Identification of a Placental-Specific Enhancer in the Rat Placental Lactogen II Gene That Contains Binding Sites for Members of the Ets and AP-1 (Activator Protein 1) Families of Transcription Factors
Yuxiang Sun and
Mary Lynn Duckworth
Department of Physiology University of Manitoba Winnipeg,
Manitoba, Canada R3E 3J7
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ABSTRACT
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We previously identified a 3-kb
proximal 5'-flanking region of the rat placental lactogen (rPLII)
gene1 that is important for reporter gene transcription in
the rat trophoblast cell line, Rcho, and targets expression to the
placentas of transgenic mice. In our current studies we have used
further deletion analysis and transfection studies in Rcho and GC cells
to map more precisely the locations of regulatory elements involved in
this placental expression. We show that sequences between -1435 and
-765 are necessary for minimal expression in Rcho cells and that there
are negative regulatory elements between -3031 to -2838 and -1729 to
-1435. Most importantly, we have identified a fragment between -1793
to -1729 that is essential for expression levels characteristic of the
complete 3-kb 5'-region. When linked to the herpes simplex thymidine
kinase minimal promoter, this fragment acts as an enhancing element in
Rcho but not GC cells. Deoxyribonuclease I (DNAse I) protection and
electrophoretic mobility shift assays with nuclear extracts and
in vitro translated proteins identify binding sites for
members of the activator protein-1 (AP-1) and Ets families of
transcription factors. Site-directed mutagenesis of the individual
AP-1- and Ets-binding sites leads to a partial loss of the enhancing
activity; a double AP-1/Ets mutation leads to a complete loss of
activity, demonstrating the functional importance of these sites. By
these criteria, putative GATA-binding sites located within the
enhancing fragment are not active. These new data suggest an important
role for this enhancing fragment in rPLII placental giant cell
expression and are the first to implicate a member of the Ets family in
the regulation of this gene family.
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INTRODUCTION
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Although the placenta is vital to mammalian fetal survival and
development, the factors that regulate placental cell-specific gene
expression have not been extensively studied. The few genes that have
been examined have come from more than one species and from a variety
of placental cell types. The activator protein-2 (AP-2) transcription
factor, originally described as the trophoblast specific
element-binding protein, has been identified as important for
the placental expression of the hCG
- and ß-subunit genes (1, 2, 3, 4)
and potentially has a role in the placental expression of some other
genes (5, 6). Its involvement in the regulation of these several genes
and its role in the developmental regulation of other cell types has
led to speculation that AP-2 may participate in a developmental cascade
during trophoblast differentiation (1). The GATA (7, 8), Fos/Jun (AP-1)
(9), and TEF (10, 11) families of transcription factors have also been
implicated in the placental expression of some genes. None of these
factors, however, are expressed only in placenta, suggesting that there
are further protein/protein interactions that give specificity. The
recently characterized basic helix-loop-helix transcription factors
Mash-2 (12) and Hand-1 (13, 14, 15, 16, 17) (previously called Hxt, eHAND, Thing-1)
have been shown to be essential for determination of specific placental
cell types; no target genes have been identified for either factor,
although there is indirect evidence that mouse placental lactogen I
(PLI) may be regulated by Hand-1 (15). The limited data available make
it difficult to decide whether there may be a specific transcription
factor or combinations of factors important for placental-specific gene
expression or whether factors will vary from gene to gene. It would be
particularly useful to be able to study the transcriptional regulation
of multiple genes expressed in a specific placental cell type, thereby
allowing direct comparisons of the cis- and
trans-acting factors involved.
The PRL-related genes expressed in the placentas of rats and mice
represent an important resource for such studies. This is a large gene
family expressed according to developmentally distinct expression
patterns involving both specific placental cell types and temporal
expression (18, 19, 20). PLI and PLII are the archetypes of these genes,
being originally identified as placental proteins that could bind to
the PRL receptor and that were highly expressed either early (PLI) or
late (PLII) in pregnancy (21, 22, 23, 24). We and others (25, 26) have shown by
in situ hybridization studies in rat placenta that, at early
times after implantation, only rPLI mRNA is expressed in the primary
and secondary giant cells of the placenta. At midpregnancy, rPLII
transcription is activated and for a brief time both mRNAs are
expressed in the same cells (25). From midpregnancy to term, only rPLII
mRNA is expressed in the basal zone giant cells and in newly
differentiated giant cells in the labyrinth region of the
chorioallantoic placenta (27). A similar developmental switch has been
shown for the mouse PLs (28, 29). Where sequence is available for
comparison, the 5'-flanking sequences of the homologs of the rat and
mouse PLI and PLII genes are highly related, suggesting that common
factors may regulate these genes in the two species. There is little
similarity, however, between the PLI and PLII 5'-flanking sequences
(Ref. 30 and M. L. Duckworth, unpublished data). The expression of
the PLI and PLII genes in the same giant cell type provides a useful
model system for investigating whether common transcription factors or
combinations of factors regulate the expression of these genes in this
placental cell type or whether different regulatory factors are used
that may reflect their different temporal expression patterns.
The factors important for mouse PLI (mPLI) expression have been studied
by transfection assays in the rat choriocarcinoma cell line, Rcho-1
(31). This cell line and the closely related Rcho line (32)
differentiate in culture to the giant cell type and are known to
express several members of the rat PRL family of placental hormones
specific to that cell type (25, 26). Mouse PLI expression has been
shown to be dependent on both AP-1 (9) and GATA 2/3 sites (7) contained
in a 274-bp 5'-flanking fragment immediately upstream of the
transcription start site. Both GATA 2 and 3 are expressed in mouse
placental giant cells (7). Although GATA 2 and 3 are also expressed in
nonplacental cell types, coexpression of either with a mPLI -274
5'-flanking/Cat reporter construct in mouse L cells, which
lack this transcription factor, is sufficient to stimulate reporter
gene expression. Targeted disruption of GATA 2/3 in mice leads to a
50% loss of mPLI mRNA, suggesting that although important there are
other factors also required for normal expression of this gene (33). We
have found that a similar 5'-flanking region of the rat PLI gene is
sufficient to regulate reporter gene expression in Rcho cells (34) and
conserves the identified GATA and AP-1 sites in essentially identical
locations (M. L. Duckworth, unpublished data).
GATA 2/3-deficient mouse embryos die in utero at day 10.5 to
11.5 from massive defects in several organ systems (35, 36), thereby
preventing any determination of possible effects on mPLII
transcription. We have recently shown that 3 kb of the rPLII gene
5'-flanking region proximal to the transcription start site are
sufficient to direct luciferase reporter gene expression in Rcho cells
and in the placentas of transgenic mice (30). In this current study, we
identify sequences within this rPLII region that act as a placental
cell enhancer. Our results suggest that placental-specific expression
of PLI and PLII are regulated differently in the same giant cell
type.
