From the Massey Cancer Center and the Departments of
§ Internal Medicine, ¶ Biochemistry and Molecular
Biophysics, and §§ Human Genetics, Medical
College of Virginia at Virginia Commonwealth University, Richmond,
Virginia 23298 and the ** Institute of Human Genetics, University of
Minnesota, Minneapolis, Minnesota 55455
Received for publication, October 17, 2000
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ABSTRACT |
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Human major histocompatibility (MHC) class I
antigen expression is important in controlling the metastatic growth of
malignant tumors. Locus-specific down-regulation of MHC class I gene
expression is frequently observed in human tumors, leading to decreased
susceptibility to cytotoxic T-cell-mediated lysis. The mechanism of
this down-regulation is incompletely understood. Here, we describe the
identification of human CCAAT displacement protein
(CDP/cut) as a locus-specific repressor of HLA-B and C gene
expression. Transient and stable transfections in HeLa and K562 cells
demonstrated the presence of a repressor element 650 base pairs
upstream of the first exon of HLA-B7. A specific binding complex with
the HLA-B7 and Cw2 repressor elements was demonstrated by EMSA.
Formation of the EMSA complex was inhibited specifically with
polyclonal antiserum to human CDP/cut, demonstrating that
CDP/cut binds the HLA-B7 repressor element. The
corresponding region of the HLA-A2 promoter neither repressed HLA-A2
gene expression nor bound CDP/cut. Overexpression of
CDP/cut in cell lines deficient in CDP/cut
resulted in a nearly 4-fold repression of reporter constructs
containing the HLA-B7 repressor element but not the corresponding
region of the HLA-A2 promoter. Repression of HLA-B and C gene
expression by CDP/cut does not involve displacement of
NF-Y, nor is CDP/cut associated with the histone
deacetylase HDAC1 when bound to the HLA-B7 repressor element. To our
knowledge, these results identify CDP/cut as the first
example of a locus-specific repressor of MHC class I gene transcription
in human tumor cells.
The human MHC1 class I
genes encode the highly polymorphic HLA class I antigen heavy chains,
membrane-spanning proteins that combine with the invariant
Down-regulation of MHC class I gene expression is observed in many
human tumors and transformed cell lines, resulting in decreased susceptibility to cytotoxic T-cell-mediated lysis (8-13). Global down-regulation of surface class I expression can arise by several mechanisms, including loss of The mechanisms that mediate locus-specific down-regulation of MHC class
I gene expression are incompletely understood. Transcriptional mechanisms appear to be involved in many cases (17-19) but have not
been well characterized. The promoter and 5'-flanking regions of most
MHC class I genes contain several highly conserved, well characterized
DNA sequence elements involved in maintenance of constitutive
expression (20-22). Down-regulation of MHC class I expression due to
decreased binding of members of the rel family of
transcription factors to the highly conserved enhancer A element has
been reported in human tumor cell lines (23) and in
adenovirus-12-transformed cell lines (24). Our laboratory has
previously shown that another highly conserved element, the
interferon-stimulated response element, functions as a locus-specific
activator of HLA-A gene expression in several hematopoietic tumor cell
lines (25). The relative contribution of these and other positive
regulatory elements in determining the steady state expression of
specific MHC class I genes in normal and malignant cells remains to be elucidated.
In addition to positive regulatory elements, DNA sequence elements that
mediate repression of classical MHC class I gene expression have been
identified in the 5'-flanking region of some MHC class I genes,
including the mouse classical class I genes H2-K and H2-L (26-31) and the swine class I gene,
PD1 (32-34). Some of these elements are tissue- and/or
differentiation-specific in their function. The factors that bind to
most of these elements have not been fully characterized, and none of
these elements has been shown to function in a locus-specific manner.
Many of the factors that bind MHC class I gene regulatory elements
produce a relatively small (2-3-fold) effect on MHC class I gene
expression when acting alone. However, even small changes in the level
of constitutive MHC class I mRNA levels have been shown to result
in much larger changes in MHC class I surface antigen expression and
cytotoxic T-cell function (35-38). It is likely that the large
locus-specific variations in MHC class I gene expression observed in
many human tumors are the result of net changes in the relative levels
of several individual MHC class I transcriptional activators and
suppressors in a given tumor type.
In the current studies, we report the identification of human
CDP/cut as a locus-specific (HLA-B and -C versus
HLA-A) repressor of MHC class I gene expression in human tumor cells.
We hypothesize that CDP/cut may contribute to the
suppression of constitutive HLA-B7 gene expression observed in some
human tumors (15).
Plasmid Construction--
All reporter constructs are based on
the promoterless chloramphenicol acetyltransferase (CAT) reporter
plasmid pCAT basic (Promega, Madison, WI). The wild type HLA-B7 CAT
reporter plasmid was constructed by inserting a PCR-generated fragment
of the HLA-B7 promoter (containing nt Cell Culture, Transfections, and Reporter Gene Assays--
HeLa
and Panc-1 cells were grown in Dulbecco's modified Eagle's medium
with 10% fetal bovine serum. K562 cells were grown in RPMI 1640 with
10% fetal bovine serum (Life Technologies, Inc.). JY cells were grown
in Iscove's medium (with L-glutamine) containing 10%
fetal bovine serum. For transfection of K562 cells, 1 × 107 cells (in log growth phase) were cotransfected with
20 µg of test plasmid and 5 µg of an internal control plasmid
containing the
HeLa and Panc-1 cells (1 × 106 cells at 70%
confluence) were transiently transfected with 5 µg of test plasmid
and 0.5 µg of CMV-
For determination of CAT activity in pooled wild type HLA-B7 CAT and
HLA-B7 del-CDP CAT stable K562 transfectants, 1 × 106
cells from each pool of transfectants was harvested and assayed for CAT
enzyme activity as described above. CAT activity was normalized to the
transfected CAT gene copy number determined by slot blotting genomic
DNA and hybridizing with a radiolabeled cRNA probe containing nt 1-214
of the CAT gene.
Preparation of Nuclear Extracts--
Nuclear protein extracts
were prepared according to the method of Dignam (43) in the presence of
the protease inhibitors leupeptin (10 µg/ml), pepstatin (10 µg/ml),
aprotinin (10 µg/ml), and phenylmethylsulfonyl fluoride (0.5 mM). Protein concentrations in extracts were determined
using a Coomassie-based protein assay (Bio-Rad).
