF-dependent and
consists of neutrophilic promyelocytes that terminally differentiate in
response to ATRA.16 We have shown that both of these cell
lines are able to express mouse NG (mNG) and mLF upon induction with
ATRA. Furthermore, a more mature myeloid cell line can be derived from
EML cells17; it is referred to as EPRO and possesses
characteristics similar to the MPRO cell line, including neutrophil
maturation17 and SGP gene expression.
In an attempt to isolate factors involved in myeloid commitment, we
used cDNA representational difference analysis (RDA)19 to
compare EML and EPRO cells (manuscript in preparation).
Among the differentially expressed genes that were isolated was a
fragment that exhibited a high degree of homology to hNC. Here, we
describe the cloning of the full-length cDNA for mNC using this
fragment. We show that the expression of this transcript is
neutrophil-specific and exhibits regulation similar to that for the
other SGP transcripts, including repression by CCAAT displacement
protein (CDP). CDP is a homeodomain protein with extensive homology to
Drosophila cut protein and has been shown to inhibit expression
of the myeloid-specific cytochrome heavy-chain gene gp91-phox, among
others.20 We have previously demonstrated CDP-mediated
repression of the SGP gene LF during G-CSF-induced maturation of 32D
C13 cells overexpressing CDP.21 Repression of both
gp91-phox22 and LF is associated with CDP binding within
their respective promoters, although no consensus binding site for CDP
has been described.20,22 Here, we demonstrate that mNC and
mNG expression are also inhibited at the mRNA level in 32D C13 cells
overexpressing CDP, a pattern similar to that of LF. CDP is thus the
first candidate for a transcriptional regulator mediating the shared
regulation of neutrophil SGP genes.
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MATERIALS AND METHODS |
Cell lines.
EML, MPRO, BHK/MKL, and HM-5 cells were kindly provided by Schickwann
Tsai (Fred Hutchinson Cancer Research Center, Seattle, WA). EML cells
were maintained in Iscove's modified Dulbecco's medium (IMDM)
containing 20% heat-inactivated horse serum and supplemented with 10%
conditioned medium from the BHK/MKL cell line as a source of rat SCF.
MPRO cells were maintained in AIM V serum-free medium (Life
Technologies Inc, Bethesda, MD) supplemented with 10% conditioned
medium from the HM-5 cell line as a source of GM-CSF. 32D C13 and
32D/CDP cells were grown in IMDM with 10% heat-inactivated fetal
bovine serum and 10% WEHI-conditioned medium as a source of
interleukin-3 (IL-3). The establishment of 32D C13/CDP cells is
described elsewhere.21 To derive the EPRO cell line, EML
cells were induced with IL-3 and 10 µmol/L ATRA (Sigma, St Louis,
MO) in the presence of SCF for 72 hours, followed by culture in IMDM containing 20% heat-inactivated horse serum
supplemented with 10% HM-5-conditioned medium.
For induction of the 32D cell lines, cells were washed twice with
phosphate-buffered saline and placed in IMDM with 10% fetal bovine
serum and 3 × 103 U/mL recombinant human G-CSF (Amgen,
Thousand Oaks, CA). MPRO cells were induced by adding ATRA to a final
concentration of 10 µmol/L in the presence of normal growth medium as
already described. All cell lines were grown at 37°C and 5%
CO2 in a humidified incubator.
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| Fig 2.
Alignment of mouse and human NC. Box 1 outlines the
cysteine switch region, and box 2 is the zinc binding domain. The
proteins share 73% identity overall. Arrow 3, signal peptidase
cleavage site; arrow 4, autolytic activation cleavage site; arrow 5, degradation site.
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Library construction.
Total RNA was isolated from uninduced EPRO cells using TRIzol reagent
(Life Technologies), and mRNA was purified using an oligo(dT) column
(Life Technologies). First-strand synthesis was initiated using an
oligo(dT) primer containing a Xho I site. After second-strand synthesis, the cDNA was blunt-ended and ligated to an
EcoRI adapter followed by directional cloning into a
phosphatased ZAP Express  vector (Stratagene, La Jolla, CA) cut with
EcoRI and Xho I and packaged using Gigapack Gold
extracts (Stratagene).
