From the Program in Molecular Biology and the
¶ Department of Pathology, University of Colorado Health Sciences
Center, Denver, Colorado 80262
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ABSTRACT |
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Mouse mammary tumor virus (MMTV) expression is
restricted primarily to mammary epithelial cells. Sequences responsible
for both the mammary-specific expression of MMTV and the activation of
cellular oncogenes are located within two enhancer elements at the
5'-end of the long terminal repeat. Whereas the Ban2 enhancer ( Infection by MMTV1 leads
to the development of mammary adenocarcinomas in laboratory mice (1,
2). The retrovirus is passed horizontally by ingestion of milk-borne
infectious viral particles or vertically, in certain strains, via the
inheritance of an active proviral copy (3). Although MMTV is latently
transforming, no oncogene is encoded in the viral genome (4).
Integration of the provirus near the int genes results in
the inappropriate expression of these cellular oncogenes and initiates
events that lead to the formation of mammary tumors (5).
Entry into cells is achieved by the virion binding to a membrane
receptor, which is expressed in many tissues (6). Low levels of MMTV
RNA are detected in the epithelial cells from several tissues, such as
salivary gland, lungs, kidney, and testes, as well as lymphoid tissue,
but expression is approximately 500-fold higher in the lactating
mammary gland (7). The MMTV LTR is widely used to direct the expression
of transgenes to the mammary gland, but the basis for this specificity
is not completely understood. The tissue specificity of MMTV expression
is not due to its inducibility by steroid hormones. There are active
and functional glucocorticoid receptors in most mouse tissues (8), yet
MMTV expression is primarily detected in the mammary gland (7).
The 5'-end of the MMTV LTR contains enhancer elements that direct MMTV
expression in a mammary-specific fashion (reviewed in Ref. 9). A
complex array of protein binding sites in this region has been
identified by gel shift and footprinting assays (10-15). One region,
termed the Ban2 enhancer, contains binding sites for four different
proteins, including AP-2, an Ets-related factor, a member of the
CTF/NF-I family, and an uncharacterized factor mp4 (11, 14, 15).
Recently, we identified a second region in the 5'-end of the MMTV LTR
that we termed the mammary-specific enhancer of MMTV (MEM element)
(16). This element is important for both the mammary-specific
expression of MMTV and the activation of cellular proto-oncogenes.
Although the MEM element acts synergistically with the Ban2 enhancer in
the MMTV LTR, multimerization of the MEM element itself can confer
mammary cell-specific transcriptional activation (16).
In this study, we show that the MEM element is a composite element
displaying at least three distinct footprinted domains that function
synergistically. Deletion or mutation of individual binding sites
decreases transcription from the MMTV promoter by up to 90% in mammary
cells. Two of the domains are bound by ubiquitous factors, one of which
is probably a member of the NF-1 family. At least one of the binding
activities, corresponding to the protected region footprint I (FpI),
appears to be restricted to mammary cells. However, multimerization of
the FpI binding domain alone cannot activate transcription in mammary
cells. Thus, it is the combination of multiple binding sites, one of
which may be mammary-specific, that contributes to the mammary tropism
of MMTV.
DNase I Footprinting--
The DNase I footprinting assay was
performed as described by Dynan (17). A 215-bp fragment of the MMTV
promoter encompassing Nuclear Extracts--
Crude nuclear extracts were prepared from
the cell lines listed in Table I according to the method of Dignam
et al. (18). Ten to 20 plates of cells (T-175 flasks or
15-cm diameter dishes) were grown to confluency and harvested to make
each extract. Protein concentrations were determined according to
Bradford (19). Typical yields ranged from 5-18 mg/ml. Extracts were
aliquoted, flash frozen in liquid nitrogen, and stored at
Plasmids--
The MMTV LTR in the MMTV-chloramphenicol
acetyltransferase (CAT) vectors is a chimera derived from the C3H and
GR strains of MMTV (GenBankTM accession numbers J02274 and
V01175, respectively). The chimeric LTR has GR sequences from
Constructs with deletions in the MMTV LTR were made by cutting the
vector with the indicated restriction enzymes, filling in the
overhangs, and religating. A double-stranded 15-bp oligomer (TGAGGTGAATTCTAG) was inserted at the Bsu36I site to alter
the spacing between the footprinted regions. The FpIIm and FpIIIm constructs were made by inserting 41-bp oligomers with the indicated base pair changes between the Bsu36I and ClaI
sites. The Amersham Pharmacia Biotech U.S.E. site-directed mutagenesis
kit was used to generate the FpI mutant constructs indicated in Fig. 2.