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RESULTS
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Deletion Analysis of the rPLII 5'-Flanking Region
We previously determined that sequences within a
SacI/PvuII (-3031 to +64) fragment of the rPLII
5'-flanking region were sufficient to direct luciferase reporter gene
expression in the rat trophoblast Rcho cell line and the placentas of
transgenic mice (30) but were inactive in the rat pituitary GC cell
line, suggesting that this region contains important sequences for
placental-specific gene expression. To identify the active region(s)
within this fragment, we constructed chimeric luciferase reporter
clones containing various deletions in this 5'-fragment and tested them
for activity using transient transfection assays in the Rcho cell line.
The 3'-end of all fragments begins at +64. The results are shown in
Fig. 1
. Sequences within both the -1729
and -1435 5'-flanking fragments produced significantly higher levels
of luciferase expression (P < 0.05) than the
promoterless control; these levels were, however, significantly lower
than the -3031 fragment itself (P < 0.05), suggesting
the presence of enhancing sequences in the region between -3031 and
-1729. The -1435 fragment showed a slight but significantly higher
activity than the -1729 fragment, suggesting the presence of
inhibitory sequences between -1729 and -1435. A construct in which an
EcoRI fragment (-2838 to -1729) was deleted from the
-3031 5'-fragment produced a further reduction in luciferase
expression, resulting in luciferase levels that were no longer
significantly different from the promoterless vector control. These
results indicated that enhancer sequences important for rPLII
expression were located within this -2838/-1729 EcoRI
fragment and that there were further negative regulatory elements
between -3031 and -2838.

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Figure 1. Deletion Analysis of the rPLII 5'-Flanking Region
A restriction enzyme map of this region is shown with the transcription
start site indicated by a bent arrow. A series of
deletion fragments were cloned into the luciferase reporter vector,
pXP2, and tested for expression in Rcho cells by transient transfection
assays. All constructs begin at nucleotide +64. Luciferase activity was
measured in light units/mg protein. All values have been corrected for
plasmid uptake as described in Materials and Methods.
Results are from two to four experiments where the number of separate
transfections is 6 for -118 and -765 and at least 12 for -3031,
-1729, -1435, deletion clone, and pXP2. Results are mean ±
SEM expressed relative to the pXP2 vector control, which
was set at 1. Statistical significance (P < 0.05)
was established by an unpaired Students t test.
Asterisks indicate constructs that are significantly
less active than the entire 3031-bp 5'-flanking fragment indicating the
loss of enhancing sequences. Double asterisks indicate
constructs that also have significantly higher activity than the
construct deleted for -2838 to -1729, suggesting the presence of
inhibitory sequences between -3031/-2838 and -1729/-1435.
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We have previously shown that sequences within a -765/+64 fragment
were insufficient to support reporter gene expression in Rcho cells
(30). Our further data suggest that there are important sequences
between -1435 and -765 that support minimum rPLII expression.
Functional Analysis of Sequences between -2838 and -1729
Identifies a Placental-Specific Enhancer Region
To test whether sequences between -2838 and -1729 act as a
placental-specific enhancer, we cloned the EcoRI fragment in
forward and reverse orientations into the luciferase vector, pT81luc
(37), which contains a minimal herpes simplex thymidine kinase (TK)
promoter. Each construct was transfected into Rcho cells grown for
either 6 or 14 days before transfection and into rat pituitary GC
cells. Cultures were assayed for luciferase expression 48 h later.
The results are shown in Fig. 2
. Both the
5'
3' and 3'
5' orientations showed significant (P
< 0.05) increases in luciferase activity over the minimal TK promoter
in the Rcho cells, but neither orientation was active in the pituitary
GC cells. These transfection data confirm that sequences within the
-2838 to -1729 fragment are important for placental cell expression.
The involvement of DNA sequences in this fragment with the
temporal-specific expression of rPLII is less clear. The enhancing
effect of the -2838/-1729 fragment in the Rcho cells was seen whether
cultures were transfected after 6 days in culture, when rPLII mRNA was
present at only very low levels, or after 14 days when rPLII mRNA was
more highly expressed (25).

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Figure 2. Sequences between -2838 and -1729 Act as a
Placental Cell-Specific Enhancer
The EcoRI fragment containing these sequences was cloned
in 5' 3' (E/EFTKpluc) and 3' 5' orientations
(E/ERTKpluc) in the luciferase vector pT81luc, which
contains the minimal herpes simplex thymidine kinase promoter.
Constructs were transfected into Rcho cells grown for either 6 days or
14 days after plating and into rat pituitary GC cells. Luciferase
expression was assayed after 48 h. Rcho cells express primarily
rPLI mRNA at day 6 and express significant levels of rPLII at about day
14. Results shown are from four separate transfections. Luciferase
activity (mean ± SEM) is expressed relative to the
pT81luc vector control, which was set at 1. Statistical significance
(*, P < 0.05) was established by an unpaired
Students t test. Both orientations of this fragment
produced significantly higher levels of luciferase activity than the
controls only in Rcho cells. There was no difference in activity
between these Rcho cells cultured for different times.
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To identify specific sequences within the -2838 to -1729 fragment
that are involved in the enhancing activity, we constructed a series of
luciferase clones containing various restriction enzyme subfragments of
the 1109-bp EcoRI fragment in pT81luc. All fragments were
assessed in the 5'
3' orientation. These constructs were transfected
into Rcho cells grown for 6 days, and cell extracts were analyzed for
luciferase activity 48 h after transfection. The constructs and
results are shown in Fig. 3
. The
enhancing activity was localized within a 65-bp
HphI/EcoRI fragment at the 3'-end of the 1109-bp
fragment. There was no significant difference between the luciferase
activities of all the constructs that contained this 3'-sequence.

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Figure 3. Deletion Analysis of the -2838 to -1729 Fragment
Identifies an Enhancing Region at Its 3'-End
A restriction enzyme map of the enhancing EcoRI fragment
indicates the sites used for subcloning into the minimal thymidine
kinase promoter vector, pT81luc. Transfections were carried out in Rcho
cells grown for 6 days. Results shown are from at least six separate
transfections. Luciferase activity (mean ± SEM) is
shown relative to the vector control. All fragments that contain the
most 3'-sequence in the EcoRI fragment (F3, F5, F7, F9)
have significantly higher activity (P < 0.05) than the vector
control, which was set at 1. There is no significant difference between
the activities of these fragments. Fragments that do not contain this
sequence (F1, F2, F4, F6, F8) are not significantly different from the
vector control.