Approximately 1.5 × 105 clones were plated and
transferred to duplicate BioTrace NT nitrocellulose membranes (Gelman
Sciences, Ann Arbor, MI). The membranes were hybridized with the
fragment EPRO 6-2 labeled with [-32P]dCTP by random
prime (Boehringer Mannheim, Indianapolis, IN). Secondary and tertiary
screens were performed as usual,23 and phagemids were
excised from isolated tertiary clones according to the manufacturer's
protocol (Stratagene). Clones were analyzed by restriction mapping and
sequenced using the dideoxy chain-termination method.24
RNA isolation and Northern analysis.
Total RNA was isolated from the cells using TRIzol reagent. Northern
analysis was performed by electrophoresis of 10 µg RNA in a 1%
agarose/formaldehyde gel, followed by transfer to nitrocellulose membrane (Gelman Sciences). Blots were probed with cDNA fragments labeled with [-32P]dCTP by random prime (Boehringer
Mannheim). Hybridization was performed overnight at 42°C in the
presence of 50% formamide and washed with 2× SSC (300 mmol/L NaCl
and 60 mmol/L sodium citrate)/0.1% sodium dodecyl sulfate (SDS) twice
at room temperature for 10 minutes, followed by two high-stringency
washes in 0.1× SSC (15 mmol/L NaCl and 3 mmol/L sodium citrate)/0.1%
SDS at 55° to 60°C for 15 minutes.
Probes.
Probes for mLF, mNG, and  -actin were prepared as previously
described.4 A probe for human p47 was kindly provided by
Steven Chanock (National Cancer Institute, Bethesda, MD). The probe
designated EPRO 6-2 was isolated from a RDA screen for differential
gene expression and cloned into the BamHI site of
pBluescript II (Stratagene). The EPRO 6-2 fragment was excised using
EcoRI and Xba I followed by agarose gel purification
and labeled as before.
RPA.
Riboprobes corresponding to -actin and mNC were prepared using the
MAXIscript transcription kit (Ambion, Austin, TX). Before transcription, a plasmid containing the full-length mNC cDNA (pBK-mNC 4-1) was linearized with BstZ17 (New England Biolabs Inc, Beverly, MA)
followed by digestion with proteinase K (final concentration, 200 µg/mL) for 30 minutes at 50°C. The linearized plasmid was then
phenol/chloroform-extracted and precipitated with ammonium acetate and
ethanol. For -actin, the pTRI-actin plasmid was used (Ambion). A
typical transcription reaction contained 1 µg linearized template and
0.5 mmol/L final concentration each for ATP, GTP, and UTP; a final CTP
concentration of 25 µmol/L was used with 1 µmol/L
32P-CTP, and the reaction was incubated at 25°C for 1 hour followed by digestion with DNAse I at 37°C for 15 minutes.
Probes were analyzed on a 6% polyacrylamide/TBE gel, exposed to film,
cut from the gel, and eluted from gel slices by soaking in 0.5 mol/L NH4OAc/1% SDS overnight at 37°C.
For the RPA, approximately 10 µg total RNA was simultaneously
precipitated with 1 × 104 cpm -actin and 1 × 105 cpm mNC probes, and the pellets were resuspended in 10 µL HybSpeed buffer (Ambion) at 95°C. All samples were normalized to
50 µg RNA with yeast RNA. As a control, yeast RNA alone was
hybridized to the indicated amounts of probe. The probe and RNA were
hybridized at 68°C for 10 minutes followed by digestion with a
mixture of RNAse A and T1 at a dilution of 1:100 for 20 minutes at
37°C. Digested samples were precipitated, and the pellets were
resuspended in loading buffer and separated on a 6% polyacrylamide/TBE
gel for 2.5 hours at 250V. The gel was then exposed to film at
 70°C with an intensifying screen.
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RESULTS |
Isolation of mNC cDNA.
Current studies in our laboratory have used the technique of cDNA
RDA19 in an attempt to isolate factors involved in
myelopoiesis. While using RDA to characterize the EPRO cell line, we
isolated a number of cDNA fragments that are differentially expressed
with respect to the multipotent cell line (EML) from which it was
derived. Sequence analysis of one of these fragments (EPRO 6-2)
revealed a high degree of homology to hNC. Therefore, we sought to
clone and characterize a full-length cDNA clone for mNC.
An oligo(dT)-primed cDNA library derived from EPRO cells was
constructed as described earlier. Approximately 1.5 × 105
plaques were screened with EPRO 6-2, resulting in 57 positive clones.