All base changes described were confirmed by sequencing.
To test enhancer activity, LTR sequences upstream of the basal MMTV
promoter ( Cell Culture, Transient Transfections, and Assays for Reporter
Gene Activity--
Transient transfections were performed in
T47D(A1-2) human breast cancer (20) and Ltk Electrophoretic Mobility Shift Assay (EMSA)--
EMSA probes
were labeled by filling in the 5'-overhangs with Klenow,
[ Multiple Binding Sites within a 107-bp Region of the MMTV
LTR--
In our previous study (16), we identified a region in the
5'-end of the MMTV LTR that plays a role in the activation of cellular
oncogenes, termed the MEM element. To begin to understand the
mechanistic basis of the tissue specificity of MMTV, DNase I
footprinting analysis was performed to identify protein binding sites
in the MEM element (Fig. 1). Nuclear
extracts from a human mammary carcinoma cell line (T47D) and a mouse
fibroblast cell line (Ltk Synergistic Function of the Three Binding Sites--
In order to
determine the functional significance of the three binding sites
delineated by footprinting analysis, a series of deletions and
mutations were introduced into the MMTV LTR (Fig. 2A). These MMTV-CAT constructs
were transiently transfected into either T47D(A1-2) or Ltk
This speculation was confirmed by making clustered point mutations in
the three binding sites individually to assess the contribution of each
subdomain. A 5-bp change in the FpIII binding site reduced activity of
the MMTV promoter in mammary cells by 70%, and a 3-bp change in
FpII/mammary cell-activating factor site reduced activity by 60% (Fig.
3B). A series of mutations
along the FpI site (FpI mut 1-4) resulted in 50-80% reductions of
promoter activity. The spatial arrangement of the three binding sites
of the MEM element is also critical for their functional synergy. A
15-bp oligonucleotide was inserted at the Bsu36I site
separating FpI from FpII/III by 1.5 turns of the DNA helix while
leaving the binding sites intact. This alteration in spacing resulted
in a 70% drop in promoter activity. It appears that all three binding
sites are needed, intact and in the correct context, in order to
achieve mammary-specific transcription.
The MEM Element Can Function as an Independent Enhancer
Unit--
In the context of the MMTV LTR, the MEM element acts in
synergy with the Ban2 element (16). To test whether the MEM element on
its own is sufficient to enhance transcription, three or four copies of
the enhancer were placed upstream of a minimal MMTV promoter (Fig.
3A). The minimal promoter ( The MEM Element Is Not Involved in the Hormone
Response--
Studies on the hormonal regulation of MMTV have provided
many of the insights that have shaped the current understanding of what
is now termed "classical" hormone response elements. MMTV has a
well characterized cluster of hormone response elements located between
Characteristics of the FpI Binding Activity--
To confirm the
mammary-specific nature of the FpI-binding protein, a panel of nuclear
extracts from 20 different cell lines (Table
I) were tested for FpI binding activity.
The extracts were bound to a probe containing the FpI binding site, and
the resultant EMSA is shown in Fig. 5.
Three DNA-protein complexes were formed with T47D extracts. Complex 1 was present in five of eight mammary extracts. In contrast, the complex
was not found in any of 12 extracts from nonmammary cells both
epithelial and nonepithelial in origin. Complex 2 was seen with all the
extracts, and the mobility of a third, faster migrating complex
(complex 3) varied among different extracts and was not always present. The presence of more than one EMSA complex using a FpI probe explains the partial footprinting seen using Ltk
Next, probes containing the four different FpI mutations (mut1-4) were
used to test for FpI binding (Fig.