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DNAse I Protection Analysis of the Enhancing Fragment Identifies
Two Protected Regions Containing AP-1- and Ets-Binding Sites
During the course of these experiments the complete sequence of
the 1109- bp EcoRI fragment was determined and examined for
the presence of known transcription factor-binding sites. Binding sites
for the GATA, Fos/Jun (AP-1), and basic helix-loop-helix families of
transcription factors, which have been implicated in the placental
expression of several genes or in the determination of placental cell
types, were highly represented throughout. Most, however, were in
regions that did not enhance luciferase expression. To identify which
site(s) in the enhancing fragment were important in the placental
expression of the rPLII gene, we carried out DNAse I protection studies
using nuclear extracts prepared from late-term dissected rat placental
labyrinth region, which represents the region of highest rPLII
expression, and the Rcho (harvested as described after 6 days), and GC
cell lines. Figure 4
shows an
autoradiogram of a representative DNAse I protection experiment. What
appear to be two separate but adjacent regions were protected in
identical patterns by the placental labyrinth and Rcho nuclear
extracts; nuclear extracts of the GC cell line, in which the enhancing
fragment was inactive, showed distinctly different protection patterns
in these regions. The sequence of the protected areas from
approximately -1765 to -1730 is underlined in Fig. 5
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Figure 4. DNAse I Protection Analysis Identifies Two Adjacent
Regions in the Enhancing Fragment That Are Protected by Rat Placental
Labyrinth and Rcho but Not GC Nuclear Extracts
The antisense strand of F5 was end labeled, incubated with increasing
amounts of placental labyrinth (2080 µg), Rcho (1040 µg), or GC
(20, 40 µg) nuclear extracts, and partially digested with DNAse I.
The labeled strand digested in the absence of protein is designated
-NE. G+A indicates a Maxam and Gilbert sequencing reaction that was
used as a marker. Two adjacent protected regions (FP1, FP2) are seen
with the labyrinth and Rcho extracts; a different pattern is seen with
the GC extract. A new hypersensitive site, marked by an
arrow, is visible in FP1 in the placental/Rcho lanes.
This is a characteristic shift in sensitivity from the adjacent
nucleotide as compared with the -NE lane. The protected regions
contain sequences for putative Ets (F1) and AP-1-binding sites (FP2).
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Figure 5. Features of the 151-bp
StuI/EcoRI Fragment (F7) Containing
Placental-Specific Enhancing Activity
Putative Ets- and AP-1-binding sites that were protected from DNAse I
digestion by rat placental labyrinth and Rcho nuclear extracts are
shown in bold. The approximate regions protected by
these extracts are underlined and labeled F1 and F2,
respectively. This region is shown as double-stranded since the
footprints were best observed on the antisense strand. The
arrow marks a new hypersensitive site. Putative GATA
sites in the context of a direct repeat sequence are also marked in
bold. Other putative AP-1 sites in this fragment are
shown in lower case. The HphI site, which
further divides this fragment into an inactive 5' >(F8) and an active 3'
(F9) fragment, is indicated below the line, separating
the GATA sites. Asterisks mark the nucleotides that were
altered by site-directed mutagenesis for functional analysis of the
binding sites.
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Placental cell footprint 1 (FP1) contained a consensus binding site for
a member of the Ets family of transcription factors (38). Footprint 2
(FP2) closely resembled an AP-1-binding site (39, 40). A distinguishing
feature of FP1 in the presence of the placental/Rcho nuclear extracts
was a shift in DNAse I sensitivity from one nucleotide in the absence
of nuclear extract to the adjacent nucleotide (marked by an
arrow in Fig. 4
). The hypersensitive nucleotide is indicated
by an arrow on the antisense strand in Fig. 5
.
Functional Significance of the Ets- and AP-1-Binding Sites
To determine whether the identified Ets- and AP-1-binding sites
were functionally important for placental giant cell expression, we
carried out site-directed mutagenesis of the core sequences of these
sites, in the context of the active 3' StuI/EcoRI
fragment (F7). The sequence of this fragment with the transcription
factor-binding sites is shown in Fig. 5
. The nucleotides that were
changed are indicated by asterisks. The PCR primers used to
create the mutants are given in the Materials and Methods.
As well as the protected Ets- and AP-1-binding sites, two sequences
closely related to GATA-binding sites (AGATAT), located within the
context of a direct repeat sequence, were also mutated, even though no
DNAse I protection had been seen with placental/Rcho nuclear extracts
in that region. Our rationale for testing the functionality of these
sites was that GATA 2/3 have been implicated in the expression of
several other placental expressed genes including mPLI (8). When the
two putative GATA sites in the active 151-bp F7 fragment were separated
by an HphI digestion, the majority of the activity remained
in the 3'-F9 fragment, which already suggested that if GATA had a role
in rPLII transcription, both sites were not required. We cloned
fragments containing the individual mutations into pT81luc, transfected
these constructs into Rcho cells, and assayed for luciferase
expression. The results are shown in Fig. 6
. Mutation of either the Ets or the AP-1
site significantly affected luciferase gene expression as compared with
the wild-type sequence (P < 0.001), but neither
mutation alone was sufficient to completely eliminate enhancing
activity. Mutation of the Ets core sequence reduced luciferase activity
by approximately 70%; a mutated AP-1 site reduced activity by
approximately 40%. The luciferase activity of the double Ets/AP-1
mutant, however, was reduced to levels comparable to the vector control
and was significantly different from either of the single mutants
(P < 0.001). Luciferase activity of the construct with
mutated GATA sites was not significantly different from the native
fragment (P > 0.05), supporting the DNAse I protection
data that a GATA factor is not necessary for the enhancing activity of
this fragment.

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Figure 6. Site-Directed Mutagenesis Indicates a Role for Ets
and AP-1 Family Members in rPLII Placental Cell Regulation
The consensus Ets- and AP-1-binding sites that were protected by
placental cell nuclear extracts were individually and doubly mutated in
the context of the StuI/EcoRI fragment as
described in Materials and Methods. Two consensus GATA
sites in the context of a direct repeat were also mutated. The
nucleotides that were altered are indicated in Fig. 5 . The mutated and
native StuI/EcoRI pT81luc clones were
transiently transfected into Rcho cultures and assayed for luciferase
activity. Results are from two experiments with at least 12 separate
transfections and are expressed as a percentage of the luciferase
activity (mean ± SEM) of the unmutated
StuI/EcoRI fragment. The individual Ets
(mEts) and AP-1 (mAP-1) mutations have significantly (*) lower activity
(P < 0.001) than the native fragment; the combined
mEts/mAP-1 mutations reduce luciferase levels to those of the vector
control. There is no significant loss of activity when the GATA sites
are mutated (P > 0.05).