From these, five clones were purified by secondary and tertiary
screens, after which four of the five clones remained positive and
three were identical in size (2 kb) while the fourth was shorter (about
1.5 kb), as determined by restriction digestion analysis (data not
shown).
Sequence analysis of the longest clone (mNC 4-1) revealed the presence
of a complete open reading frame encoding 465 amino acid residues (Fig
1; Genbank Accession No. U96696). There appears to be a
relatively short 5 untranslated region (UTR) of 73 nucleotides (nt)
and a 3 UTR of about 500 nt. We have not determined if there are
additional 5 untranslated sequences; however, the hNC 5 UTR is
approximately 95 nt, so it is likely that the clone we isolated
represents the full-length mRNA.
At the amino acid level, the human and mouse proteins share 73%
identity, although several functional domains exhibit much higher
similarity (Fig 2). The hNC zymogen is normally latent and can be activated autolytically in response to various
stimuli.25,26 An important region required for latency in
all MMPs is the cysteine switch region found within the propeptide
domain that is thought to interact with the zinc atom bound to the
active site.27 Box 1 in Fig 2 highlights the residues that
comprise the cysteine switch region; the sequence within this region is
identical in hNC and mNC. Box 2 shows that the sequence of the Zn
binding domain/active site is also identical when comparing the two
proteins.
Other sites within hNC that are important for regulation of enzyme
activity include the autolytic activation and degradation sites, as
well as the signal peptidase.25 Each of these residues is
indicated in Fig 2. The residues corresponding to the signal peptidase
(arrow 3) and autolytic activation (arrow 4) cleavage sites are
conserved between the two proteins, whereas the second residue at the
degradation site (arrow 5) is proline rather than leucine.
Expression of mNC during neutrophil maturation.
The MPRO cell line can be induced to undergo neutrophil maturation in
response to superphysiologic concentrations of ATRA.16 By
72 hours in the presence of 10 µmol/L ATRA, greater than 90% of the
cells exhibit neutrophil morphology and express SGP transcripts such as
LF and NG.
To determine if mNC is induced upon neutrophil maturation, we analyzed
mNC levels after exposure of MPRO cells to 10 µmol/L ATRA. By RPA
analysis, uninduced MPRO cells express mNC at a low level (Fig 3, lane
2). Upon initial exposure to ATRA, levels
of mNC transcript increase slightly (lanes 3 and 4) and peak at 72 hours (lane 5), a time at which more than 90% of the cells are neutrophils (data not shown). As a positive control, uninduced EPRO
mRNA (500 ng) was included in the assay (lane 1). Lanes 6 and 7 show
the probes hybridized to yeast RNA with and without RNAse digestion to
confirm the digestion of single-stranded probe while confirming the
absence of hybridization to yeast RNA and to confirm the size and
integrity of undigested probe, respectively. As a control for the
target RNA integrity and amount, a -actin riboprobe was
simultaneously hybridized to all samples (lanes 1 to 7).
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| Fig 3.
RPA of mNC expression in MPRO cells. RNA was isolated
from uninduced and ATRA-induced cells at the indicated time points and subjected to RPA using riboprobes for mNC and -actin. The mNC riboprobe is approximately 500 nt and protects a fragment of 424 nt.
The -actin probe is 330 nt, and the protected fragment is 250 nt.
Lane 1, EPRO polyA RNA (500 ng); lane 2, uninduced MPRO; lane 3, MPRO
induced with ATRA for 24 hours, lane 4, 48 hours, and lane 5, 72 hours;
lane 6, yeast RNA digested with RNase; lane 7, yeast RNA alone,
indicating size of undigested riboprobes.
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To further show that the cDNA we isolated was a neutrophil-specific
gene, we assayed for its expression in a variety of mouse tissues and
cell lines. A number of tissues and murine hematopoietic cell lines
were analyzed for expression of mNC. All mouse tissues assayed do not
express a transcript corresponding to mNC (Fig 4, lanes 1 to
7). Furthermore, uninduced MEL and 32D C13
cells fail to express mNC; the -actin control is absent for the
uninduced 32D C13 sample, indicating probable degradation of this
sample, although we have confirmed nonexpression in uninduced 32D C13 cells by Northern blot analysis (Fig 5).Uninduced MPRO cells (Fig 3, lane 9) express a low level of mNC which
is upregulated upon induction with ATRA (lane 10). After induction with
G-CSF, 32D C13 cells undergo terminal maturation to neutrophils, with
corresponding upregulation of SGP transcripts.4,14 As
expected, mNC is also induced after treatment of 32D C13 cells with
G-CSF (lane 12). As a control for the RNA amount and integrity,
-actin was included in the hybridization (lanes 1 to 14). As
previously described, both riboprobes were hybridized with yeast RNA in
the presence (lane 13) and absence (lane 14) of RNase.