6A). Compared with a wild-type
FpI sequence, none of the four mutants were able to bind complex 1 well. Additionally, mut2 did not form complex 2, and mut3/mut4 could
not form complex 3. Because all four mutations lead to a reduction in
the transcriptional activation by the MEM element (Fig. 2B),
complex 1 exhibits the best correlation between FpI binding activity
and transcriptional activity.
In order to determine the specificity of binding to the FpI probe, a
competition EMSA was performed with a variety of competitor DNAs (Fig.
6B). A 10-fold molar excess of cold wild-type FpI
oligonucleotide efficiently competed for binding of all the complexes.
The FpI mutants could only compete for binding of the complexes they
formed in Fig. 6A. Thus, mut2 was unable to compete well for
complex 2 binding, and neither mut3 nor mut4 could compete for complex 3. The FpI mutants, with the exception of mut1, could not compete for
complex 1 very efficiently. Two unrelated oligonucleotides could not
compete for the binding of any of the complexes, demonstrating the
specificity of the nuclear extracts for binding the FpI sequences.
The FpI-binding Protein Alone Cannot Activate
Transcription--
To test whether the FpI binding region was itself
sufficient to activate transcription in mammary cells, reporter gene
constructs containing either a wild-type or mutant 35-bp
oligonucleotide spanning the FpI binding site were multimerized and
cloned upstream of a minimal MMTV promoter (Fig.
7A). As before, the minimal
MMTV promoter ( Homology to a C/EBP Binding Site--
The sequence of the FpI
binding site shows similarity to the consensus binding sequence for the
C/EBP family of transcription factors, including two CAAT-box motifs
(Fig. 8A). A set of reciprocal competition gel shift assays revealed some similarities between FpI
binding and C/EBP binding. EMSA experiments using T47D nuclear extracts
and either a 33-bp FpI probe or a 20-bp C/EBP probe were performed and
a 50-fold molar excess of competitor DNA was added. In Fig.
8B, both the 33-bp FpI oligonucleotide and a shorter 21-bp FpI oligonucleotide competed for binding to the FpI probe (lanes 2 and 3). Interestingly, a C/EBP binding site was also
able to efficiently compete for the binding of complex 1 and partially for complex 2 (Fig. 8B, lane 4). The binding was specific,
as a mutant C/EBP oligonucleotide could not compete for the complexes (lane 5). When a C/EBP binding site was used as a probe,
both FpI oligonucleotides competed for binding almost as well as the C/EBP oligonucleotide (lanes 7-9). Again, binding to the
C/EBP probe was specific because a mutant C/EBP binding site did not compete for the complexes (lane 10). These findings raise
the possibility that a C/EBP family member contributes to the
mammary-specific expression of MMTV and are discussed more fully
below.
The 5'-end of the MMTV LTR is responsible for both the
mammary-specific expression of the virus and the activation of cellular oncogenes (16). The fact that MMTV induces tumors primarily in the
mammary gland appears to be linked with mammary-specific expression of
the virus. A number of binding sites for nuclear proteins have been
reported in this region of the LTR. These fall within two regions
defined as having mammary-specific transcriptional activity: the Ban2
enhancer and the MEM element. When the MEM element is multimerized
upstream of a minimal MMTV promoter, it can enhance transcription in a
mammary-specific fashion. Similar experiments with the Ban2 enhancer
show that it, too, can enhance transcription on its own (10, 15).
However, we have shown that inactivation of either the Ban2 enhancer or
the MEM element in the context of the LTR abrogates both the MMTV
promoter and its ability to activate a nearby proto-oncogene promoter
(16). Thus, the Ban2 and the MEM elements function synergistically when contained in a full-length LTR.