Electrophoretic Mobility Shift Assays
(EMSAs)--
Double-stranded DNA fragments containing nt
For detection of NF-Y binding activity in JY nuclear extracts, probes
were incubated with 10 µg of nuclear extract under previously described conditions (44) and run on 3.5% polyacrylamide gels in 0.5×
TBE. A 27-bp oligonucleotide containing a consensus NF-Y binding site
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as a positive
control. Goat polyclonal antibodies to NF-Y subunits A, B, and C or
preimmune goat polyclonal IgG (Santa Cruz Biotechnology) were added (2 µg each) to reactions as indicated and incubated at room temperature
for 45 min prior to the addition of probe.
EMSA to detect methylcytosine-binding protein (MeCP1) binding activity
in HeLa nuclear extracts was performed exactly as described (45).
Depletion of HDAC1 from Nuclear Extracts--
Goat monoclonal
antibody to the C-terminal portion of HDAC1 (15 µg; Santa Cruz
Biotechnology) or goat preimmune IgG (0.8 µg; Santa Cruz
Biotechnology) was immobilized to 200 µl of protein G-agarose beads
(Pierce) as described (46). The beads were incubated with HeLa nuclear
extract for 3 h at 4 °C. Aliquots of HDAC1-depleted nuclear
extract were used in EMSA as described above.
The HLA-B7 Promoter Contains an Upstream Repressor Element--
We
initially identified two closely spaced potential binding sites for the
transcriptional repressor CDP/cut in the HLA-B7 promoter,
~650 nt upstream of exon I (Table I).
Both of these AT-rich segments are homologous to the consensus binding
site for the cut repeat 1 and 2 (CR-1 and CR-2) domains of
CDP/cut (47), although only the 5' site contains the CCAATA
core sequence, which appears to be important for efficient
CDP/cut binding (47, 48) (Table I). To determine whether
these elements mediate repression of HLA-B7 gene expression,
heterologous CAT reporter constructs containing a 664-nt fragment of
the HLA-B7 promoter (Fig. 1A)
were transiently transfected into HeLa cells, which express high levels
of endogenous CDP/cut (42). Deletion of a 120-nt region
containing the putative HLA-B7 CDP/cut binding sites (HLA-B7
del CDP construct, Fig. 1A) resulted in a statistically significant (p
Sequence information for the corresponding regions of other MHC class I
loci and alleles is limited. A GenBankTM search revealed a
total of only three sequence variations in the regions directly
corresponding to the two potential HLA-B7 promoter CDP/cut
binding sites (Table I). As shown in Fig. 1B, an HLA-B7
promoter reporter construct in which the HLA-B7 repressor element was
converted to the corresponding region of the HLA-Cw2 promoter also
mediated repression of reporter gene expression. In contrast to HLA-B7
and HLA-Cw2, deletion of the homologous region of the HLA-A2 promoter
resulted in a significant decrease rather than an increase in
expression (Fig. 1B, upper portion), demonstrating that this element represses MHC class I gene expression in a locus-specific manner.
To determine whether the HLA-B7 repressor element functions when stably
integrated into chromosomal DNA, the wild type HLA-B7 CAT and HLA-B7
del CDP CAT constructs were stably transfected into K562 cells.
Deletion of the HLA-B7 repressor element resulted in ~3-fold higher
CAT activity (when corrected for gene copy number) compared with the
wild type HLA-B7 CAT construct (data not shown).
Binding of CDP/cut to the HLA-B7 Repressor Element Is
Locus-specific--
A double-stranded DNA probe containing the HLA-B7
repressor element identified in Fig. 1 was used in EMSA to determine
whether any specific binding activity was associated with this element. When this probe was incubated with nuclear extracts from HeLa cells
(Fig. 2), a specific, low mobility
complex was formed. An excess of unlabeled probe (Fig. 2) effectively
competed with the wild type probe for binding. Analogous to the results
obtained in the functional studies (Fig. 1), specific DNA competitors
that contained mutations in either the upstream CCAAT box or the
downstream AT-rich sequence were able to partially compete with the
wild type HLA-B7 probe for binding (Fig. 2). The corresponding region of the HLA-Cw2 promoter also competed effectively for binding (Fig.
3C). In contrast, competitor
DNA in which both potential CDP/cut binding sites in the
HLA-B7 promoter were disrupted was not able to compete for binding
(Fig. 2). Finally, in agreement with the functional data in Fig. 1, the
corresponding region of the HLA-A2 promoter competed for binding much
less effectively than the HLA-B7 WT specific competitor (Fig. 3,
A, B, and D).
The addition of a polyclonal guinea pig antibody to human
CDP/cut resulted in complete ablation of the specific
complex (Fig. 4), demonstrating that the
protein complex that binds to the HLA-B7 repressor element contains
human CDP/cut. Preimmune serum from the same animal had no
effect on CDP/cut binding to the HLA-B7 repressor element
(Fig. 4).
Overexpression of CDP/cut in a Cell Line Deficient in CDP/cut
Represses the HLA-B7 Promoter, but Not the HLA-A2 Promoter--
To
further establish that CDP/cut represses HLA-B7 expression
by binding to the HLA-B7 repressor element, the HLA-B7 CAT and the B7
del-CDP CAT reporter constructs were cotransfected with a full-length
CDP/cut expression vector in CDP/cut-deficient
Panc-1 cells (49). EMSA was performed on nuclear extract prepared from Panc-1 cells to confirm decreased CDP/cut DNA-binding
activity in these cells (Fig.
5A). The empty expression
vector was cotransfected as a control. As shown in Fig. 5B,
overexpression of CDP/cut resulted in a 3.5-fold suppression
of HLA-B7 promoter reporter gene expression, whereas CDP/cut
overexpression had no effect on reporter gene expression when the
CDP/cut binding sites were deleted from the construct. In
the absence of CDP/cut expression, the HLA-B7 repressor element did not mediate repression of the HLA-B7 promoter in
CDP/cut-deficient Panc-1 cells (Fig. 5B, compare
the two experiments with the empty expression vector PMT2 only).
Finally, overexpression of CDP/cut did not repress a
reporter construct containing the corresponding region of the HLA-A2
promoter (Fig. 5B). Similar experiments were attempted in
CDP/cut-deficient JY cells, but CDP/cut
overexpression resulted in unacceptable toxicity in these cells (data
not shown).