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| Fig 4.
RPA of mNC expression in mouse tissues and cell lines.
RNA from a variety of tissues was subjected to RPA. Lane 1, brain; lane
2, embryo; lane 3, kidney; lane 4, pancreas; lane 5, spleen; lane 6, liver; lane 7, muscle; lane 8, mouse erythroleukemia cells; lane 9, uninduced MPRO cells; lane 10, MPRO cells induced with ATRA for 72 hours; lane 11, uninduced 32D C13 cells; lane 12, 32D C13 induced for
10 days with G-CSF; lane 13, yeast RNA digested with RNase; lane 14, yeast RNA alone.
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| Fig 5.
Northern analysis of 32D cells overexpressing CDP.
32D/CDP and 32D/neo cells were induced with G-CSF, and RNA was
harvested at the indicated time points. Northern analysis was
performed, and the blot was sequentially hybridized with probes for
mLF, mNG, mNC, human p47, and -actin. Lane 1, uninduced 32D/neo
cells; lane 2, 32D/neo induced for 4 days with G-CSF; lane 3, uninduced 32D/CDP cells; lane 4, 32D/CDP induced with G-CSF for 2 days and 4 days
(lane 5); lane 6, 32D C13 induced with G-CSF for 10 days.
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Inhibition of mNC expression by CDP.
CDP has been found to silence the expression of a number of genes,
including the myeloid-specific oxidase component
gp91phox.20,22,28 Recently, we have found that CDP binds to
an element within the LF promoter and serves to inhibit transcription
in uninduced 32D C13 cells.21 Upon G-CSF-induced
neutrophil differentiation, CDP no longer binds to this site and LF
expression increases. We have established a 32D C13 cell line that
overexpresses CDP and fails to induce expression of LF upon induction
with G-CSF while undergoing normal morphologic
differentiation.21 In an effort to provide further evidence
for common regulation of SGP transcript expression, we performed
Northern analysis using 32D/CDP cells and probes for the known mouse
SGP homologs. 32D/CDP and 32D/neo cells were induced with G-CSF, and
RNA was isolated for Northern blot analysis on days 0, 2, and 4. Figure
5 shows a representative Northern blot of bulk cell populations
sequentially probed with mLF, mNG, mNC, and human p47 cDNA fragments.
In 32D/neo cells, mLF, mNG, and mNC are upregulated by day 4 in
response to G-CSF (lane 2), whereas in cells overexpressing CDP, both
of these transcripts fail to be upregulated (Fig 5, lane 2 v
5). In contrast, the oxidase component p47, which is known to be
unaffected by CDP,22 does not exhibit repression in the
presence of excess CDP (lane 5). The blot was probed with -actin to
confirm equal loading of sample RNA. This pattern of SGP gene
expression has been noted in clonal 32D C13 cell lines overexpressing
CDP (data not shown).
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DISCUSSION |
Neutrophils are crucial both for the host responses to bacterial
infections and as critical mediators of inflammation. Secondary granules are secretory granules that fuse with the plasma membrane and
release their contents upon neutrophil activation.3
Although the functions of SGPs are not fully understood, they are
thought to have both bactericidal and proinflammatory functions. LF is hypothesized to sequester iron away from bacteria, contributing to
bacterial killing,29,30 and metalloproteases such as NG and
NC are postulated to contribute to matrix modification at the site of
inflammation.
The development of secondary granules is a late event during neutrophil
maturation, and the detection of SGP expression correlates with the
transition from the promyelocyte stage to later stages of neutrophil
differentiation.3 Stage-specific expression of SGP mRNA can
be detected in primary bone marrow from healthy donors and during in
vitro differentiation of G-CSF-induced CD34+ progenitor
cells.31 In contrast, leukemic cell culture models of
myeloid differentiation, such as HL60 and NB4, show a defect in SGP
gene expression2,12,13,32 and fail to express SGP transcripts even after induction of neutrophil maturation by chemical agents such as DMSO or ATRA.10,12 Therefore, to study the
control of SGP gene transcription, it is necessary to use murine
factor-dependent cell lines that have a more normal pattern of
differentiation; this requires isolation of the corresponding murine
SGP genes.