The Ban2 enhancer contains binding sites for four different proteins,
including AP-2, an Ets-related factor, a member of the CTF/NF-I family,
and an uncharacterized factor mp4 (11, 14, 15). The Ban2 fragment
( The MEM element has only recently been recognized as a distinct
functional element (16). We have now characterized the functional interplay of the binding sites within the MEM element that constitute the active, tissue-specific enhancer. DNase I footprinting analysis revealed three protected regions within this element in extracts from
T47D mammary carcinoma cells (Fig. 1). All three sites contribute to
the mammary-specific expression of the virus. Deletions that removed
one, two, or all three of the binding sites reduced transcriptional activity by 85-96%, and base substitutions in individual sites reduced transcriptional activity by 50-80%. These results with the
MEM element contrast with the Ban2 enhancer, in which clustered point
mutations in all four binding sites were required to reduce the
activity by 80% (14). The spatial context of the MEM element binding
sites with respect to one another is also important. When the three
binding sites are left intact but a 15-bp insertion is introduced, the
LTR can no longer support transcription in mammary cells. Thus, not
only are all three binding activities of the MEM element important for
the mammary-specific expression of MMTV, it may be that physical
interaction between the factors or a common coactivator is required for
MEM element activity.
In dissecting the MEM element, we have focused on the FpI protected
region, because DNase I footprinting analyses revealed a difference in
binding activity in extracts from T47D mammary carcinoma cells and
fibroblasts. EMSA studies indicated three activities in T47D cell
extracts that bound a FpI oligonucleotide. In order to be certain that
there was a mammary-specific binding activity associated with the FpI
factor, we assayed the binding activity of nuclear extracts from 20 cell lines representing mammary carcinomas, nontumor mammary
epithelium, nonmammary epithelial cells, and nonepithelial cells (Table
I). Complex 1 was found exclusively in mammary cells and was present in
the majority of the mammary lines examined. A second complex, complex
2, was found in all cell types. Finally, a third, faster migrating
complex was present in many cases, but its mobility varied depending on the cell line. Four different clustered point mutations within the FpI
binding site decreased MEM element activity. Complex one correlated
best with this functional data, as it was the only one of the three
complexes of which the binding was decreased by all four mutations.
The FpI region cannot mediate mammary-specific transcription on its
own, however, even in multiple copies. When the FpI binding site is
multimerized upstream of a minimal MMTV promoter and separated from
other two MEM element-binding proteins, there is no increase in
transcriptional activity in a mammary cell line. Thus, the MEM element
behaves as a composite enhancer, the function of which is dependent on
functional synergy between tissue-specific (or at least
tissue-restricted) and nonspecific factors.
The binding of multiple transcription factors to a composite enhancer
element to allow cell-type specific transcription has been observed in
a number of tissues, including the mammary gland, liver, lymphoid
cells, heart, and muscle (28-32). This combinatorial effect
circumvents the requirement for tissue-specific transcription factors
and instead mediates tissue-restricted gene expression by combining
both ubiquitous and tissue-enriched transcription factors in a novel
way. No factor specific to the mammary gland has been identified,
although a couple of binding proteins were named for that putative
property. Mammary gland factor is identical to Stat5 and is expressed
in many tissues (33). Mammary gland factor/Stat5 does, however, mediate
the prolactin signal and is important for the expression of milk
proteins, such as 1075
to
978) has been well characterized, the mammary-specific enhancer of
MMTV from
956 to
862 has only recently been recognized as a key
determinant of mammary-specific oncogene activation by MMTV. The
present study identifies and characterizes three binding sites located
within this element. Transient transfection of deletion and mutation
constructs shows that all three sites contribute to the basal
expression of MMTV in mammary cells. One of the binding activities
(footprint I) is restricted to mammary cells, whereas the other two
sites bind factors found in both mammary and nonmammary cells. The
multimerized mammary-specific enhancer of MMTV on its own can enhance a
minimal promoter in a mammary-specific fashion. However, the FpI
binding site alone cannot mediate this effect. Thus, it is the binding
of multiple factors in a combinatorial fashion that mediates the
mammary-restricted expression of MMTV.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
969 to
754 of the MMTV LTR was uniquely
labeled at the 5'-end of the noncoding strand. Approximately 80,000 cpm
of probe were incubated with increasing amounts of crude nuclear
extracts and 2 µg of poly[d(I·C)] in a final reaction volume of
50 µl for 15 min on ice. Fifty µl of a solution containing 5 mM CaCl2 and 10 mM
MgCl2 were added to each tube. A variable amount of DNase I was then added to each reaction and incubated for 1 min at room temperature. The reactions were terminated by the addition of 90 µl
of stop solution (200 mM NaCl, 30 mM EDTA, 1%
SDS, and 100 µg/ml yeast tRNA), extracted with phenol/chloroform, and
ethanol-precipitated. DNA pellets were resuspended in 5 µl of 0.1 N NaOH:formamide (1:2, v/v) containing xylene cyanol and
bromphenol blue. The samples were boiled for 2 min, loaded onto a thin
8% urea polyacrylamide gel, and run in 1× TBE for ~2 h at 1600 V. Following electrophoresis, the gel was dried and subjected to autoradiography.
70 °C.
291 to
+83 and C3H sequences from
1194 to
292 and from +84 to +99
108 to +99) in the MMTV-CAT reporter plasmid were replaced
by three or four copies of a fragment (
969 to
862) encompassing the
MEM element. Alternatively, a 33-bp oligonucleotide containing only the
FpI site was multimerized and cloned upstream of the basal MMTV
promoter. Constructs that contained four or six wild-type copies of the
FpI site and a construct with six mutant copies were chosen and
confirmed by sequencing.
mouse fibroblast cell
lines using the DEAE-dextran/Me2SO shock method, as
described previously (16). In T47D(A1-2) cells, CAT reporter gene
activity was normalized using pCH110, a plasmid expressing
-galactosidase from an SV40 promoter (21), and in Ltk
cells using
RSVL, a luciferase reporter gene driven by the Rous sarcoma virus LTR
(22). Cells were harvested 70 h after transfection for
determination of protein concentration, CAT,
-galactosidase, and
luciferase activity in the extracts (16, 19, 23, 24).
-32P]dCTP, and cold nucleotides. A 20-µl reaction
containing 15-25 µg of crude nuclear extract and 1-2 µg of
poly[d(I·C)] was incubated for 15 min at 4 °C in binding buffer
(50 mM KCl, 10 mM Tris, pH 7.5, 5% glycerol, 1 mM EDTA, 2 mM MgCl2, 0.8 µg of
gelatin, 1.3 mM dithiothreitol). In some cases, cold
competitor was then added and incubated for 10 min at 4 °C. Finally,
labeled probe (30,000 cpm) was added to each reaction and incubated 15 min at 4 °C. The samples were loaded on a precooled 4%
polyacrylamide gel and run in 0.25× TBE at 250 volts for 2 h at
4 °C. The gel was dried and subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) were compared. FpIII was the 3'-most
binding site and contained a consensus NF-I binding site of
TTGGCN5GCCAA (25). FpIII binding activity was seen with
both mammary and fibroblast extracts. The next binding activity, FpII,
was also seen with both extracts. This small footprint corresponded to
a previously identified binding site for an uncharacterized activity.
Despite the fact that the binding activity is present in both mammary and nonmammary cells, it has been termed mammary cell-activating factor
(10). A difference in binding activity between mammary and fibroblast
extracts was observed in the third protected region, FpI. Although the
protection was present most clearly with the T47D nuclear extracts,
there was partial protection of this region using Ltk
extracts.
However, the footprint seen with the mammary extracts was accompanied
by a DNase I hypersensitive site. Thus, the MEM element is composed of
multiple binding sites, one of which appears to bind a
tissue-restricted, if not mammary-specific, factor.
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Fig. 1.
DNase I footprinting analysis of the MEM
element. A fragment of the MMTV LTR from 969 to
862 was
uniquely labeled at the 5'-end of the noncoding strand and incubated
with increasing amounts (50-200 µg) of crude nuclear extracts from
either T47D human mammary carcinoma cells or Ltk
mouse fibroblast
cells. The left lane (G/A) represents a chemical
sequencing reaction to identify G and A residues. Control lanes
(0) contain no nuclear extract. The black
rectangles to the right of the autoradiograph depict
the three protected footprints and their boundaries relative to the
start of transcription. The arrow denotes a hypersensitive
site seen only with the T47D mammary cell extracts.
cells to
test their transcriptional activity. The largest deletion (
76),
which removed 76 bp that included FpII, FpIII, and much of the FpI
region, had the greatest effect, virtually ablating the basal
expression of MMTV in mammary cells. This same deletion had no effect
on transcription in fibroblast cells (compare Fig. 2B with
Fig. 2C). Deletion of either much of FpI (
BB,
938 to
903) or of FpII/III (
BC,
903 to
862) resulted in a loss of
promoter activity in mammary cells, suggesting that all three binding
sites may be necessary for transcriptional activity.
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Fig. 2.
Effect of deletions or mutations in the MEM
element on MMTV promoter activity. A depicts the
MMTV-CAT expression vector and the deletions or mutations that were
introduced into the MEM element. The three protected regions identified
by DNase I footprinting are indicated at the top in the
schematic of the wild-type MEM element. Substitution mutations are
indicated by the black stars, with the base pair changes
shown to the side. T47D(A1-2) mammary carcinoma (B) and
Ltk mouse fibroblast (C) cells were transiently
transfected with 2 µg of the reporter construct and 0.2 µg of the
internal control plasmid. Bar graphs represent the averages
of three to six experiments in which each condition was performed in
duplicate, with the error bars representing the S.E. Results
are expressed as a percentage of the activity measured for the control
MMTV construct, which was set at 100%. The average raw CAT value for
the control MMTV construct was 1.49 pmol/min, with an assay background
of 0.15 pmol/min in T47D(A1-2) cells and 1.54 ± 0.13 pmol/min in
Ltk
cells.
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Fig. 3.
A multimerized MEM element has enhancer
activity. Schematics of the constructs used in transient
transfections are shown in A. The shaded box
spanning from HinfI to ClaI represents the 107-bp
MEM element containing the three protected regions (FpI, FpII, and
FpIII). Three or four copies of the MEM element were placed upstream of
a minimal MMTV promoter, with the direction of the arrow
denoting the orientation of the MEM element. The constructs were tested
by transient transfection of either T47D(A1-2) cells (B) or
Ltk cells (C). Results are expressed as a percentage of
the activity measured for the full-length MMTV construct included in
each experiment.
Sst) had no activity in
T47D(A1-2) cells. A single MEM element had little effect on this
promoter. However, adding back three or four copies of the MEM element
rendered the minimal promoter 5-10 times more active than the
full-length MMTV LTR in mammary cells (Fig. 3B). In
fibroblast cells (Fig. 3C), there was no change in the
transcriptional activity of any of the constructs. These data support
the hypothesis that the MEM element is a key determinant in the
mammary-specific expression of MMTV.
80 and
200 in the MMTV LTR (26) that mediate the prodigious
induction of promoter activity resulting from exposure to the
appropriate steroid hormones. We wanted to test whether the MEM element
was involved in the hormone responsiveness of the MMTV LTR. The
constructs diagrammed in Fig. 2A were transiently transfected into T47D(A1-2) and Ltk
cells and treated with 40 nM dexamethasone, a synthetic glucocorticoid, for 20 h
before harvest. As shown in Fig. 4, none
of the deletions or mutations in the MEM element significantly affected
the hormone response of MMTV in either cell type. Thus, the MEM element
is necessary for the basal transcription of MMTV but is not involved in
the hormone response.
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Fig. 4.
The MEM element is not involved in the
steroid hormone responsiveness of the MMTV LTR. T47D(A1-2)
(A) or Ltk (B) cells were transfected with
MMTV-CAT or the indicated mutant MMTV-CAT vectors and then treated with
40 nM dexamethasone for 20 h before harvest. The data
represent three to seven experiments ± 1 S.E. Results are
expressed as a percentage of the activity measured for the full-length
MMTV construct included in each experiment. The average raw CAT value
for the full-length MMTV construct was 79.0 pmol/min, with an assay
background of 0.16 pmol/min in T47D(A1-2) cells and 72.3 ± 0.13 pmol/min in Ltk
cells.
fibroblast extracts (Fig. 1).
However, there is a mammary-restricted binding activity associated with
the FpI protected region, corresponding to complex 1.
Cell lines used to make nuclear extracts for EMSA
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Fig. 5.
The complex 1 FpI binding activity is found
exclusively in mammary cell lines. EMSA experiments were performed
using nuclear extracts made from the cell lines listed in Table I and a
33-bp probe containing the FpI binding site. Three specific complexes
were identified and are indicated on the left. A fourth,
faster migrating complex, is a nonspecific band that could be largely
eliminated by the inclusion of poly[d(I·C)] during the incubation.
Lanes 1-8 depict results with extracts from the indicated
mammary cell lines, and lanes 9-20 represent nonmammary
cell lines.
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Fig. 6.
Specificity of FpI binding activities.
EMSA experiments were performed using 20 µg of nuclear extract from
T47D cells. In A, five different labeled probes were used to
compare the binding activity of the wild-type FpI site with that of the
four mutant sites. In B, a 33-bp wild-type FpI probe was
used in all reactions, and different cold competitor oligonucleotides
were added in a 10-fold molar excess as indicated above each lane.
Lane 1 shows binding in the absence of competitor. The
sequences of the oligonucleotides used in these experiments are shown
in C.
Sst) showed almost background levels of
transcriptional activity in T47D(A1-2) cells (Fig. 7B).
Adding back four or six copies of the wild-type FpI binding site failed
to enhance the activity of the minimal promoter further. Therefore, the
FpI domain requires the other domains of the MEM element to enhance
transcription in mammary cells, even though the FpI domain alone
appears to have mammary cell-specific binding activity.
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Fig. 7.
Multimerized FpI binding site cannot activate
a minimal MMTV promoter. A, either four or six copies
of the FpI binding site or six copies of a mutant FpI site were placed
upstream of a basal MMTV promoter. The sequences of the
oligonucleotides used to construct these vectors are shown.
B, the constructs were transiently transfected into
T47D(A1-2) cells to assess their transcriptional activity relative to
the full-length (FL) reference MMTV promoter.
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Fig. 8.
Comparison of FpI and C/EBP binding
activities. The homology between the FpI binding site and a
consensus C/EBP binding site is shown in A. EMSA experiments
were performed using T47D nuclear extracts and either a 33-bp FpI probe
(B, left), or a 20-bp C/EBP probe (B, right). The
indicated unlabeled competitor DNA was added at a 50-fold molar excess.
The sequences of the oligonucleotides used are shown in
C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1075 to
978) upstream of the thymidine kinase promoter is active
only in mammary cells and not in HepG2 liver cells (10, 14). A 180-bp
fragment (
1166 to
987) containing this enhancer can also target a
transgene to the mammary gland of mice when linked to the SV40 promoter
(27). Even when present as a single copy and independent of the MEM
element, the Ban2 enhancer was able to direct mammary-specific
transcription. This may be because the Ban2 enhancer is separated from
the negative regulatory elements contained in the full-length MMTV LTR,
or due to the use of a stronger heterologous promoter, rather than the
very weak MMTV basal promoter. However, there were higher levels of
transgene expression when the Ban2 and MEM elements were both present
(10).