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In addition to mutation of the Ets- and AP-1-binding sites, we tested
the effect of overexpression of Ets2 and c-Fos/c-Jun on the
F9/luciferase construct in Rcho cells (Fig. 7
). Ets2 was chosen as a representative
of the Ets family of transcription factors since we had detected Ets2,
but not Ets1, mRNA in both placenta and Rcho cells (data not shown).
Ets2 has been implicated in the placental expression of other genes
(41, 42) and is known to interact with AP-1 proteins (43, 44).
Cotransfection of the c-Fos/c-Jun expression vectors with the reporter
construct produced a 2-fold increase in luciferase activity over the
luciferase vector alone, as did cotransfection of the Ets2 expression
clone. When Ets2/c-Fos/c-Jun were cotransfected, there was a further
increase in activity, which appeared to be additive.

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Figure 7. Cotransfection of Ets2 and c-Fos/c-Jun Expression
Vectors Increases Activity from the F9 Fragment
The human Ets2, rat c-Fos, and rat c-Jun cDNAs were cloned in the
expression vector pcDNA3 for these experiments. Five micrograms each of
Ets2 or c-Fos/c-Jun or Ets2/c-Fos/c-Jun were cotransfected with 10 µg
of the F9/TKpluc clone into Rcho cells. The total amount of transfected
plasmid was kept constant at 25 µg in all cases by the addition of
pcDNA3 vector. Results shown are from three experiments with 10
separate transfections; luciferase activities are expressed relative to
the F9/pT81luc clone alone, which was set at 1. Cotransfection of
either Ets2 or c-Fos/c-Jun increased luciferase activity approximately
2-fold. When all three clones were cotransfected, levels were increased
by approximately 4-fold. These levels were all significantly (*) higher
(P < 0.05) than the F9 clone alone.
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Electrophoretic Mobility Shift Studies of the F9 Enhancing
Fragment
Although these mutagenesis and overexpression studies strongly
implicated members of the Fos/Jun and Ets families of transcription
factors in the function of the rPLII enhancer in trophoblast cells, we
undertook to assess more directly the identity of the proteins that
bound to the DNAse I-protected sequences. Electrophoretic mobility
shift assays were carried out using in vitro translated
c-Fos, c-Jun, Ets1, and Ets2 proteins. Results are shown in Fig. 8A
. Using the labeled 65-bp F9 fragment
as a probe, a specific retarded complex was formed with a mixture of
recombinant c-Fos/c-Jun proteins, which was competed with both a
consensus AP-1-binding site oligonucleotide and Jun-specific antisera
but not by an unrelated oligonucleotide. A 41-bp oligonucleotide
containing mutations in the AP-1 and Ets sites also did not
compete. There was no shift of the fragment with recombinant
c-Jun alone, suggesting that only Fos/Jun dimers were bound.

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Figure 8. Assessment of AP-1 and Ets Factor Binding to the F9
Enhancer Fragment
A, The F9 fragment binds in vitro translated AP-1, but
not Ets1 or Ets2 proteins. Lane 1 contains the free probe. Lane 2 shows
the complexes formed when in vitro translated c-Fos and
c-Jun dimers bind to the labeled 65-bp F9 fragment. Addition of a
consensus AP-1-binding site at 100-fold molar excess (lane 3) or AP-1
antisera (lane 5) specifically compete the upper band, marked by an
arrow. Neither an unrelated oligonucleotide (lane 4) nor
an oligonucleotide containing a mutated AP-1 site (lane 6) competes the
specific complex at 500-fold molar excess. C-Jun protein alone does not
produce the specific complex (lane 7). No specific complexes are formed
in the presence of in vitro translated Ets1 (lane 8) or
Ets2 (lane 9). B, The F9 fragment forms different complexes with
placental and GC nuclear extracts. Lanes 16 show complexes formed
with rat placenta nuclear extract. A number of complexes are produced
(lane 1); those complexes that are competed with 200-fold molar excess
of the probe (lane 4) are marked with arrows and numbered.
None of the complexes appear to be competed by a 500-fold excess of an
Ets2-binding site (lane 2), but complex 2 is competed by a 150-fold
excess of a consensus AP-1 oligonucleotide (lane 3). A 41-bp
oligonucleotide containing mutations in the AP-1- and Ets-binding sites
at 500-fold excess does not compete for complexes 2, 3, or 4, but does
compete complex 1 (lane 5). An unrelated oligonucleotide at 500-fold
excess does not compete any complexes (lane 6). Complexes formed with
GC nuclear extracts are shown in lanes 712. Neither an Ets2 nor an
AP-1 consensus binding site competes at 500-fold excess for any of the
complexes (lanes 8 and 9). All complexes are partially competed by
unlabeled probe at 400-fold excess (lane 10), as well as by a 500-fold
excess of the oligonucleotide containing mutations in the AP- and
Ets-binding sites (lane 11). There is no competition by an unrelated
oligonucleotide at 500-fold excess (lane 12).
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We were unable to detect a specific shift of the F9 fragment, however,
with Ets2 or the related Ets1. When either Ets protein was incubated
together with c-Fos/c-Jun in the binding reaction, only the AP-1
complex was formed (data not shown), suggesting that the lack of Ets
factor binding was not due to a requirement for initial formation of an
AP-1 complex. Although the Ets consensus sequence always contains
GGA(A) at its core and there is a degree of promiscuity among the
binding sites for different Ets proteins in vitro, the
specificity of binding in this family is thought to involve sequences
outside the core region (38). The sequence of the protected FP1 region
is related but not identical to that of the Ets1 and -2-binding sites
(38) and must specify a binding site for another member of this large
family.
When the F9 fragment was incubated with placental nuclear extracts,
several specific complexes were formed (Fig. 8B
). One of these was
competed by both the 65-bp F9 fragment and the consensus AP-1
oligonucleotide, suggesting that it represented an interaction with
c-Fos/c-Jun proteins. This complex was not competed by the
oligonucleotide that contained mutations in the AP-1 and Ets consensus
binding sites. At least one other complex was specifically competed
with the F9 fragment, but not by the oligonucleotide with the mutated
binding sites, suggesting that it may represent a complex containing an
Ets family-binding protein. Neither this nor any other complex,
however, was competed by a consensus Ets2 oligonucleotide. A further
larger complex was specifically competed by both the F9 fragment and
the oligonucleotide containing the mutations, but not by the
nonspecific competitor. The composition of this complex is unclear.
The complexes formed with nuclear extracts from GC cells were markedly
different from those seen with placental extracts (Fig. 8B
). Neither
the consensus AP-1 or Ets2 oligonucleotide was able to compete any of
the GC cell complexes even at 500-fold molar excess. Unlike in the
placental extracts, both the native F9 fragment and the oligonucleotide
with the mutated AP-1- and Ets-binding sites partially competed all the
complexes, suggesting that the binding to the F9 fragment in the GC
extracts does not involve the core AP-1- or Ets-binding sites.