The HLA-B7 CDP/cut Binding Site Represses Activated Transcription
from a Non-MHC Class I Promoter--
To determine whether binding of
CDP/cut to the HLA-B7 repressor element is able to repress
transcription from a promoter other than the HLA-B7 or HLA-Cw2
promoters, the HLA-B7 CDP/cut binding site was inserted 800 nt upstream of a CAT reporter construct containing 156 nt of the
HSV tk promoter (Fig. 6).
Overexpression of CDP/cut repressed expression of this
construct nearly 6-fold in HeLa cells relative to the wild type HSV
tk promoter reporter construct (Fig. 6). Similar experiments
in the CDP/cut-deficient cell lines Panc-1 and JY were not
successful due to extremely low basal expression of constructs driven
by the HSV tk promoter (data not shown).
Repression of HLA-B7 Gene Expression by CDP/cut Is Not Mediated by
Displacement of the Transcriptional Activator NF-Y and Does Not Involve
Association of CDP/cut with HDAC1--
Previous studies have shown
that CDP/cut represses transcription by some promoters by
displacing the positive regulatory factor NF-Y, which binds CCAAT box
elements (50-52). To determine whether NF-Y binds the HLA-B7 repressor
element, nuclear extracts from JY cells, a lymphoblastoid B-cell line
with decreased expression of CDP/cut (Fig. 5A),
were used in EMSA with an HLA-B7 repressor element probe. EMSA was
carried out under conditions that have been shown to promote NF-Y
binding (44). As shown in Fig.
7A, the addition of goat
polyclonal antibodies to NF-Y subunits A, B, and C did not ablate or
supershift any HLA-B7 repressor element binding complexes, suggesting
that this element does not bind NF-Y. As a positive control, a
double-stranded oligonucleotide containing an NF-Y consensus binding
site was incubated with the same JY nuclear extracts (Fig.
7B), demonstrating the ability of this assay to detect NF-Y
binding activity in these extracts.
Results from recent studies suggest that CDP/cut may
actively repress expression of some genes by associating with the
histone deacetylase HDAC1 (49, 53). To determine whether
CDP/cut is associated with HDAC1 in the HLA-B7 repressor
element binding complex, EMSA was carried out with HeLa nuclear
extracts in which HDAC1 had been immunodepleted. As shown in Fig.
8A, depletion of HDAC1 had no
effect on the formation or mobility of the
CDP/cut-containing complex. Depletion of HDAC1 from the
extracts was confirmed by demonstrating inhibition of MeCP1 complex
formation (Fig. 8B). Association of MeCP1 with HDAC1 is
required for MeCP1 to bind DNA (46). Moreover, treatment of HeLa cells
with the histone deacetylase inhibitor trichostatin A (in experiments
analogous to those shown in Fig. 1B) failed to relieve
repression of HLA-B7 wild type reporter gene expression (data not
shown).
In these studies, we have identified what is to our knowledge the
first example of a locus-specific (HLA-B and -C versus
HLA-A) repressor element in an MHC class I gene promoter. These
experiments also identify the protein that binds to this novel
repressor element as CDP/cut, the mammalian homologue of the
Drosophila protein cut (54). CDP/cut
overexpression in Panc-1 cells results in a 3.5-fold repression of the
HLA-B7 promoter (Fig. 5B). This degree of repression is
similar to that reported for several other genes containing one or two
CDP/cut binding sites (55-57). CDP/cut has not
been shown previously to function in a locus or allele-specific manner.
While complete sequence information in this region of the promoter is
available for only a few MHC class I genes, many of the alleles in
Table I occur in vivo with high frequency. Therefore, it is
likely that the most frequently occurring HLA-B and C alleles are
susceptible to repression by CDP/cut, whereas most HLA-A
alleles and nonclassical MHC class I genes are not. In contrast, none
of the murine MHC class I genes (H2-D, H2-K, and
H2-L) contain any regions that are homologous to the
HLA-B7 repressor element.
CDP/cut is a ubiquitous transcriptional repressor first
identified as a negative regulator of the sperm-specific sea urchin histone H2-B-1 gene (58). Since then,
CDP/cut has been shown to repress transcription of a number
of genes, including myeloid cytochrome gp91-phox (59, 60),
lactoferrin (61), c-myc (62), p21 (63), histone H4
(64), tryptophan hydroxylase (65), the transforming growth factor- CDP/cut contains a distinctive homeodomain and three highly
conserved CRs. Each of the three CRs and the homeodomain of
CDP/cut are able to bind DNA independently with broad
binding specificities that are distinct but overlapping (47, 69). While
the overall consensus binding sequence contains significant
variability, the CCAATA core has been shown to be important for
efficient CDP/cut binding (47, 48). As shown in Table I, the
HLA-B7 repressor element contains two closely spaced regions which are
homologous to the consensus binding sequences for the CR-1 and CR-2
domains of CDP/cut (47, 48). The EMSA and immunoablation
experiments in Figs. 2-4 demonstrate that CDP/cut binds
selectively to this region upstream of HLA-B and C promoters. Both the
CCAAT and TATT motifs in the HLA-B7 promoter must be altered
simultaneously to disrupt CDP/cut binding and to relieve
transcriptional repression (Figs. 1 and 2), suggesting that
CDP/cut-binding to the HLA-B7 repressor occurs through
either site. In the HLA-A2 promoter, both the CCAAT and TATT motifs
contain sequence variations that have been shown to disrupt binding of
CDP/cut to other targets (47, 48). Although the 5' CCAAT
motif of the HLA-A2 promoter differs from the HLA-B7 promoter CCAAT
motif by only one base pair (Table I), the mutation alters the
important CCAATA core sequence. This sequence variation significantly
interferes with CDP/cut binding to the HLA-A2 promoter (Fig.
3, B and D) and results in the inability of
CDP/cut to repress expression from the HLA-A2 promoter
(Figs. 1B and 5B). In contrast, the corresponding
region of the HLA-Cw2 promoter contains two CAATA motifs, consistent with the apparent higher affinity of this site for CDP/cut
(Fig. 3C). A recent study demonstrated that cooperative
binding of isolated recombinant CDP/cut CR-1 and CR-2
domains is enhanced by the presence of two juxtaposed CAAT sites,
compared with a single site (48). The same study also showed that the
ATCAAT motif, present in the HLA-Cw2 promoter (Table I), is a high
affinity site for cooperative binding of the CDP/cut CR-1
and HD domains. Despite increased affinity of the HLA-Cw2 promoter for
CDP/cut compared with the HLA-B7 promoter, both of these
elements repress reporter gene expression to a similar extent (Fig.