In this study, we have described the cloning and characterization of
expression of mNC. While performing a RDA screen for differentially
expressed genes, we isolated a fragment with significant homology to
hNC. Sequence analysis of the corresponding cDNA showed a high degree
of homology at the nucleotide level to hNC (data not shown). The mNC
clone contains a 1,395-bp open reading frame (Fig 1), and the
hypothetical translation of this sequence reveals 73% identity with
hNC (Fig 2). Not surprisingly, the functional domains common to MMPs,
ie, the zinc binding and cysteine switch regions, are identical between
the mouse and human proteins, as are the signal peptidase and autolytic
activation sites. Interestingly, the autolytic degradation site is not
conserved between the two proteins (Fig 2, arrow 3): degradation occurs
in hNC at the G262-L263 bond, but the L residue is a P residue in the
mouse protein. Although the hydrophobic character of this residue is
maintained, the potential structural ramifications and their likely
impact on the rate of degradation remain unclear.
We have characterized mNC expression in a variety of tissues and
hematopoietic cell lines. The RPA studies presented here show no
evidence for expression of mNC in nonhematopoietic tissues. Unlike
interstitial collagenase, which is widely expressed, NC expression has
been thought to be restricted to neutrophils. Recent studies have
suggested that hNC may also be expressed by chondrocytes,33 especially following stimulation with IL-1,34 and that
it may play a role in articular destruction in arthritis. If this is also a characteristic of mNC, then isolation of the cDNA described here
may have potential value in the investigation of mouse models of
arthritis.
Our primary interest in mNC is focused on the pattern of coordinate
tissue- and stage-specific expression that it may share with other
SGPs. The studies presented here provide support for the hypothesis
that the positive and negative regulation of these genes is linked.
Within hematopoietic cell lines, our studies confirm that mNC follows
the predicted pattern of expression shared by other SGP genes. The MPRO
cell line is arrested at the promyelocyte stage and undergoes
neutrophil maturation16 with expression of SGP transcripts
upon induction with ATRA. The transcript for mNC is expressed at low
levels in uninduced MPRO cells, in contrast to both mLF and mNG, while
all three SGP genes are upregulated in response to ATRA. Furthermore,
mNC is also expressed in G-CSF-induced 32D C13 cells in concert with
the other SGP genes.
As already noted, expression of SGP genes is impaired in leukemic cell
lines induced to undergo differentiation toward neutrophils, such as
NB4 and HL60. A similar defect in SGP expression has been described in
myelodysplasia and in the residual neutrophils of patients with newly
diagnosed leukemia.35,36 We therefore hypothesize that the
pathogenesis of these malignancies involves an event that disrupts the
differentiation program even in cells that retain a capacity for
limited maturation, as reflected in the coordinate absence of
expression of SGP mRNA.
In this regard, the pattern of SGP gene repression in cells
overexpressing CDP is striking. CDP was initially identified as a
repressor of sperm H2B transcription in sea urchin
embryos.28 It has since been shown to repress a wide
variety of genes in many species, including gp91-phox, the  -globin
genes, and mouse NCAM and myosin heavy-chain.20,22,37,38
Studies from our laboratory have shown that CDP binds to a region
within the LF promoter that possesses silencing activity.21
Furthermore, overexpression of CDP in the 32D C13 cell line results in
inhibition of LF induction without blocking morphologic
differentiation. We show here that expression of the mNC gene,
characterized in this study, as well as mNG, is similarly repressed at
the mRNA level by CDP overexpression. The cloning and characterization
of mNC has thus allowed us to add further credence to the hypothesis
that shared trans-regulatory elements may be responsible for
both positive and negative regulation of SGP genes. Furthermore, we
have identified CDP as the first transcriptional regulator that is a
candidate for mediating coordinate repression of these genes, although
potential shared activating factors remain to be found.
| |
FOOTNOTES |
Submitted September 22, 1997;
accepted November 14, 1997.
Supported by National Institutes of Health (NIH) Grants No. DK48053 and
HD33184 (N.B.), a Scholar Grant from the Leukemia Society of America
(N.B.), and NIH Training Grant No. GM07499 (N.D.L.).
Address reprint requests to Nancy Berliner, MD, Department of Internal
Medicine, Section of Hematology WWW428a, Yale University School of
Medicine, 333 Cedar St, New Haven, CT 06510.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract