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DISCUSSION
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We previously identified a 3-kb proximal 5'-flanking region of the
rPLII gene that is important for gene transcription in the trophoblast
Rcho cell line and the placentas of transgenic mice but that does not
function in the rat pituitary GC cell line (30). In our current study
we have examined this rPLII 5' region in detail to identify the
regulatory elements that are involved in placental cell expression.
Using deletion analysis and transfection assays with luciferase
reporter constructs in Rcho cell cultures, we have identified an
EcoRI fragment from -2838 to -1729, which when deleted
results in a significant decrease in luciferase activity. Consistent
with the presence of enhancing sequences within this fragment is its
ability to activate luciferase gene expression in either a forward or
reverse orientation from the herpes simplex thymidine kinase minimal
promoter. Enhancement occurs in Rcho trophoblast cells but not in rat
pituitary GC cells, suggesting that specific placental protein factors
or complexes are binding to sequences within this fragment. The
enhancement occurs whether the Rcho cultures have been grown for 6 days
or 14 days before transfection. We have previously noted that our Rcho
cultures express rPLI mRNA shortly after plating, essentially as soon
as giant cells differentiate in these cultures, but that rPLII mRNA is
expressed at only low levels until about 14 days after plating,
suggesting that further developmental changes may be occurring in these
cultures (25). The fact that both early and late cultures show
comparable enhancement of reporter gene expression suggests that we may
have identified a placental cell enhancer that is not directly involved
in the temporal regulation of the rPLII gene. Nonetheless, since rPLII
mRNA is still detectable in RNA blots of the earlier Rcho cultures, we
cannot rule out that sufficient amounts of factors are present in these
cultures to interact with the rPLII enhancer sequences and bring about
increased expression. The role of these sequences for the normal
developmental expression of rPLII mRNA will require further
investigation.
Detailed deletion analysis of the EcoRI fragment indicates
that the enhancing activity is located at -1793 to -1729 on a 3'
HphI/EcoRI fragment (F9). DNAse I protection
studies identify two adjacent regions between approximately -1765 and
-1730 that show similar patterns of protection by nuclear extracts
from late-term rat placental labyrinth and Rcho cells. FP1 appears to
be protected more by the placental nuclear extracts, while footprint 2
(FP2) is protected more by the Rcho extracts at comparable protein
concentrations, suggesting that the cognate factors are present in
different proportions in the two nuclear extracts and that they can
bind independently to the DNA. FP1 lies over a region that contains a
consensus sequence for the Ets family of transcription factors
(C/AGGAA/T) (38), while FP2 is related to an AP-1-binding site (39, 40). A distinctive feature of FP1, in the placental cell extracts, is
the appearance of a new hypersensitive site on the noncoding strand at
the nucleotide immediately 5' of the consensus Ets-binding sequence.
Although nuclear extracts from GC cells show protection of the FP1 and
FP2 regions, the patterns of protection are different. With GC
extracts, new hypersensitive sites appear in the FP2 region and
immediately 3' of the region designated FP1, neither of which are seen
with the placental or Rcho cell nuclear extracts. Gel mobility shift
studies also show that the complexes formed by GC extracts on the F9
fragment are markedly different from those seen with placental nuclear
extract. These data strongly suggest that the complexes formed in GC
cells are distinct from those in placental cells and, from our
transfection studies, inactive.
Electrophoretic mobility shift studies using in vitro
transcribed and translated rat c-Fos and c-Jun proteins (45) confirm
that the 65-bp F9 fragment binds Fos/Jun dimers but not Jun dimers
alone; a consensus AP-1 oligonucleotide-binding site is specifically
able to compete this complex as well as a complex formed between the F9
fragment and placental nuclear extract. This latter complex is not
competed by an oligonucleotide containing a mutant version of the F9
AP-1-binding site.
Although AP-1 transcription factors have been implicated in the
placental expression of several placental genes including the mouse and
rat PLI, the involvement of the Ets family of transcription factors in
placental gene expression is less well documented. Ets proteins form a
large family (now >33 members), which is defined by a highly conserved
DNA-binding domain of about 85 amino acids (38). All family members
bind DNA elements with the core consensus sequence GGAA/T (38). The
specificity of these proteins appears to lie in the sequences that
surround this core region, and although there is evidence of
overlapping binding specificity, not all identified Ets sites bind all
members of the family (46, 47). Ets1 and Ets2 have both been implicated
in the placental expression of the matrix metalloprotease genes,
stromelysin (MMP3) and collagenase (MMP1), in the human and have been
shown to cooperate with AP-1 factors in this role (41, 43). Very
recently the targeted mutation of the mouse Ets2 gene was found to have
profound effects on early placental development primarily mediated
through deficient expression of the MMP9 (gelatinase B) gene, which
resulted in failed implantation (42). It was also observed that in the
absence of Ets2, mPLI levels were elevated in the very early
placenta.
Since Ets2 and Ets1 have been implicated in the expression of several
of the MMP genes in placenta and we were able to detect Ets2 (although
not Ets1) mRNA in developing rat placenta and Rcho cells (data not
shown) we tested these in vitro translated proteins for the
ability to bind the 65-bp F9 fragment in gel mobility shift studies.
Neither recombinant Ets2 or Ets1 protein binds the F9 fragment; they
both shift a consensus Ets2-binding site (48). This consensus
Ets2-binding site does not compete any of the complexes formed between
placental nuclear extract and the F9 fragment. There is, however, a
specific placental complex that is competed by the native sequence but
is not competed by an oligonucleotide containing a mutation of the GGA
core of the Ets-binding site. These results strongly suggest that Ets2
is not the family member that binds the rPLII enhancer GGA sequence
directly in vivo, and the identity of that family member
remains to be determined. We cannot rule out, however, that Ets2 could
still have a role through protein-protein interactions with another Ets
proteins. Such interactions have been previously reported for the human
stromelysin gene (43).
The physiological importance of the AP-1 and Ets-binding sites in rPLII
placental expression is supported by the effect of mutations of each
core sequence on the enhancing activity of the active fragment.
Mutation of the Ets site alone reduces activity to approximately 30%
of that of the native -1880 to -1729 F7 fragment, while mutation of
the AP-1 site reduces luciferase activity to approximately 60% of the
nonmutagenized fragment. The double Ets/AP-1 mutant eliminates the
enhancing activity of the fragment indicating that both the Ets and
AP-1 sites are important in this activity. The fact that neither
mutation alone causes complete loss of enhancing activity again
suggests that the binding of each protein at these adjacent sites may
be independent. Cotransfection of c-Fos/c-Jun or Ets2 expression clones
with the F9/luciferase construct in Rcho cells results in an increase
in reporter gene expression in both cases. When c-Fos, c-Jun, and Ets2
are co-transfected together, there is a further increase in reporter
gene expression that appears to be additive. Given that our gel shift
data do not demonstrate binding of Ets2 to the F9 fragment, it appears
that its overexpression may be acting indirectly. In this regard, it
has been reported that Ets2 binds to and activates the jun-B
and c-Fos promoters (49, 50).
Unlike the PLI gene, and some other placental genes, members of the
GATA family of transcription factors do not have a role in the
enhancing activity of this rPLII fragment. There is no protection by
the placental nuclear extracts of two putative GATA sites found in this
active fragment that are adjacent and occur within the context of a
direct repeat (AGATATGTAGATATGT) at -1799 to
-1785. Mutation of both core GATA sequences has a slight but not
significant effect (P > 0.05) on luciferase activity,
supporting our conclusion from the DNAse I protection data.
Very recently, work on the 5'-flanking portion of the mouse PLII gene
has also identified a region containing activating DNA sequences (51).
This region from -1471 to -1340 has a marked similarity to the
segment of the rPLII gene immediately 3' of our reported sequence and
overlaps our F9 fragment by 27 bp at its 5'-end. A comparison of the
rat and mouse PLII sequences is shown in Fig. 9
. It is intriguing that although the
most 5'-region of the mPLII gene, designated region m1 and shown by
mutagenesis studies to contain an enhancing sequence, is highly related
to our FP1 region, it does not contain an Ets-binding site. Gel
mobility shift studies with Rcho extracts showed the formation of
specific complexes on the m1 sequence, but this sequence does not
represent a binding site for a known transcription factor. The more 5'
AP-1-binding site we identified in the rat sequence is not represented
in the activating mouse PLII sequence. The region of the mPLII gene
designated m7, which was also shown to contain enhancing sequences, is
less highly conserved between the rat and mouse, while the activating
m8 and m9 regions are strongly conserved, including the consensus GATA
site, which was found not to bind a GATA factor. It therefore appears
that, in spite of the overall high degree of relatedness between the
PLII genes in these species, somewhat different regulatory mechanisms
have evolved in the rat and mouse, and it will be interesting to
determine the precise identity of the factors responsible for enhancing
the transcription of these two genes.

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Figure 9. A Comparison of rPLII and mPLII 5'-Enhancing
Sequences
The mPLII sequence is from Lin and Linzer (51 ). The mPLII fragment
overlaps at its 5'-end with the most 3'-end of the rPLII F9 fragment as
indicated in the different type. Gaps have been added to the sequences
to increase homology; mismatches in the mPLII sequence are shown in
lowercase letters. The brackets indicate
the regions of the mPLII gene that were systematically mutated. The
mPLII fragment has been reported to contain positive regulatory
elements in the regions designated m1 and m7m9. The m1 region
corresponds to the region of a consensus Ets-binding site in the rPLII
gene that is underlined. When the m1 region of the mouse
sequence is mutated, there is a loss in enhancing activity. A consensus
GATA site that was found not to bind a GATA factor is
underlined in both sequences.
|
|
The DNAse I protection patterns and gel mobility shift complexes formed
between GC nuclear extracts and the 65-bp fragment are distinct from
those seen with the placental extracts and indicate the binding of
markedly different protein complexes in these two tissues. The factors
involved in the GC complexes are unclear. An AP-1 consensus binding
site does not compete any F9/GC complex, and a oligonucleotide
containing mutant AP-1- and Ets-binding sites competes similarly to the
native sequence. Combined with our previous study in transgenic mice
(30), our new data strongly suggest that the enhancing region we have
identified has an important role in the placental cell-specific
expression of the rPLII gene. The complete developmental expression of
the rPLII gene, however, may be dependent on the presence of further
regulatory sequences. Our initial deletion experiments of the 3-kb
5'-flanking fragment identified other potentially important but as yet
uncharacterized regions. Sequences between -1435 to -765 are
essential for expression in Rcho cells, although at a low level; other
sequences appear to have negative effects on reporter gene
transcription as demonstrated by a marked decrease in activity when
included in the absence of the enhancing region (-3031 to -2838) or
by a marked increase in expression when deleted (-1729 to
-1435).
A comparison of DNA sequences and transcription factors that have been
reported to be important in placental-specific gene expression, now
including the rPLII enhancer, suggests that there may not be a single
combination of factors that marks a gene for placental expression. The
sequence we have described does not contain an AP2 (TSE) site, a
functional GATA site, or a basic helix-loop-helix binding site,
all of which have been previously associated with placental gene
expression. In particular, the rPLII enhancer is different from the
sequences that were identified as important for the expression of the
mPLI and the mPLII genes, suggesting that even the same placental cell
type may use different combinations of transcription factors for the
regulation of expression of different genes.
 |
MATERIALS AND METHODS
|
---|
Materials
Some restriction enzymes, Klenow DNA polymerase, and T4 DNA
ligase were from Pharmacia (Baie dUrfé, Québec, Canada.);
FBS, RPMI 1640 medium, DMEM, sodium pyruvate, HEPES buffer,
penicillin/streptomycin, trypsin/EDTA, NUNC culture dishes and flasks,
agarose, some restriction enzymes, NACS columns, and
custom-synthesized oligonucleotides were from Life Technologies
(Burlington, Ontario, Canada); NCTC 135 medium was from Sigma-Aldrich
(Oakville, Ontario, Canada); random prime DNA labeling kits and
Sequenase kits were from Amersham (Oakville, Ontario, Canada);
luciferase assays kits, reporter lysis buffer, PCR sequencing, and
in vitro transcription/translation kits and DNAse I (RQ)
were from Promega (Madison, WI); AP-1 consensus oligo, AP-1, and Ets2
antisera were from Santa Cruz Biotechnology (Santa Cruz, CA); Nitroplus
membrane, guanidine isothiocyanate, and general laboratory chemicals
were from Fisher Scientific (Nepean, Ontario, Canada);
[32P]dCTP, [35S]dATP, and
[32P
]ATP were from Mandel Scientific (Guelph, Ontario,
Canada); Bio-Rad protein assay reagent was from Bio-Rad Laboratories
(Mississauga, Ontario, Canada); Qiagen DNA plasmid columns were from
Qiagen Inc. (Mississauga, Ontario, Canada); Kodak XAR film was from
Intersciences Inc. (Markham, Ontario, Canada).
Cell Lines
The rat choriocarcinoma Rcho cell line (32) was kindly provided
by Drs. A. Verstuyf and M. Vandeputte (Rega Institute for Medical
Research, Catholic University of Louvain, Louvain, Belgium). The rat
pituitary GC cell line was a gift from Dr. P. A. Cattini
(Department of Physiology, University of Manitoba, Winnipeg,
Canada).
Clones and Vectors
The rPLII genomic clone, GC I, and some subclones are described
by Shah et al. (30). Other vectors and clones were
generously provided as follows: luciferase vector pXP2 (37), a
cytomegalovirus promoter/luciferase construct (CMVp.luc), and an herpes
simplex thymidine kinase promoter/luciferase vector, pT81luc (37), Dr.
R. J. Matusik, Department of Urological Surgery, Vanderbilt
University, Nashville, TN; a cytomegalovirus promoter/chloramphenicol
transacetylase construct (pcDNA3.cat), Dr. R. P. C. Shiu,
Department of Physiology, University of Manitoba, Winnipeg, Canada;
hEts1 and hEts2 cDNA clones (52), Dr. P. A. Cattini; rat c-Fos and
rat c-Jun (45), Dr. T. Curran, St Jude Childrens Research Hospital,
Memphis, TN.
rPLII 5'-Flanking Plasmid Clones
5'-Flanking plasmid subclones were constructed from an rPLII
-genomic clone, GC I (30). We have shown that a -3031 5'-flanking
fragment proximal to the transcription start site is sufficient to
direct reporter gene expression in the rat trophoblast Rcho cell line
and the placenta of transient transgenic mice. All plasmid DNAs were
prepared from overnight bacterial cultures using Qiagen DNA plasmid
columns according to the suppliers protocol. DNA sequencing of the
clones was carried out by the dideoxy method (53) using Sequenase or a
PCR sequencing kit and [35S]dATP. All restriction enzyme
fragments were purified by agarose gel electrophoresis followed by
electroelution and ethanol precipitation.
Chimeric rPLII 5'-flanking/luciferase clones containing the native
rPLII promoter were constructed in the vector pXP2. Clones are named
according to the 5'-end of the fragment; the 3'-end of these clones
originates at position +64. The clones -765rPLIIp.luc and
-3031rPLIIp.luc have been previously described (30). Further rPLII
native promoter luciferase clones were constructed as follows: a 4.5-kb
5'-rPLII fragment cloned into the HindIII/BamHI
sites in pBspSK (Stratagene, La Jolla, CA) was digested with
EcoRI/BamHI, and the isolated fragment was
recloned into EcoRI/BamHI cut pBspSK to remove
sequences between -3031 to -1729 and conserve a 5'-HindIII
cloning site; a HindIII/BamHI fragment of this
clone was isolated and ligated into
HindIII/BglII-digested pXP2 to form
-1729rPLIIp.luc. -1729rPLIIp.luc was digested with
BamHI/BglII and religated to form
-1435rPLIIp.luc. A pBspSK clone containing the PvuII
fragment from -765 to +64 was digested with
EcoRV/BamHI and cloned into the
SmaI/BglII site of pXP2 to form
-118-rPLIIp.luc. To produce the clone,
ErPLIIp.luc, deleted for
-2838 to -1729, the -4.5-kb 5'flanking pBspSK clone, was digested
with EcoRI and religated; this deletion clone was further
digested with SacI/BamHI and cloned into
SacI/BglII-cut pXP2.
The analysis of the -2838 to -1729 EcoRI fragment for
placental-specific enhancing activity in the context of a heterologous
promoter was carried out in the luciferase reporter vector pT81luc,
which contains a minimal (-81 to +52) herpes simplex thymidine kinase
promoter. Clones were constructed as follows: the EcoRI
fragment was cloned into EcoRI-digested pBspSK to produce
E/EpBspSK in both 5'
3' and 3'
5' directions as determined by
sequencing; this fragment was removed from the 5'
3'
(E/EF) clone by HindIII/SmaI
digestion and cloned into HindIII/SmaI-digested
pT81luc to produce E/EFTKpluc (5'
3'); the fragment from
the 3'
5'pBspSK (E/ER) clone was digested with
BamHI/EcoRV and ligated into
BamHI/SmaI-cut pT81luc to give
E/ERTKpluc. Subfragments of the EcoRI fragment
were ligated into the luciferase reporter vector, pT81luc, as follows,
moving from the 5'- to 3'-end of the fragment: E/EFpBspSK
was digested with HindIII/DraI, and the
5'-HindIII/DraI fragment (F1) was isolated and
ligated to HindIII/SmaI cut vector; an internal
DraI/DraI fragment (F2) was isolated and ligated
to SmaI cut vector. E/EFpBspSK was also digested
with DraI/SacI, and the
3'-DraI/SacI fragment (F3) was isolated and
ligated to SmaI/SacI cut vector. A
HindIII/SacI fragment isolated from the F3 clone
was further digested with BsaBI. The
5'-HindIII/BsaBI fragment (F4) was cloned into
HindIII/SmaI cut vector; the
3'-BsaBI/SacI fragment (F5) was cloned into
SmaI/SacI cut vector. The F5 clone was digested
with HindIII and StuI, and the 5'
HindIII/StuI fragment (F6) was isolated and
ligated into HindIII/SmaI cut vector; the F5
clone was also digested with StuI/SacI, and the
3'-StuI/SacI fragment (F7) was isolated and
ligated into SmaI/SacI cut vector. A
BamHI fragment from F7TKpluc (polylinker sites) was digested
with HphI and blunt-ended with Klenow polymerase. This
fragment was further digested with HindIII and
SacI; the 5'-HindIII/HphI fragment
(F8) was cloned into HindIII/SmaI cut vector; the
3'-HphI/SacI fragment (F9) was cloned into
SmaI/SacI cut vector.
All constructs were sequenced across the newly created boundaries to
determine correct ligations.
Cell Culture and Transient Transfection Assays
The rat choriocarcinoma Rcho cell line was grown routinely on
RPMI 1640 medium containing HEPES buffer, supplemented with
heat-inactivated 20% FBS, 1 mM sodium pyruvate, 50
µM ß-mercaptoethanol, 50 U/ml streptomycin, and 50
µg/ml penicillin. Medium was changed every other day, and cells were
split before confluency, every 3 days, using trypsin/EDTA. When grown
for transfections and nuclear extract preparations, cells were changed
after 4 days to NCTC 135 medium with 10% FCS and supplements that
stimulate giant cell differentiation (31). The rat anterior pituitary
GC cell line was grown in DMEM as described by Cattini and Eberhardt
(54).
The Rcho cells were transfected using the calcium phosphate method
essentially as described by Vuille et al. (55) at the times
indicated. Transfections were routinely carried out in 10-cm dishes
using 10 µg of test plasmids and 1 µg of CMVp.cat for
determining transfection efficiency. The rat anterior pituitary GC
cells were grown to 4050% confluency and transfected as described by
Nickel et al. (56).
Cell extracts were prepared as previously described (30) except that
reporter lysis buffer (Promega, Madison, WI) was used. Luciferase
assays were carried out immediately after lysate preparation using a
Promega Luciferase Assay kit according to suppliers instructions.
Activity was measured in relative light units using a TROPIX
luminometer (Bio/Can Scientific, Mississauga, Ontario, Canada). The
chloramphenicol acetyltransferase activity was measured by the
two-phase fluor diffusion assay as described by Nickel et
al. (56). To standardize for variations in plasmid uptake, all
luciferase activities were normalized to the CAT assay data for the
same sample. Protein determinations were carried out using a Bio-Rad
protein assay reagent according to the suppliers protocol.
Nuclear Extract Preparation
Nuclear extracts were prepared from Rcho and GC cell lines, and
rat placental labyrinth regions collected and dissected at days 14 to
16, according to published protocols (57) with the following
modifications. Rcho cultures were grown for 6 days as described;
remaining small undifferentiated cells were first removed by a short
0.25% trypsin pretreatment before giant cells were collected for
nuclei (15). Frozen placentas were ground to a fine powder in a mortar
and pestle placed on dry ice; placental nuclear extracts were prepared
as for the cell culture extracts with the initial volume being taken as
the volume of powdered tissue. All procedures involving animals were
carried out according to protocols approved by the University of
Manitoba Animal Care Committee.
DNAse I Protection Assays
DNAse I protection assays of the 329-bp rPLII 5'-flanking
BsaBI/EcoRI fragment (F5) described above were
carried out according to standard protocols (58). The F7TKpluc clone
was digested at an XhoI site in the pT81luc polylinker and
treated with calf intestinal phosphatase, and the antisense strand was
end-labeled with T4 polynucleotide kinase and
[32P
]ATP. The fragment was released by a
HindIII digestion and purified by agarose gel
electrophoresis, followed by electroelution and ethanol precipitation.
Binding reactions were carried out in a final volume of 20 µl
containing approximately 20,000 cpm (510 fmol) of fragment and 0.5
µg dI:dC. Increasing amounts of placental labyrinth and Rcho nuclear
extracts (1080 µg) or GC nuclear extracts (20 and 40 µg) were
incubated with the probe on ice for 15 min followed by digestion with
0.05 U of DNAse I for 90 sec at 26 C. After phenol/chloroform
extraction and precipitation, the digested products were fractionated
on a denaturing 6% polyacrylamide/urea gel and exposed to Kodak XAR
film for autoradiography.
Electrophoretic Mobility Shift Assays
The 65-bp F9 fragment that had been cloned into the
SmaI/EcoRI sites of pBspSK was cut out with
EcoRI, electroeluted from an agarose gel, and end-labeled
with Klenow fragment and
-dATP. Recombinant c-Fos, c-Jun, Ets1, and
Ets2 proteins were synthesized using an in vitro
transcription/translation kit according to the suppliers protocol.
Nuclear extracts were made as described. Four microliters each of one
or more of the in vitro translated mixtures, 20 µg of rat
placental nuclear extract, or 5 µg of GC extract were used in a
binding reaction. Reactions were carried out in 40 µl of binding
buffer containing 20 mM HEPES buffer, pH 7.9, 100
mM KCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 5 mM MgCl2, 1 mM
phenylmethylsulfonylfluoride, 0.5 µg dI:dC. Specific or nonspecific
competitors were added for 15 min before the labeled probe; Jun or Ets2
antisera were added 45 min before the probe; 10 to 20 fmoles of labeled
probe (12 x 104 dpm) were incubated for a further
30 min. All binding reactions were at room temperature. Complexes were
separated by electrophoresis on 4% nondenaturing polyacrylamide gels.
Gels were dried and exposed for autoradiography.
Coding strands of specific competitor oligonucleotides used:
AP-1 consensus: CGCTTGATGACTCAGCCGGAA
Ets2 consensus (48): CTAGGACCGGAAGTGGGAGT
mAP-1/mEts: CCAGGGTTATTTctagAAGGGTAAACAttcAGTAGGGCTTG
Site-Directed Mutagenesis
A PCR strategy was used to mutate transcription factor-binding
sites that had been identified by DNAse I protection studies and
sequence analysis. These sites included a consensus Ets-binding site, a
putative AP-1-binding site, and a pair of putative GATA-binding sites
found within a direct repeat sequence. The
StuI/EcoRI fragment that had been shown to retain
activity in transfection assays was cloned into the
SmaI/SacI sites of pBspKS. Three mutagenic
primers were synthesized as follows:
mEts:
GCGCGAGCTCGAATTCAAGCCCTACTgaaTGTTTACCCTTGAGCA
mAP-1:
GCGCGAGCTCGAATTCAAGCCCTACTTCCTGTTTACCCTTctagAAATAACCCTGGAAATG
mGATA:
GCGCGAGCTCGAATTCAAGCCCTACTTCCTGTTTACCCTTGAGCAAATAACCCTGGAAATGCGTAAAACACATcggTACATAcgTTACTCACC
Sequences are shown 5'
3'; mEts mutates a putative Ets-binding site,
mAP-1 mutates a putative AP-1 site; and mGATA mutates two putative GATA
sites all shown in lower case. In addition to the mutated
sequences, each primer included a SacI site and a 4-bp GCGC
extension at the 3'-end of the primers (underlined) to
facilitate directional cloning. The M13 reverse primer was used with
the mutagenic primers in PCR reactions. PCR fragments were isolated by
agarose gel electrophoresis and electroelution, digested with
HindIII/SacI, and cloned into these sites in
pBspKS for sequencing to confirm that only the expected mutations had
been introduced. The mutated HindIII/SacI
fragments were cloned into HindIII/SacI-digested
pT81luc for testing in transfection assays. The double Ets/AP-1 mutant
was generated by PCR as described using the mAP-1 primer with the mEts
pBsp clone. PCR product was cloned into pBspKS as described above for
the single mutants to facilitate sequencing, followed by cloning of the
correct fragment into pT81luc.
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Agnes Fresnoza for her excellent technical
assistance. We thank Dr. Peter Cattini for his helpful and stimulating
discussions and for the generous use of his luminometer. We also thank
the members of the Cattini laboratory for their advice on nuclear
extract preparation and setting up DNAse I protection and gel
electrophoretic mobility shift assays.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Mary Lynn Duckworth, Department of Physiology, University of Manitoba, Room 421 Basic Medical Sciences Building, 730 William Avenue, Winnipeg, Manitoba, R3E 3J7 Canada. E-mail: mdckwth{at}cc.umanitoba.ca
This work was supported by grants from the Medical Research Council of
Canada, the Manitoba Medical Services Foundation, and the Manitoba
Health Research Council.
Received for publication March 3, 1998.
Revision received October 2, 1998.
Accepted for publication November 11, 1998.
 |
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