1B). Since the degree of repression observed with either
element is relatively small, it is possible that any difference may not
be detectable. Alternatively, maximal repression may occur only when
one CDP/cut site is occupied due to interference from a
closely bound second CDP/cut molecule at the adjacent site
or due to conformational restraints resulting from a single
CDP/cut molecule bound at both sites.
The mechanism of transcriptional repression of HLA-B and -C locus gene
expression by CDP/cut remains to be elucidated.
CDP/cut was initially identified as a protein that directly
displaces the transcriptional activator CCAAT-binding protein in the
histone H2-B-1 gene (58) and the gp91-phox
gene (59). In the immunoglobulin heavy chain gene, CDP/cut
represses transcription by displacing the B-cell-specific Bright
transcription factor from its binding site in the intronic enhancer
(67). Displacement of the CCAAT-binding transcriptional activator NF-Y
by CDP/cut has been demonstrated for a few genes (50, 52).
In CDP/cut-deficient Panc-1 cells, the HLA-B7 repressor
element functions as a weak activator of the HLA-B7 promoter (see Fig.
5B, experiments with the empty expression vector PMT2),
which suggests that CDP/cut may be displacing a positive
transcriptional activator from an overlapping binding site. However,
our EMSA results using nuclear extracts that contain NF-Y, but very
little CDP/cut, suggest that NF-Y does not bind the HLA-B7
repressor element (Fig. 7). A TRANSFAC analysis (70) using the 32-nt
HLA-B7 and Cw2 repressor elements failed to identify other known
transcription factors that could potentially bind these regions.
Therefore, if CDP/cut represses HLA-B and -C gene expression
by displacement of positive regulatory factors, the identity of these
factors remains unknown. Alternatively, the 120-nt DNA sequence used in
the reporter constructs in this study may contain an unidentified
positive regulatory element for which activity is only observed in the
absence of CDP/cut expression.
Other evidence suggests that the C-terminal domain of
CDP/cut functions at a distance as a direct transcriptional
repressor of some genes (71), possibly by association with histone
deacetylase activity (49, 53). However, our results indicate that
neither DNA binding nor transcriptional repression of HLA-B7
gene expression by CDP/cut appear to require HDAC1. Taken
together, these observations suggest that CDP/cut represses
transcription by multiple, promoter-specific mechanisms. Whatever the
mechanism, transcriptional repression mediated by CDP/cut
binding to the HLA-B or -C repressor elements does not appear to
involve interactions that are specific for MHC class I promoters. As
seen in Fig. 6, activated transcription driven by the HSV tk
promoter is repressed nearly 6-fold when the HLA-B7 repressor element
is inserted upstream of the tk promoter.
While MHC class I repressor elements have been previously described,
none of these elements are locus-specific. Only one of these elements,
a silencer identified in the miniature swine class I gene
PD1, is homologous to the HLA-B locus repressor
element (34). The PD1 silencer element was among the first
negative elements to be described in an MHC class I gene. Given the
similarity of the swine PD1 silencer element to the human HLA-B
repressor element, it is possible that swine CDP/cut also
binds the PD1 silencer element.
Most reported regulatory factors have a relatively small effect on MHC
class I gene transcription, depending on the cell type and the specific
MHC class I locus (25, 72-74). However, even small changes in MHC
class I gene expression (i.e. 2-fold) can have a profound
effect on MHC class I antigen cell surface expression and the resultant
effect on cytotoxic T-cell function (35-38). For this reason, it is
likely that overexpression of CDP/cut in tumor cells, which
produces up to a 3.5-fold decrease in expression driven by the HLA-B7
promoter (Fig. 5B), has important consequences on MHC class
I antigen surface expression.
In summary, we have identified a repressor element that mediates
locus-specific down-regulation of HLA-B and -C (versus
HLA-A) gene expression in human tumor cell lines. We have also shown that the transcriptional repressor CDP/cut binds this
element in a locus-specific manner and mediates the observed
repression. It is interesting to speculate that overexpression of
CDP/cut contributes to the frequent observation of selective
HLA-B locus down-regulation in melanoma, colon cancer, and other human
tumors. Understanding the mechanisms that control locus-specific
regulation of MHC class I gene expression has potentially important
implications for the development of vaccine-based cancer therapeutics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-microglobulin to form the HLA class I surface antigen
(1). Classical MHC class I (HLA-A, -B, and -C) antigen expression is
essential for processing and presentation of peptide antigens to
cytotoxic CD8+ lymphocytes (2). In addition, recent evidence suggests
that certain classical and nonclassical class I antigens play an
important role in inhibition of natural killer (NK) cell function
(3-6) and certain subsets of cytotoxic T-cells (7). Therefore, changes
in MHC class I gene expression can have profound effects on the overall
susceptibility to NK cell and cytotoxic T-cell-mediated lysis, the net
effect depending on the specific MHC Class I genes involved.
2-microglobulin or peptide
transporter gene expression (9, 14). More frequently, loss of
MHC class I antigen expression in tumor cells occurs at a single locus. For example, selective down-regulation of HLA-B locus gene expression has been observed in many cell lines derived from patients with metastatic melanoma (15) and colon cancer (16). The specific MHC class
I alleles in a particular tumor that activate cytotoxic T-cells may be
different from those that inhibit NK cell function. Therefore,
understanding how MHC class I antigen expression is controlled at the
level of specific loci and alleles is an important goal with possible
implications for the development of vaccine-based cancer therapeutics.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
672 to
8, numbered with
respect to the beginning of exon 1) into the
HindIII/PstI site of the polylinker region of
pCAT basic. An HLA-B7 genomic clone kindly provided by Dr. Sherman
Weissman (39) was used as a template for PCR. The plasmids HLA-B7
del-CDP, B7 CCAAT mutant, B7 AT mutant, B7 double mutant (Fig.
1A), and B7 Cw2 mutant were generated from wild type HLA-B7
pCATb using PCR overlap extension techniques (40). The plasmid A2 pCATb
wild type (WT) was constructed by inserting a PCR-generated fragment
containing 687 nt of the HLA-A2 promoter (using genomic DNA from the
HLA-A2 homozygous cell line, JY, as a template) into the polylinker
region of pCAT basic. The plasmid A2 del CDP pCATb contains only the
first 525 nt of the HLA-A2 promoter (25). Sequences of all
PCR-generated fragments and mutations were verified by the dideoxy
chain termination method. For stable transfections, the neomycin
resistance gene was amplified by PCR, using the plasmid pTarget
(Promega, Madison, WI) as a template, and ligated into the
BamHI sites of wild type HLA-B7 pCATb and HLA-B7 del-CDP
pCATb. The plasmid tk-CAT-CDP was constructed from
tk pCAT basic (41), which contains nt
105 to +51 of the herpes simplex virus thymidine kinase (HSV tk) promoter in
pCAT basic. A 120-nt fragment of the HLA-B7 promoter containing the HLA-B7 repressor element (nt
672 to
553) was generated by PCR, with
the appropriate restriction enzyme sites incorporated into the primers,
and inserted 800 bp upstream of the tk promoter in tk pCAT basic (at the AlwNI site). The plasmid
PMT2-CDP+, an adenovirus major late promoter expression vector
containing full-length CDP/cut cDNA, and the empty
expression vector, PMT2, were kindly provided by Dr. Ellis Neufeld
(42). The CAT cRNA vector for slot blotting (see below) was constructed
by inserting nt 1-214 of the CAT gene into the
EcoRI/HindIII site of pGem3zf(
) (Promega).
After linearizing the plasmid with HindIII, T7 RNA
polymerase and [
-32P]ATP were used to generated the
CAT cRNA. All plasmids were purified by double cesium chloride banding.
-galactosidase gene driven by the cytomegalovirus
promoter (CMV-
-Gal) using a Gene Pulser (Bio-Rad) (250 V, 950 microfarads). For stable transfection, the cells were selected with
G418 (0.5 mg/ml) beginning 48 h after transfection. For transient
transfection, cells were harvested after 48 h, and lysates were
prepared for determination of CAT enzyme activity according to the CAT
enzyme assay system protocol (Promega) with slight modifications.
Briefly, cells were washed twice with phosphate-buffered saline,
pelleted, and resuspended in 0.1 ml of 0.25 M Tris-HCl (pH
7.8). Cells were then subjected to three freeze/thaw cycles (5 min
each) and centrifuged at 4 °C for 5 min. The supernatants were
heated to 65 °C for 10 min prior to assaying CAT enzyme activity
using n-butyryl-coenzyme A (Promega) and
[14C]chloramphenicol (Amersham Pharmacia Biotech)
followed by extraction with mixed xylenes and liquid scintillation
counting.
-Galactosidase activity in lysates was measured using an
assay from Promega. CAT activity was normalized to
-galactosidase
activity for all transient transfection samples.
-Gal (10 µg for Panc-1 cells) using calcium
phosphate coprecipitation. Panc-1 cells were also cotransfected with 20 µg of PMT2 or PMT2-CDP+. After 48 h, cells were harvested and
assayed for CAT and
-galactosidase activity as above. In some
experiments, trichostatin A was added to a final concentration of 100 nM 6 h after transfection.
666 to
547 of the HLA-B7 promoter region were generated by PCR using the
following plasmids as templates: wild type HLA-B7 CAT, B7 CCAAT mutant
CAT, B7 AT mutant CAT, B7 double mutant CAT, and B7 Cw2 mutant CAT. The
corresponding region of the HLA-A2 promoter (nt
666 to
546) was
generated by PCR using HLA-A2 pCATb WT as a template. The PCR products
were gel-purified and used directly as specific competitors for EMSA.
The gel-purified wild type B7 promoter PCR product was end-labeled with
[
-32P]ATP using T4 polynucleotide kinase (New England
Biolabs, Beverly, MA) and used as a probe. Except where indicated, EMSA
was carried out by incubating nuclear extract (10-20 µg of protein),
6 µg of poly(dI-dC), ATP (1 mM), 0.5 µl of guinea pig
anti-CDP/cut immune serum or preimmune serum (a kind gift
from Dr. Ellis Neufeld) when indicated, and specific DNA competitors,
when indicated, in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 5% glycerol, and 0.2 mM
dithiothreitol in a final volume of 20 µl on ice for 15 min.
Radiolabled probe (25 fmol, 50,000-100,000 cpm) was added, and the
incubation on ice was carried out for an additional 30 min. The binding
reaction was then separated on a 4% nondenaturing polyacrylamide gel
in 0.5× Tris borate-EDTA at 4 °C. Gels were then dried and
visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.05), greater than 2-fold increase
in reporter gene expression (Fig. 1B), demonstrating that
this region of the HLA-B7 promoter contains a repressor element.
Similar results were observed in K562 cells (data not shown).
Constructs containing mutations of either of these AT-rich regions were
still able to mediate repression of reporter gene expression (Fig.
1B). Simultaneous mutation of both of these regions resulted
in a statistically significant 2-fold increase in reporter gene
expression equivalent to that observed with deletion of the entire
region containing the putative CDP/cut binding sites (Fig.
1, A and B).
Potential CDP/cut binding sites in MHC class I promoters
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Fig. 1.
HLA-B and -C promoters contain a
locus-specific repressor element located 650 nt upstream of exon
1. A, HLA-B7 promoter reporter constructs based on the
promoterless CAT reporter plasmid pCATbasic. Nucleotide
positions are numbered relative to the beginning of HLA-B7 exon 1. The
regions of the wild type HLA-B7 promoter that are homologous to the
CDP/cut CR-2 consensus binding site (Table I) are shown in
boldface type. Nucleotides that differ from the
wild type HLA-B7 promoter are underlined in each of the
mutant constructs. Both potential CDP/cut binding sites were
deleted in the HLA-B7 del CDP construct. B, transient
transfections of the HLA-B7 CAT constructs in A were carried
out in HeLa cells as described under "Experimental Procedures." The
plasmid HLA-A2 WT pCATb contains 687 bp of the HLA-A2 promoter. HLA-A2
del-CDP pCATb contains 525 bp of the HLA-A2 promoter (the potential
CDP/cut binding sites have been deleted, analogous to the B7
del CDP construct). In the plasmid B7 Cw2 mutant pCATb, the HLA-B7
repressor element has been converted to the corresponding sequence in
the HLA-Cw2 promoter (see Table I). Results, normalized to
-galactosidase activity, are reported as the mean of at least three
independent experiments. Error bars indicate S.E.
values for each set of experiments. Asterisks indicate
values that are significantly different from the HLA-B7 WT construct
(p
0.05).
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Fig. 2.
A specific complex binds the HLA-B7 repressor
element. EMSA was performed using HeLa nuclear extracts and a
120-bp double-stranded radiolabeled DNA probe containing the HLA-B7
repressor element (nt 666 to
547). The specific competitors are
described under "Experimental Procedures," and their sequences are
shown in Fig. 1A and Table I. The molar -fold excess of each
competitor is indicated.
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Fig. 3.
The HLA-B7 repressor element binding complex
is locus-specific. A-C, EMSA was carried out with a
HLA-B7 WT repressor element probe, as in Fig. 2, with specific
competitors as indicated in each image. The molar -fold
excess of each competitor is shown at the top of each
image. D, binding to the HLA-B7 WT probe was
quantitated by PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA) analysis for the experiments shown in A and
B.
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Fig. 4.
CDP/cut binds the HLA-B7
repressor element. EMSA was performed as in Fig. 2 in the presence
of guinea pig preimmune serum or polyclonal CDP/cut immune
serum.
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Fig. 5.
Overexpression of CDP/cut in
the CDP/cut-deficient cell line Panc-1 represses
expression from the HLA-B7 promoter but not the HLA-A2 promoter.
A, EMSA carried out as in Fig. 2 using equivalent protein
amounts of nuclear extracts from the indicated cell lines to
demonstrate decreased CDP/cut expression in JY and Panc-1
cells. B, transient cotransfections of the indicated
reporter constructs with a CDP/cut expression vector
(PMT2CDP) or the empty expression vector (PMT2) were carried out in
CDP/cut-deficient Panc-1 cells. Results, normalized to
-galactosidase activity, are presented as the mean of at least four
independent experiments. Error bars indicate the
S.E. for each set of experiments.
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Fig. 6.
The HLA-B7 repressor element mediates
repression of a non-MHC class I promoter. A wild-type HSV
tk-CAT reporter construct and the same construct containing
the HLA-B7 repressor element inserted 800 bp upstream of the HSV
tk promoter were transiently cotransfected with a
CDP/cut expression vector (PMT2CDP) in HeLa cells. Results,
normalized to cotransfected -galactosidase activity, are presented
as the mean of at least three independent experiments. Error
bars indicate the S.E. value for each set of
experiments.
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Fig. 7.
NF-Y does not bind the HLA-B7 repressor
element. A, EMSA was carried out using JY nuclear
extracts (which contain reduced levels of CDP/cut) under
conditions that promote binding of NF-Y (see "Experimental
Procedures"). Polyclonal antibodies to NF-Y subunits or preimmune
polyclonal IgG were added as indicated. B, EMSA was carried
out as in Fig. 7A except that an NF-Y consensus binding site
probe was used.
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Fig. 8.
CDP/cut does not associate
with HDAC1 when bound to the HLA-B7 repressor element.
A, HeLa nuclear extract was depleted of HDAC1 by incubation
with agarose-conjugated HDAC1 antibody or treated with
agarose-conjugated goat preimmune IgG. EMSA was carried out using a
wild type HLA-B7 repressor element probe. B, HeLa nuclear
extract, treated as in A, was used in EMSA with a methylated
MeCG11 probe. The binding of MeCP1 to MeCG11 has been previously shown
to require association with HDAC1 (see "Results"),
confirming depletion of HDAC1 in the nuclear extracts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor (57), the mouse mammary tumor virus long terminal repeat (66),
several human papillomavirus genes (53, 56), the cystic fibrosis
transmembrane conductance regulator (49), the immunoglobulin heavy
chain (67), and
-globin (68). CDP/cut expression is high
in many undifferentiated cell types and decreases when the cells are
induced to differentiate (60). CDP/cut expression also
appears to be increased in most tumor cell lines (63). Therefore,
CDP/cut overexpression may down-regulate the expression of
several developmentally important genes during malignant transformation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Ellis J. Neufeld for generously providing CDP/cut immune serum and CDP/cut expression vectors.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant NIH RO1-CA45634 (to G. G.) and aided by American Cancer Society Grant IN-105, the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust, and the Medical Research Endowment Trust and the A. D. Williams Trust funds at Virginia Commonwealth University (to S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
804-828-9723; Fax: 804-828-8453; E-mail: ssnyder@hsc.vcu.edu.
Present address: Abbott Laboratories, Dept. 463, 100 Abbott
Park Rd., Abbott Park, IL 60064-3500.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009454200
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ABBREVIATIONS |
---|
The abbreviations used are:
MHC, major
histocompatibility complex;
NK, natural killer;
CDP, CCAAT
displacement protein;
CAT, chloramphenicol acetyltransferase;
HSV, herpes simplex virus;
tk, thymidine kinase;
CMV--Gal,
-galactosidase gene driven by the cytomegalovirus promoter;
EMSA, electrophoretic mobility shift assay;
CR, cut repeat;
NF-Y, nuclear
factor Y;
HDAC1, histone deacetylase 1;
MeCP1, methylcytosine binding
protein 1;
HD, homeodomain;
PCR, polymerase chain reaction;
nt, nucleotide(s);
WT, wild type;
bp, base pair(s).
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REFERENCES |
---|
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---|
1. | Ploegh, H. L., Orr, H. T., and Strominger, J. L. (1981) Cell 24, 287-289[Medline] [Order article via Infotrieve] |
2. | Cowan, E. P., Coligan, J. E., and Biddison, W. E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4490-4494[Abstract] |
3. | Braud, V. M., Allan, D. S., O'Callaghan, C. A., Soderstrom, K., D'Andrea, A., Ogg, G. S., Lazetic, S., Young, N. T., Bell, J. I., Phillips, J. H., Lanier, L. L., and McMichael, A. J. (1998) Nature 391, 795-799[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Brooks, A. G.,
Borrego, F.,
Posch, P. E.,
Patamawenu, A.,
Scorzelli, C. J.,
Ulbrecht, M.,
Weiss, E. H.,
and Coligan, J. E.
(1999)
J. Immunol.
162,
305-313 |
5. |
Lee, N.,
Llano, M.,
Carretero, M.,
Ishitani, A.,
Navarro, F.,
Lopez-Botet, M.,
and Geraghty, D. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5199-5204 |
6. | Leibson, P. J. (1998) Immunity 9, 289-294[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Speiser, D. E.,
Pittet, M. J.,
Valmori, D.,
Dunbar, R.,
Rimoldi, D.,
Lienard, D.,
MacDonald, H. R.,
Cerottini, J. C.,
Cerundolo, V.,
and Romero, P.