-casein and whey acidic protein (28). Recently, a
functional Stat5 binding site has been identified in the middle portion
of the MMTV LTR and is important for the mammary-specific expression of
MMTV (34). The mammary cell-activating factor was found to be a member
of the Ets family of transcription factors and is not restricted to the
mammary gland (35). Its activity has not been well characterized, but
because of such a binding site in the Ban2 enhancer, it does play some
role in the mammary-specific regulation of MMTV. Fig. 9 outlines the binding sites located
within the two enhancer elements in the 5'-end of the MMTV LTR. Of the
eight binding sites, six binding activities are found in both mammary
and nonmammary cells. The uncharacterized mp4 factor was reported to
show binding activity in mammary cell lines only (11), and the FpI
complex 1 binding activity was shown to be restricted to mammary cell
lines in this study. We found that the FpI region binds ubiquitous
factors as well. An earlier study (10) that termed this region F4
observed nonspecific binding activities but not a mammary-specific
complex as we have seen. Because we did not see the mammary-specific
complex (complex 1) in all mammary cell lines, this may explain the
discrepancy.
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Fig. 9.
Composite elements direct the mammary
specific expression of MMTV. Shown is a schematic of the binding
sites identified in the 5'-end of the MMTV LTR that participate in the
mammary-specific expression of MMTV. The sites are clustered in two
independently defined regions, the Ban2 enhancer and the MEM element.
Boundaries of the binding sites are shown, as are alternate names or
the putative factors that bind individual sites.
The competition EMSA data (Fig. 8) raise the interesting possibility
that the FpI-binding protein may be a member of the C/EBP family of
transcription factors (36). The 3'-half of the binding site is well
conserved, and it has been documented that C/EBP binding sites may show
significant homology to only one half of the palindrome (37). Three of
the six isoforms, C/EBP ,
, and
, are expressed in the mouse
mammary gland and are important for its development and function
(38-40). C/EBP
is important for the mammary-specific expression of
-casein and whey acidic protein, in conjunction with other
ubiquitous transcription factors, such as Stat5, YY1, NF-I, and the
glucocorticoid receptor (28). Although the sequence of the FpI binding
site, along with EMSA data, is consistent with a member of the C/EBP
family, other data weigh against this conclusion. In contrast to the
marked heat stability characteristic of C/EBP proteins, the FpI binding
activity is heat labile at temperatures above 37 °C. When nuclear
extracts were heated to 60 °C for 1, 2, 3, 4, or 5 min and used in
EMSA analysis, the extracts retained the ability to form a complex with
a C/EBP probe but not with a FpI probe (data not shown). Furthermore,
the inclusion of antibodies against C/EBP
, C/EBP
, or C/EBP
neither upshifted any of the complexes binding an FpI oligonucleotide
nor disrupted the complexes. Finally, none of these three forms of
C/EBP exhibit a cell-type distribution as restricted as does the
complex 1 binding activity. Initial attempts to enrich for complex 1 binding and study its biochemical properties have been hampered by its
apparent lability to extensive manipulation.
Our previous results and the results from this study firmly establish
the importance of the MEM element in the mammary-specific expression of
MMTV. Further experiments to characterize the poorly defined binding
activities of the mammary-specific MEM and Ban2 elements need to be
carried out. It will be critical to determine whether the FpI and mp4
factors binding these elements are truly restricted to the mammary
gland and whether, at least in the case of FpI, it is related to the
C/EBP family of transcription factors. It is apparent that a complex
interaction of many transcription factors in a combinatorial fashion is
required to allow the mammary-specific expression of MMTV. The
mechanism by which combinatorial interactions are translated into
tissue-specific, developmentally appropriate activity is a key question
in the regulation of transcription.
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FOOTNOTES |
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* 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.
§ Present address: Baylor College of Medicine, Dept. of Cell Biology, One Baylor Plaza, Houston, TX 77030.
To whom correspondence should be addressed: Dept. of
Pathology, 4200 E. Ninth Ave., Box B216, Denver, CO 80262. Tel.:
303-315-5463; Fax: 303-315-6721; E-mail:
Steve.Nordeen{at}uchsc.edu.
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ABBREVIATIONS |
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The abbreviations used are: MMTV, mouse mammary tumor virus; LTR, long terminal repeat; MEM element, mammary-specific enhancer of MMTV; CAT, chloramphenicol acetyltransferase; bp, base pair(s); EMSA, electrophoretic mobility shift assay; C/EBP, CCAAT/enhancer-binding protein; mut, mutation; FpI, footprint I.
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REFERENCES |
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