(1999)
J. Exp. Med.
190,
775-782 |
8. | Travers, P. J., Arklie, J. L., Trowsdale, J., Patillo, R. A., and Bodmer, W. F. (1982) Monogr. Natl. Cancer Inst. 60, 175-180 |
9. | Restifo, N. P., Kawakami, Y., Marincola, F., Shamamian, P., Taggarse, A., Esquivel, F., and Rosenberg, S. A. (1993) J. Immunother. 14, 182-190[Medline] [Order article via Infotrieve] |
10. | Zijlstra, M., and Melief, C. J. M. (1983) Nature 305, 776-778[Medline] [Order article via Infotrieve] |
11. | Tanaka, K., Issselbacher, K. J., Khoury, G., and Jay, G. (1985) Science 228, 26-30[Medline] [Order article via Infotrieve] |
12. | Moller, P., Hermann, G., Moldenhauer, G., and Momburg, F. (1987) Intl. J. Cancer 40, 32-39[Medline] [Order article via Infotrieve] |
13. | Doyle, A., Martin, W. J., Funa, K., Gazdar, A., Carney, D., Martin, S. E., Linnoila, I., Cuttitta, F., Mulshine, J., Bunn, P., and Minna, J. (1985) J. Exp. Med. 161, 1135-1151[Abstract] |
14. | Johnsen, A., France, J., Sy, M. S., and Harding, C. V. (1998) Cancer Res. 58, 3660-3667[Abstract] |
15. |
Marincola, F. M.,
Shamamian, P.,
Axexander, R. B.,
Gnarra, J. R.,
Turetskaya, R. L.,
Nedospasov, S. A.,
Simonis, T. B.,
Taubenberger, J. K.,
Yannelli, J.,
and Mixon, A.
(1994)
J. Immunol.
153,
1225-1237 |
16. | Browning, M. J., Krausa, P., Rowan, A., Hill, A. B., Bicknell, D. C., Bodmer, J. G., and Bodmer, W. F. (1993) J. Immunol. 14, 163-168 |
17. |
Soong, T. W.,
and Hui, K. M.
(1992)
J. Immunol.
149,
2008-2020 |
18. | Soong, T. W., and Hui, K. M. (1991) Int. J. Cancer. 6 (Suppl.), 131-137 |
19. | Henseling, U., Schmidt, W., Scholer, H. R., Gruss, P., and Hatzopoulos, A. K. (1990) Mol. Cell. Biol. 10, 4100-4109[Medline] [Order article via Infotrieve] |
20. | van den Elsen, P. J., Gobin, S. J. P., van Eggermond, M. C., and Peijnenburg, A. (1998) Immunogenetics 48, 208-221[CrossRef][Medline] [Order article via Infotrieve] |
21. | Le Bouteiller, P. (1994) Crit. Rev. Immunol. 14, 89-129[Medline] [Order article via Infotrieve] |
22. | Singer, D. S., and Maguire, J. E. (1990) Crit. Rev. Immunol. 10, 235-257[Medline] [Order article via Infotrieve] |
23. | van't Veer, L. J., Beijersbergen, R. L., and Bernards, R. (1993) EMBO J. 12, 195-200[Abstract] |
24. | Schouten, G. J., van der Eb, A. J., and Zantema, A. (1995) EMBO J. 14, 1498-1507[Abstract] |
25. |
Waring, J. F.,
Radford, J. E.,
Burns, L. J.,
and Ginder, G. D.
(1995)
J. Biol. Chem.
270,
12276-12285 |
26. | Liu, X., Ge, R., Westmoreland, S., Cooney, A. J., Tsai, S. Y., Tsai, M. J., and Ricciardi, R. P. (1994) Oncogene 9, 2183-2190[Medline] [Order article via Infotrieve] |
27. | Proffitt, J. L., Sharma, E., and Blair, G. E. (1994) Nucleic Acids Res. 22, 4779-4788[Abstract] |
28. | Mavria, G., Hall, K. T., Jones, R. A., and Blair, G. E. (1998) Biochem. J. 330, 155-161[Medline] [Order article via Infotrieve] |
29. | Katoh, S., Ozawa, K., Kondoh, S., Soeda, E., Israel, A., Shiroki, K., Fujinaga, K., Itakura, K., Gachelin, G., and Yokoyama, K. (1990) EMBO J. 9, 127-135[Abstract] |
30. | Flanagan, J. R., Murata, M., Burke, P. A., Shirayoshi, Y., Appella, E., Sharp, P. A., and Ozato, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3145-3149[Abstract] |
31. | Miyazaki, J., Appella, E., and Ozato, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9537-9541[Abstract] |
32. | Maguire, J. E., Frels, W. I., Richardson, J. C., Weissman, J. D., and Singer, D. S. (1992) Mol. Cell. Biol. 12, 3078-3086[Abstract] |
33. |
Saji, M.,
Shong, M.,
Napolitano, G.,
Palmer, L. A.,
Taniguchi, S. I.,
Ohmori, M.,
Ohta, M.,
Suzuki, K.,
Kirshner, S. L.,
Giuliani, C.,
Singer, D. S.,
and Kohn, L. D.
(1997)
J. Biol. Chem.
272,
20096-20107 |
34. | Weissman, J. D., and Singer, D. S. (1991) Mol. Cell. Biol. 11, 4217-27[Medline] [Order article via Infotrieve] |
35. |
Fromm, S. V.,
Mey-Tal, S. W.,
Coligan, J. E.,
Schechter, C.,
and Ehrlich, R.
(1998)
J. Biol. Chem.
273,
15209-15216 |
36. | Rivoltini, L., Barracchini, K. C., Viffiano, V., Kawakami, Y., Smith, A., Mixon, A., Restifo, N. P., Topalian, S. L., Simonis, T. B., Rosenberg, S. A., and Marincola, F. M. (1995) Cancer Res. 55, 3149-3157[Abstract] |
37. |
Anichini, A.,
Molla, A.,
Mortarini, R.,
Tragni, G.,
Bersani, I.,
Di Nicola, M.,
Gianni, A. M.,
Pilotti, S.,
Dunbar, R.,
Cerundolo, V.,
and Parmiani, G.
(1999)
J. Exp. Med.
190,
651-668 |
38. | Cormier, J. N., Panelli, M. C., Hackett, J. A., Bettinotti, M. P., Mixon, A., Wunderlich, J., Parker, L. L., Restifo, N. P., Ferrone, S., and Marincola, F. M. (1999) Int. J. Cancer 80, 781-790[CrossRef][Medline] [Order article via Infotrieve] |
39. | Srivastava, R., Duceman, B. W., Biro, P. A., Sood, A. K., and Weissman, S. M. (1985) Immunol. Rev. 84, 93-121[Medline] [Order article via Infotrieve] |
40. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Gustafson, K. S.,
and Ginder, G. D.
(1996)
J. Biol. Chem.
271,
20035-20046 |
42. | Neufeld, E. J., Skalnik, D. G., Lievens, P. M., and Orkin, S. H. (1992) Nat. Genet. 1, 50-55[Medline] [Order article via Infotrieve] |
43. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
44. | Dorn, A., Bollekens, J., Staub, A., Benoist, C., and Mathis, D. (1987) Cell 50, 863-72[Medline] [Order article via Infotrieve] |
45. |
Singal, R.,
Ferris, R.,
Little, J. A.,
Wang, S. Z.,
and Ginder, G. D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13724-13729 |
46. | Ng, H. H., Zhang, Y., Hendrich, B., Johnson, C. A., Turner, B. M., Erdjument-Bromage, H., Tempst, P., Reinberg, D., and Bird, A. (1999) Nat. Genet. 23, 58-61[CrossRef][Medline] [Order article via Infotrieve] |
47. | Aufiero, B., Neufeld, E. J., and Orkin, S. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7757-7761[Abstract] |
48. |
Moon, N. S.,
Berube, G.,
and Nepveu, A.
(2000)
J. Biol. Chem.
275,
31325-31334 |
49. |
Li, S.,
Moy, L.,
Pittman, N.,
Shue, G.,
Aufiero, B.,
Neufeld, E. J.,
LeLeiko, N. S.,
and Walsh, M. J.
(1999)
J. Biol. Chem.
274,
7803-7815 |
50. | Kim, E. C., Lau, J. S., Rawlings, S., and Lee, A. S. (1997) Cell Growth Differ. 8, 1329-1338[Abstract] |
51. | Mantovani, R. (1999) Gene (Amst.) 239, 15-27[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Marziali, G.,
Perrotti, E.,
Ilari, R.,
Coccia, E. M.,
Mantovani, R.,
Testa, U.,
and Battistini, A.
(1999)
Blood
93,
519-526 |
53. |
O'Connor, M. J.,
Stunkel, W.,
Koh, C. H.,
Zimmermann, H.,
and Bernard, H. U.
(2000)
J. Virol.
74,
401-410 |
54. | Blochlinger, K., Bodmer, R., Jack, J., Jan, L. Y., and Jan, Y. N. (1988) Nature 333, 629-635[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Antes, T. J.,
Chen, J.,
Cooper, A. D.,
and Levy-Wilson, B.
(2000)
J. Biol. Chem.
275,
26649-26660 |
56. |
Ai, W.,
Toussaint, E.,
and Roman, A.
(1999)
J. Virol.
73,
4220-4229 |
57. | Jackson, R. J., Antonia, S. J., Wright, K. L., Moon, N. S., Nepveu, A., and Munoz-Antonia, T. (1999) Arch. Biochem. Biophys. 371, 290-300[CrossRef][Medline] [Order article via Infotrieve] |
58. | Barberis, A., Superti-Furga, G., and Busslinger, M. (1987) Cell 50, 347-359[Medline] [Order article via Infotrieve] |
59. |
Skalnik, D. G.,
Strauss, E. C.,
and Orkin, S. H.
(1991)
J. Biol. Chem.
266,
16736-16744 |
60. |
Lievens, P. M.,
Donady, J. J.,
Tufarelli, C.,
and Neufeld, E. J.
(1995)
J. Biol. Chem.
270,
12745-12750 |
61. |
Khanna-Gupta, A.,
Zibello, T.,
Kolla, S.,
Neufeld, E. J.,
and Berliner, N.
(1997)
Blood
90,
2784-2795 |
62. | Dufort, D., and Nepveu, A. (1994) Mol. Cell. Biol. 14, 4251-4257[Abstract] |
63. |
Coqueret, O.,
Berube, G.,
and Nepveu, A.
(1998)
EMBO J.
17,
4680-4694 |
64. | Last, T. J., Birnbaum, M., van Wijnen, A. J., Stein, G. S., and Stein, J. L. (1998) Gene (Amst.) 221, 267-277[CrossRef][Medline] [Order article via Infotrieve] |
65. | Teerawatanasuk, N., Skalnik, D. G., and Carr, L. G. (1999) J. Neurochem. 72, 29-39[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Zhu, Q.,
Gregg, K.,
Lozano, M.,
Liu, J.,
and Dudley, J. P.
(2000)
J. Virol.
74,
6348-6357 |
67. |
Wang, Z.,
Goldstein, A.,
Zong, R. T.,
Lin, D.,
Neufeld, E. J.,
Scheuermann, R. H.,
and Tucker, P. W.
(1999)
Mol. Cell. Biol.
19,
284-295 |
68. | Superti-Furga, G., Barberis, A., Schreiber, E., and Busslinger, M. (1989) Biochim. Biophys. Acta 1007, 237-242[Medline] [Order article via Infotrieve] |
69. | Harada, R., Berube, G., Tamplin, O. J., Denis-Larose, C., and Nepveu, A. (1995) Mol. Cell. Biol. 15, 129-140[Abstract] |
70. |
Wingender, E.,
Chen, X.,
Hehl, R.,
Karas, H.,
Liebich, I.,
Matys, V.,
Meinhardt, T.,
Pruss, M.,
Reuter, I.,
and Schacherer, F.
(2000)
Nucleic Acids Res.
28,
316-319 |
71. | Mailly, F., Berube, G., Harada, R., Mao, P. L., Phillips, S., and Nepveu, A. (1996) Mol. Cell. Biol. 16, 5346-5357[Abstract] |
72. | Gobin, S. J., Peijnenburg, A., Keijsers, V., and van den Elsen, P. J. (1997) Immunity 6, 601-611[Medline] [Order article via Infotrieve] |
73. |
Mansky, P.,
Brown, W. M.,
Park, J. H.,
Choi, J. W.,
and Yang, S. Y.
(1994)
J. Immunol.
153,
5082-5090 |
74. | Driggers, P. H., Elenbaas, B. A., An, J. B., I. J., L., and Ozato, K. (1992) Nucleic Acids Res. 20, 2533-2540[Abstract] |